A NOTCH3 homozygous nonsense mutation in familial Sneddon syndrome with pediatric stroke.

Sneddon syndrome is a rare disorder affecting small and medium-sized blood vessels that is characterized by the association of livedo reticularis and stroke. We performed whole-exome sequencing (WES) in 2 affected siblings of a consanguineous family with childhood-onset stroke and identified a homozygous nonsense mutation within the epidermal growth factor repeat (EGFr) 19 of NOTCH3, p.(Arg735Ter). WES of 6 additional cases with adult-onset stroke revealed 2 patients carrying heterozygous loss-of-function variants in putative NOTCH3 downstream genes, ANGPTL4, and PALLD. Our findings suggest that impaired NOTCH3 signaling is one underlying disease mechanism and that bi-allelic loss-of-function mutation in NOTCH3 is a cause of familial Sneddon syndrome with pediatric stroke.

Tildrakizumab for the treatment of psoriasis.

Psoriasis is a chronic skin disorder driven by IL-23 and the downstream T-helper cell 17 (Th17) pathway. Tildrakizumab is a humanized monoclonal antibody selectively targeting the p19 subunit of IL-23, a key cytokine for Th17 cells. Here, we provide an overview of IL-23 in the context of psoriasis pathogenesis and review the results of the Phase I, II and III clinical trials for tildrakizumab in patients with moderate-to-severe chronic plaque psoriasis in order to assess its efficacy, safety and clinical usefulness. In all clinical trials, tildrakizumab demonstrated significant clinical improvement and a favorable safety profile. In Phase III trials, 75% of tildrakizumab-treated patients reached a Psoriasis Area and Severity Index 75 at week 28 demonstrating superior efficacy as compared with etanercept treatment. The tildrakizumab-induced reduction in skin inflammation proves the important pathogenic role of IL-23 in psoriasis and further supports the utility of drugs targeting the IL-23/Th17 pathway. Targeting IL-23p19 with tildrakizumab augments the therapeutic repertoire for patients with moderate-to-severe chronic plaque psoriasis.

Ustekinumab treatment in severe atopic dermatitis: Down-regulation of T-helper 2/22 expression.

BACKGROUND: It has recently been suggested that patients with moderate to severe atopic dermatitis (AD) may profit from anti-interleukin (IL)-12/-23 p40 therapy. OBJECTIVE: We sought to assess the immunologic effects of ustekinumab treatment on AD skin and to correlate them with the clinical efficacy of this drug. METHODS: We investigated the course of 3 patients with severe AD who were administered 45 mg of subcutaneous ustekinumab over a period of 16 weeks. Clinical scores and skin biopsy specimens, taken at baseline and at week 8, were used to assess changes in disease severity. RESULTS: All patients showed a gradual improvement of the disease, achieving a 50% reduction in the Eczema Area and Severity Index score by week 16. Immunohistology of skin biopsy specimens revealed a significant decrease in the degree of epidermal hyperplasia/proliferation and the number of infiltrating dermal T cells, dendritic cells, and mast cells after treatment. Using quantitative real-time polymerase chain reaction of lesional skin, we found a clear reduction of T-helper 2-/22-associated molecules after therapy. LIMITATIONS: The small number of patients (n = 3) limits efficacy analysis and warrants prospective placebo-controlled studies in larger patient cohorts. CONCLUSION: Blocking IL-12/-23 p40 could be beneficial for a subgroup of patients with severely infiltrated AD.

Surgical Elimination of the Gastric Digestion by Roux-en-Y Gastric Bypass Impacts on Food Sensitisation-a Pilot Study.

BACKGROUND: Impairment of gastric digestion due to pH elevation increases the risk for food allergy induction. As patients after Roux-en-Y gastric bypass (RYGB) surgery have lower gastric acidity and less gastric gland secretion, we aimed to analyse in a prospective study the effect of limiting gastric digestion capacity by surgical intervention on the immune response towards allergens. METHODS: Nine patients undergoing RYGB surgery for morbid obesity and one control patient having undergone surgery for treatment of an incisional hernia were enrolled in the study. Before and 1, 3, 6, 9 and 12 months after surgery, blood was collected for analysis of specific IgE antibodies, and patients were subjected to skin prick testing with 16 food and 18 aeroallergens. RESULTS: Skin prick test results revealed an increase of positive reactions indicating sensitisations towards the tested food and aeroallergens in 77.8 and 88.9 % of the patients, respectively, after surgical elimination of gastric digestion. These results were in line with elevated titers of food- and aeroallergen-specific IgE antibodies in 7 out of 9 (7/9) and 5/9 patients, respectively, after RYGB surgery. Serum cytokine levels revealed a mixed response for IFN-γ and were mostly beneath detection limit for IL-4. CONCLUSION: A change of IgE reactivity pattern occurred after impairment of gastric digestion due to surgical elimination underlining the important gastric gatekeeping function during oral sensitisation. Even though this study indicates an increased allergy risk for gastric bypass patients, further studies are needed to investigate in-depth the immunological changes associated with RYGB surgery.

Clinical improvement in psoriasis with specific targeting of interleukin-23.

Psoriasis is a chronic inflammatory skin disorder that affects approximately 2-3% of the population worldwide and has severe effects on patients' physical and psychological well-being. The discovery that psoriasis is an immune-mediated disease has led to more targeted, effective therapies; recent advances have focused on the interleukin (IL)-12/23p40 subunit shared by IL-12 and IL-23. Evidence suggests that specific inhibition of IL-23 would result in improvement in psoriasis. Here we evaluate tildrakizumab, a monoclonal antibody that targets the IL-23p19 subunit, in a three-part, randomized, placebo-controlled, sequential, rising multiple-dose phase I study in patients with moderate-to-severe psoriasis to provide clinical proof that specific targeting of IL-23p19 results in symptomatic improvement of disease severity in human subjects. A 75% reduction in the psoriasis area and severity index (PASI) score (PASI75) was achieved by all subjects in parts 1 and 3 (pooled) in the 3 and 10 mg kg(-1) groups by day 196. In part 2, 10 out of 15 subjects in the 3 mg kg(-1) group and 13 out of 14 subjects in the 10 mg kg(-1) group achieved a PASI75 by day 112. Tildrakizumab demonstrated important clinical improvement in moderate-to-severe psoriasis patients as demonstrated by improvements in PASI scores and histological samples.

Specific roles for dendritic cell subsets during initiation and progression of psoriasis.

Several subtypes of APCs are found in psoriasis patients, but their involvement in disease pathogenesis is poorly understood. Here, we investigated the contribution of Langerhans cells (LCs) and plasmacytoid DCs (pDCs) in psoriasis. In human psoriatic lesions and in a psoriasis mouse model (DKO* mice), LCs are severely reduced, whereas pDCs are increased. Depletion of pDCs in DKO* mice prior to psoriasis induction resulted in a milder phenotype, whereas depletion during active disease had no effect. In contrast, while depletion of Langerin-expressing APCs before disease onset had no effect, depletion from diseased mice aggravated psoriasis symptoms. Disease aggravation was due to the absence of LCs, but not other Langerin-expressing APCs. LCs derived from DKO* mice produced increased IL-10 levels, suggesting an immunosuppressive function. Moreover, IL-23 production was high in psoriatic mice and further increased in the absence of LCs. Conversely, pDC depletion resulted in reduced IL-23 production, and therapeutic inhibition of IL-23R signaling ameliorated disease symptoms. Therefore, LCs have an anti-inflammatory role during active psoriatic disease, while pDCs exert an instigatory function during disease initiation.

Association of the chromosome 11q13.5 variant with atopic dermatitis in Austrian patients.

BACKGROUND: Recently, the two single nucleotide polymorphisms, rs7927894 on chromosome 11q13.5 and rs877776 within the region of the hornerin gene, were identified as novel susceptibility variants for atopic dermatitis in the first genome wide association study in atopic dermatitis. OBJECTIVE: The aim of our study was to evaluate the influence of these two genetic variants on atopic dermatitis and disease-related phenotypes in the Austrian population. METHODS: 275 atopic dermatitis patients and 243 controls were genotyped for the two variants rs7927894 and rs877776 by using Taqman based allelic discrimination assays. RESULTS: When comparing patients with controls we found a significant association of the rs7927894 variant on chromosome 11q13.5 with atopic dermatitis (OR: 1.71; CI 1.14-2.59; p=0.010). Subgroup analysis revealed no significant association of rs7927894 with early age of onset of the disease, concomitant asthma and allergic rhinoconjunctivitis, total serum IgE levels and family history of atopy. The analysis of the rs877776 variant showed neither a relevant difference in the allelic distribution between patients and controls nor a statistically significant association with any of the analyzed atopic dermatitis phenotypes. CONCLUSIONS: In summary our data show a statistically significant association of the rs7927894 variant on chromosome 11q13.5 with atopic dermatitis but not with other disease-related phenotypes. Therefore, we assume that the rs7927894 single nucleotide polymorphism selectively influences eczema development. More investigations in distinct study populations are needed to assess the role of this interesting polymorphism in atopic dermatitis.

Establishing an allergic eczema model employing recombinant house dust mite allergens Der p 1 and Der p 2 in BALB/c mice.

The major house dust mite allergens Der p 1 and Der p 2 are prevalent inducers of eczema. Der p 1 is a cysteine protease disrupting epithelial barriers, whereas Der p 2 functionally mimics the LPS-binding compound MD-2 within the TLR4 complex. In this work, we tested the percutaneous sensitizing capacity of recombinant (r) Der p 1 and Der p 2 in BALB/c mice. Mice were sensitized by percutaneous application of low (10 µg/application) and high dose (100 µg) rDer p 1 or rDer p 2, or with rDer p 1 followed by rDer p 2. Allergen-specific and total IgE antibodies were determined by ELISA. Eczema of BALB/c was classified by the itching score and corresponded to erosions. Infiltrating immune cells were identified by haematoxylin/eosin and Giemsa staining for eosinophils or mast cells, CD3 staining for T lymphocytes. Percutaneous treatments with rDer p 1, but not rDer p 2-induced specific IgG1. However, cotreatment with rDer p 1 led to increase in anti-Der p 2 IgG titres. Both allergens elicited skin erosions because of scratching, thickening of the epidermis, and eosinophil and T-cell infiltration. Our data indicate that recombinant mite allergens in the absence of adjuvant are sufficient for inducing eczema in BALB/c mice. As the enzymatic activity of an allergen might be an important cofactor for specific sensitization via the skin, Der p 1 may act as adjuvant for other allergens too. The presented mouse model is suitable for investigating the mechanisms of allergic eczema.

Clinical and cytological effects of pimecrolimus cream 1% after resolution of active atopic dermatitis lesions by topical corticosteroids: a randomized controlled trial.

BACKGROUND: Topical pimecrolimus may maintain remissions of atopic dermatitis (AD) by inhibiting subclinical inflammation. OBJECTIVE: To evaluate clinical and cytological effects of pimecrolimus in topical corticosteroid-treated and resolved AD lesions. METHODS: Patients (n=67) with resolved AD lesions were randomized to 3-week double-blind treatment with either pimecrolimus cream 1% or vehicle cream. Outcome measures were reduction in Eczema Area and Severity Index (EASI) and number of leukocytes in skin biopsies in all randomized patients who were evaluable at the end of study. RESULTS: The proportion of patients with a localized EASI<2 at the end of study was higher with pimecrolimus cream 1% than with vehicle cream (73.5 vs. 39.4%, respectively). There was a significant decrease in the number of infiltrating CD45+ cells in pimecrolimus cream 1% compared with placebo cream (-88.2 vs. 43.2 cells/mm(2), respectively, p=0.047) and a slight but nonsignificant reduction in the number of dermal dendritic cells, Langerhans cells, T cells and macrophages with pimecrolimus versus vehicle cream. LIMITATIONS: This was an exploratory study. CONCLUSION: Topical pimecrolimus was effective at maintaining betamethasone-17α-valerate-induced AD remission by inhibiting recurrences of the inflammatory infiltrate in the skin.

The PKC inhibitor AEB071 may be a therapeutic option for psoriasis.

PKC isoforms tau, alpha, and beta play fundamental roles in the activation of T cells and other immune cell functions. Here we show that the PKC inhibitor AEB071 both abolishes the production of several cytokines by activated human T cells, keratinocytes, and macrophages in vitro and inhibits an acute allergic contact dermatitis response in rats. To translate these findings into humans, single and multiple ascending oral doses of AEB071 were administered to healthy volunteers and patients with psoriasis, respectively. AEB071 was well tolerated with no clinically relevant laboratory abnormalities. Ex vivo stimulation of lymphocytes from subjects exposed to single doses of AEB071 resulted in a dose-dependent inhibition of both lymphocyte proliferation and IL2 mRNA expression. Clinical severity of psoriasis was reduced up to 69% compared with baseline after 2 weeks of treatment, as measured by the Psoriasis Area Severity Index (PASI) score. The improvement in psoriasis patients was accompanied by histological improvement of skin lesions and may be partially explained by a substantial reduction of p40+ dermal cells, which are known to mediate psoriasis. These data suggest that AEB071 could be an effective novel treatment regimen for psoriasis and other autoimmune diseases, and that AEB071 warrants long-term studies to establish safety and efficacy.

Serum IgE autoantibodies target keratinocytes in patients with atopic dermatitis.

Previous studies have shown that sera of patients with severe atopic dermatitis (AD) contain IgE specific for self-proteins, supporting the hypothesis of autoreactivity as a pathogenic factor in AD. In this study, we screened a large panel of AD patients (n=192) by western blotting (WB) for IgE reactivity not only against the human epithelial cell line A431 but also against primary keratinocytes (KCs). To investigate autoantigenic cell structures in detail, normal human skin and primary KCs were incubated with sera from both WB-reactive patients and, for control purposes, healthy individuals, and analyzed by immunohistology, confocal laser microscopy, and flow cytometry. Our analysis revealed that 28% of AD patients, but not healthy individuals, display serum IgE autoreactivity by WB analysis. The individual IgE reaction patterns of the sera pointed to the existence of unique as well as common specificities against epidermal or A431-derived proteins. Immunostainings identified cytoplasmic and, occasionally, also cell membrane-associated moieties as targets for autoreactive IgE antibodies. Interestingly, in certain autoreactive patients, the surface-staining pattern was accentuated at cellular contact sites. We conclude that IgE autoreactivity is common, particularly among severe AD patients, and that non-transformed primary cells are needed for characterization of the entire spectrum of IgE-defined autoantigens.

The IgE-reactive autoantigen Hom s 2 induces damage of respiratory epithelial cells and keratinocytes via induction of IFN-gamma.

Hom s 2, the alpha-chain of the nascent polypeptide-associated complex, is an intracellular autoantigen that has been identified with IgE autoantibodies from atopic dermatitis patients. We investigated the humoral and cellular immune response to purified recombinant Hom s 2 (rHom s 2). rHom s 2 exhibited IgE reactivity comparable to exogenous allergens, but did not induce relevant basophil cell degranulation. The latter may be attributed to the fact that patients recognized single epitopes on Hom s 2 as revealed by IgE epitope mapping with rHom s 2 fragments. In contrast to exogenous allergens, rHom s 2 had the intrinsic ability to induce the release of IFN-gamma in cultured peripheral blood mononuclear cells from atopic as well as non-atopic individuals. IFN-gamma-containing culture supernatants from Hom s 2-stimulated peripheral blood mononuclear cells caused disintegration of respiratory epithelial cell layers and apoptosis of skin keratinocytes, which could be inhibited with a neutralizing anti-IFN-gamma antibody. Our data demonstrate that the Hom s 2 autoantigen can cause IFN-gamma-mediated cell damage.

Naturally occurring hypoallergenic Bet v 1 isoforms fail to induce IgE responses in individuals with birch pollen allergy.

BACKGROUND: Engineered hypoallergens are currently being investigated for specific immunotherapy of allergic diseases in preclinical and clinical studies. Naturally occurring hypoallergens have by and large not been considered as a source of vaccine candidates. OBJECTIVE: Evaluation of the antibody response in atopic individuals induced by birch pollen containing isoforms of the major birch pollen allergen Bet v 1. METHODS: Isoform-specific antibody isotype responses for Bet v 1.0101, Bet v 1.0401, and Bet v 1.1001 were determined for 35 sera of individuals with birch pollen allergy. Isoform structures were compared and related to IgE-binding inhibitory capacities and induction of mediator release in human Fcvarepsilon receptor transformed rat basophilic leukemia cells. RESULTS: Bet v 1.0101 induced a predominant IgE response, whereas the significant highest levels of IgG(4) antibodies were directed against Bet v 1.0401. Bet v 1.1001 induced only a minimal antibody response. Structural comparisons revealed that most of the amino acid differences between the isoforms were located on the protein surfaces. IgE induced by Bet v 1.0101 only partly cross-reacted with the 2 other isoforms and bound to them with notably lower affinity. Bet v 1.0401 and Bet v 1.1001 also were poor inducers of mediator release. CONCLUSION: Bet v 1 isoforms possess highly variant immunogenic and allergenic properties. Bet v 1.0101 acts as the sensitizing agent, whereas Bet v 1.0401 and Bet v 1.1001 can induce only a minimal IgE response.

Tumoricidal activity of TLR7/8-activated inflammatory dendritic cells.

Imiquimod (IMQ), a synthetic agonist to Toll-like receptor (TLR) 7, is being successfully used for the treatment of certain skin neoplasms, but the exact mechanisms by which this compound induces tumor regression are not yet understood. While treating basal cell carcinoma (BCC) patients with topical IMQ, we detected, by immunohistochemistry, sizable numbers of both myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) within the inflammatory infiltrate. Surprisingly, peritumoral mDCs stained positive for perforin and granzyme B, whereas infiltrating pDCs expressed tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The biological relevance of this observation can be deduced from our further findings that peripheral blood-derived CD11c(+) mDCs acquired antiperforin and anti-granzyme B reactivity upon TLR7/8 stimulation and could use these molecules to effectively lyse major histocompatibility complex (MHC) class I(lo) cancer cell lines. The same activation protocol led pDCs to kill MHC class I-bearing Jurkat cells in a TRAIL-dependent fashion. While suggesting that mDCs and pDCs are directly involved in the IMQ-induced destruction of BCC lesions, our data also add a new facet to the functional spectrum of DCs, ascribing to them a major role not only in the initiation but also in the effector phase of the immune response.

Sphingosine 1-phosphate phosphatase 2 is induced during inflammatory responses.

Sphingosine 1-phosphate (S1P) levels in cells and, consequently, its bioactivity as a signalling molecule are controlled by the action of enzymes responsible for its synthesis and degradation. In the present report, we examined alterations in expression patterns of enzymes involved in S1P-metabolism (sphingosine kinases including their splice variants, sphingosine 1-phosphate phosphatases, and sphingosine 1-phosphate lyase) under certain inflammatory conditions. We found that sphingosine kinase type 1 (SPHK1) mRNA could be triggered in a cell type-specific manner; individual SPHK1 splice variants were induced with similar kinetics. Remarkably, expression and activity of S1P phosphatase 2 (SPP2) was found to be highly upregulated by inflammatory stimuli in a variety of cells (e.g., neutrophils, endothelial cells). Bandshift analysis using oligonucleotides spanning predicted NFkappaB sites within the SPP2 promoter and silencing of NFkappaB/RelA via RelA-directed siRNA demonstrated that SPP2 is an NFkappaB-dependent gene. Silencing of SPP2 expression in endothelial cells, in turn, led to a marked reduction of TNF-alpha-induced IL-1beta mRNA and protein and to a partial reduction of induced IL-8, suggesting a pro-inflammatory role of SPP2. Notably, up-regulation of SPP2 was detected in samples of lesional skin of patients with psoriasis, an inflammatory skin disease. This study provides detailed insights into the regulation of SPP2 gene expression and suggests that SPP2 might be a novel player in pro-inflammatory signalling.

IgA pemphigus--occurrence of anti-desmocollin 1 and anti-desmoglein 1 antibody reactivity in an individual patient.

BACKGROUND: IgA pemphigus is a rare pustular autoimmune disease with exclusive IgA anti-keratinocyte cell surface antibody reactivity. Two subtypes have been discerned: in the subcorneal pustular dermatosis type, desmocollin 1 has been identified as a targeted autoantigen, while in few cases of the intraepidermal neutrophilic type, IgA anti-desmoglein 1 or IgA anti-desmoglein 3 reactivity has been demonstrated. PATIENTS AND METHODS: A 48-year-old white male presented with generalized large confluent pustules. Skin pathology was assessed by histology and direct immunofluorescence analysis. IgG/IgA autoantibodies against desmoglein 1/3 and desmocollin 1 were measured by ELISA and indirect immunofluorescence using desmocollin 1 cDNA-transfected COS7 cells, respectively. RESULTS: Histopathology revealed subcorneal pustules and direct immunofluorescence microscopy exclusively showed in vivo bound IgA with an intercellular pattern in the epidermis. Desmocollin 1 was identified as a target of IgA autoantibodies by indirect immunofluorescence microscopy utilizing desmocollin 1 cDNA-transfected COS7 cells. In addition, IgA anti-desmoglein 1 reactivity was demonstrated by ELISA. Neither IgA anti-desmoglein 3 nor IgG anti-desmoglein 1/3 autoantibodies were present. CONCLUSIONS: Both desmocollin 1 and desmoglein 1 were autoantigens in this patient with IgA pemphigus and a distinct clinical presentation. To our knowledge, this is the first IgA pemphigus case with dual autoantibody reactivity.

3D parallel coordinate systems--a new data visualization method in the context of microscopy-based multicolor tissue cytometry.

BACKGROUND: Presentation of multiple interactions is of vital importance in the new field of cytomics. Quantitative analysis of multi- and polychromatic stained cells in tissue will serve as a basis for medical diagnosis and prediction of disease in forthcoming years. A major problem associated with huge interdependent data sets is visualization. Therefore, alternative and easy-to-handle strategies for data visualization as well as data meta-evaluation (population analysis, cross-correlation, co-expression analysis) were developed. METHODS: To facilitate human comprehension of complex data, 3D parallel coordinate systems have been developed and used in automated microscopy-based multicolor tissue cytometry (MMTC). Frozen sections of human skin were stained using the combination anti-CD45-PE, anti-CD14-APC, and SytoxGreen as well as the appropriate single and double negative controls. Stained sections were analyzed using automated confocal laser microscopy and semiquantitative MMTC-analysis with TissueQuest 2.0. The 3D parallel coordinate plots are generated from semiquantitative immunofluorescent data of single cells. The 2D and 3D parallel coordinate plots were produced by further processing using the Matlab environment (Mathworks, USA). RESULTS: Current techniques in data visualization primarily utilize scattergrams, where two parameters are plotted against each other on linear or logarithmic scales. However, data evaluation on cartesian x/y-scattergrams is, in general, only of limited value in multiparameter analysis. Dot plots suffer from serious problems, and in particular, do not meet the requirements of polychromatic high-context tissue cytometry of millions of cells. The 3D parallel coordinate plot replaces the vast amount of scattergrams that are usually needed for the cross-correlation analysis. As a result, the scientist is able to perform the data meta-evaluation by using one single plot. On the basis of 2D parallel coordinate systems, a density isosurface is created for representing the event population in an intuitive way. CONCLUSIONS: The proposed method opens new possibilities to represent and explore multidimensional data in the perspective of cytomics and other life sciences, e.g., DNA chip array technology. Current protocols in immunofluorescence permit simultaneous staining of up to 17 markers. Showing the cross-correlation between these markers requires 136 scattergrams, which is a prohibitively high number. The improved data visualization method allows the observation of such complex patterns in only one 3D plot and could take advantage of the latest developments in 3D imaging.

Biodegradable PLGA particles for improved systemic and mucosal treatment of Type I allergy.

Although allergen immunotherapy is basically a story of success, it still needs improvement. The goal of this study was to optimize parenteral and oral allergen formulations through using the biocompatible polymer of lactic and glycolic acid (PLGA). Subcutaneous application of birch pollen allergen Bet v 1 encapsulated in nanoparticles biased the immune response toward Th1 in allergic mice and did not elicit granuloma formation in mice and in human volunteers. When oral immunotherapy of mice was tried with birch pollen-filled PLGA microparticles, mucosal targeting was indispensable for achieving any immune response, and targeting of M-cells was necessary for modulating an ongoing allergic response toward Th1. The authors suggest that biocompatible PLGA nano- or microparticles can be useful tools for upgrading therapy of type I allergy.

Dimerization of the major birch pollen allergen Bet v 1 is important for its in vivo IgE-cross-linking potential in mice.

In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.

Dendritic cells in atopic dermatitis: expression of FcepsilonRI on two distinct inflammation-associated subsets.

BACKGROUND: Dendritic cells (DCs) represent a major portion within the infiltrate of atopic dermatitis (AD) lesions. As antigen-presenting cells they have the ability to regulate both the quantity and quality of T-cell responses and, thus, are likely to play a key role in the pathogenesis of T-cell-dominated skin diseases such as AD. Thus we sought to identify the DC repertoire occurring in AD patients. METHODS: For this purpose, we phenotypically analyzed various defined DC subsets of AD patients and healthy controls in skin biopsies and peripheral blood by immunofluorescence staining. RESULTS: In AD lesions, two inflammation-associated DC subsets with varying expression of costimulatory molecules occurred besides epidermal Langerhans cells (LCs) and dermal myeloid DCs (dmDCs) indigenously residing in normal skin: (1) CD1a+/CD1c+/FcepsilonRI+/IgE+/CD207- myeloid DCs (mDCs) in the epidermis and dermis and (2) CD123+/BDCA-2+/CD45RA+/CD68+ plasmacytoid DCs (pDCs) in the dermis. In the peripheral blood of the patients, these cells exhibited an immature phenotype. Interestingly, we found FcepsilonRI and cell-bound IgE to be expressed not only on myeloid, but also on plasmacytoid DCs from both the skin and peripheral blood of AD patients. CONCLUSIONS: It is tempting to speculate that the disease-regulating role of inflammatory DCs in AD is influenced by both FcepsilonRI occupancy and their degree of maturity.