Clinical characteristics of pediatric cutaneous lupus erythematosus: experience from a tertiary referral center in Korea.

The clinical characteristics of cutaneous lupus erythematosus (CLE) are well delineated in adults, but pediatric data, particularly in Asian populations, are limited. Therefore, we evaluated the characteristics of pediatric cases by retrospectively reviewing the medical records of children with CLE during a 15-year period in a tertiary care dermatology clinic in South Korea. The study included 21 children (8 males and 13 females), 4 of whom had neonatal lupus erythematosus (NLE). Among 17 patients with CLE, discoid lupus erythematosus (DLE) was most common (47.1%), followed by acute CLE (ACLE, 35.3%). All ACLE cases had systemic lupus erythematosus (SLE). Female predominance was conspicuous in ACLE/SLE (6/11 females versus 0/6 males), as was older age, whereas DLE and NLE showed near-equal sex distributions. The median age at the diagnosis of CLE was significantly higher in females than in males (15 years versus 4.5 years, p = 0.02). All patients with ACLE/SLE simultaneously showed skin and systemic symptoms from onset. The kidney was the most commonly involved organ. This study revealed unique characteristics of pediatric CLE, further warranting a comprehensive review among various ethnicities to understand the wide spectrum of CLE in the pediatric population.

Oral tolerance modulates the skin transcriptome in mice with induced atopic dermatitis.

Defective gut immune reactions have been implicated in the development of atopic dermatitis (AD), whereas oral tolerance (OT), that is, the immune unresponsiveness induced by oral antigen administration, protects mice against AD. To investigate this protective role of OT, the transcriptomic profiles of skin were obtained by RNA sequencing from mice that were epicutaneously sensitized, orally tolerized prior to epicutaneous sensitization, or neither (control). Oral tolerance inhibited the upregulation of keratin- and allergic inflammation-associated genes that occurred in the epicutaneously sensitized group. Compared to the controls, mice that were orally tolerized and epicutaneously sensitized showed an upregulation of genes that regulate inflammation or keratinocyte differentiation. Knocking down two of those genes, SCGB1A1 and TSC22D3, upregulated Th2 inflammatory mediators and downregulated a cornified cell envelope-related gene. Based on our findings, OT may protect skin against allergic inflammation by promoting the expression of genes that regulate Th2 inflammatory responses and skin barrier function.

Oral tolerance inhibits atopic dermatitis-like type 2 inflammation in mice by modulating immune microenvironments.

BACKGROUND: Oral tolerance is immune unresponsiveness induced by oral administration of innocuous antigens. Oral administration of allergens has been shown to be effective for suppressing IgE production in allergic responses. However, whether oral tolerance has a role in protection from allergic skin inflammation has not been fully investigated. Here, we evaluated the potential protective role of oral tolerance in a murine model of atopic dermatitis (AD) and investigated the underlying immunologic mechanisms. METHODS: Mice were fed with ovalbumin (OVA) in drinking water then epicutaneously sensitized by repeated application of OVA to tape-stripped skin. Skin biopsies were analyzed for immunohistopathologic features. Levels of antibodies in sera and intestinal washes were measured by ELISA. Flow cytometry and real-time PCR analysis of the skin and mesenteric lymph nodes (MLN) were performed to investigate the immunologic effects of oral tolerance in epicutaneous (EC) sensitization-induced allergic responses. RESULTS: Induction of oral tolerance effectively inhibited inflammatory responses provoked by EC sensitization. Tolerogenic immune mediators were significantly increased in the skin and MLN of EC-sensitized mice following induction of oral tolerance. A marked increase in Il5 and Il13 expression and infiltration of eosinophils and type 2 innate lymphoid cells (ILC2) in the skin of EC-sensitized mice were significantly inhibited by oral tolerance. CONCLUSIONS: Oral tolerance plays a protective role in the development of AD in a murine model by modulating immune microenvironments to be more favorable for immune regulation. This modulation involves inhibition of ILC2 infiltration in skin lesions.

Expression and characterization of a recombinant Cry1Ac crystal protein with enhanced green fluorescent protein in acrystalliferous Bacillus thuringiensis.

AIMS: To investigate fusion expression between Bacillus thuringiensis crystal protein and a foreign protein, the expression of a fusion protein comprised of Cry1Ac, and enhanced green fluorescent protein (EGFP) in B. thuringiensis Cry(-)B strain was examined. METHODS AND RESULTS: The N-terminal fusion expression of EGFP in Cry1Ac was attempted under the control of the native cry1Ac promoter. The EGFP gene was cloned into pProMu and named pProMu-EGFP. The transformant, ProMu-EGFP/CB produced parasporal inclusions that were of bipyramidal-shaped crystals in size ranging from 200 to 300 nm. The fusion protein was approximately 150 kDa and identified by the immunoblot analysis using a Cry1Ac antibody and also a GFP antibody. The LC(50) of the ProMu-EGFP/CB was twofold higher when compared with that by the ProAc/CB. However, the crystal protein produced by the ProMu-EGFP/CB was effective on Plutella xylostella larvae. CONCLUSIONS: The ProMu-EGFP/CB produced bipyramidal shaped and insecticidal crystals comprising fusion proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: Through the N-terminal fusion expression of EGFP and Cry1Ac, expression and crystallization between the B. thuringiensis crystal protein and a foreign protein were validated.

Baculovirus expression vectors that incorporate the foreign protein into viral occlusion bodies.

Current baculovirus expression systems typically produce soluble proteins that accumulate within the infected insect cell or are secreted into the growth medium. A system has now been developed for the incorporation of foreign proteins, along with the matrix protein, polyhedrin, into baculovirus occlusion bodies. Initial studies showed that a recombinant virus expressing a translational fusion between polyhedrin and GFP did not form occlusion bodies. However, a baculovirus coexpressing native polyhedrin and the polyhedrin-GFP fusion protein formed occlusion bodies that fluoresced under UV light, demonstrating that they included the polyhedrin-GFP fusion protein. This was confirmed by immunoblot analysis. Thus, incorporation of a foreign protein into occlusion bodies depends on an interaction between native polyhedrin and the polyhedrin fusion protein. Electron microscopy demonstrated that the occlusion bodies containing GFP also incorporated virions as expected. These ColorPol occlusion bodies were as infectious to insect larvae as occlusion bodies produced by wild-type virus. This new system expands the capabilities for foreign gene expression by baculoviruses, which has implications for biopesticide design, novel vaccine delivery systems, and fusion protein purification applications.

Isolation and characterization of a strain of Bacillus thuringiensis ssp. kurstaki containing a new delta-endotoxin gene.

A strain of Bacillus thuringiensis that showed significantly high toxicity to Plutella xylostella and Spodoptera exigua was isolated from a Korean soil sample and characterized. The isolate, named B. thuringiensis K1, was determined to belong to ssp. kurstaki (H3a3b3c) type by an H antisera agglutination test and produced bipyramidal inclusions. Plasmid pattern of K1 was different from that of the reference strain, ssp. kurstaki HD-1, but the parasporal inclusion protein profile of K1 had two major bands that were similar in size to those of ssp. kurstaki HD-1. To verify the delta-endotoxin gene types of K1, PCR analysis with specific cry gene primers was performed to show that K1 contained a new cry gene in addition to cry1Aa, cry1Ab, cry1Ac, cry1E and cry2 genes. PCR-amplified region of the new cry gene, cryX, showed 79% similarity to cry1Fa1 gene (GenBank Accession No. M63897). In an insect toxicity assay, K1 had higher toxicity against Plutella xylostella and S. exigua than ssp. kurstaki HD-1.

Isolation and characterization of a Bacillus thuringiensis ssp. kurstaki strain toxic to Spodoptera exigua and Culex pipiens.

A strain of Bacillus thuringiensis with dual toxicity was isolated from Korean soil samples and named K2. K2 was determined as ssp. kurstaki (H3a3b3c) by serological test and produced bipyramidal-shaped parasporal inclusions. The plasmid and protein profiles of B. thuringiensis K2 were different from those of the reference strain, ssp. kurstaki HD-1. To verify gene type of B. thuringiensis K2, PCR analysis with specific cry gene primers was performed. The result showed that B. thuringiensis K2 had cry1Aa, cry1Ab, cry1C, and cry1D type genes, whereas ssp. kurstaki HD-1 had cry1Aa, cry1Ab, cry1Ac, and cry2 type genes. In addition, B. thuringiensis K2 had high toxicity against Spodoptera exigua and Culex pipiens, whereas B. thuringiensis ssp. kurstaki HD-1 does not have high toxicity against these two insect species.

The 120-kDa soluble ectodomain of type XVII collagen is recognized by autoantibodies in patients with pemphigoid and linear IgA dermatosis.

BACKGROUND: Type XVII collagen promotes adhesion of basal keratinocytes to epidermal basement membrane, and is the target of disease in patients with certain inherited or acquired blistering diseases. Two forms of type XVII collagen are produced by cultured human keratinocytes: a 180-kDa full-length, transmembrane protein, and a recently identified 120-kDa soluble fragment that corresponds to its collagenous ectodomain. OBJECTIVES: We aimed to determine the incidence and pattern of reactivity of autoantibodies against the 180- and 120-kDa forms of type XVII collagen in sera from 40 patients with bullous pemphigoid (BP), pemphigoid gestationis or cicatricial pemphigoid (CP), as well as six patients with linear IgA dermatosis (LAD). METHODS: Various immunochemical techniques were used. RESULTS: These studies found that the 120-kDa fragment of type XVII collagen was bound by circulating autoantibodies in 13 of 38 patients with BP or CP and all six patients with LAD. While many pemphigoid sera had specific reactivity against one but not both forms of this protein, autoantibodies from patients with LAD bound only the soluble ectodomain. CONCLUSIONS: These findings are consistent with the presence of both neoepitopes and cross-reactive epitopes on the ectodomain of type XVII collagen. The finding that sera from patients with LAD showed specific reactivity to epidermal basement membrane suggests that such neoepitopes are present in human skin and that their targeting by autoantibodies may contribute to disease pathogenesis.

Isolation of a strain of Bacillus thuringiensis ssp. kurstaki HD-1 encoding delta-endotoxin Cry1E.

A strain of Bacillus thuringiensis, STB-1, toxic against Spodoptera exigua, was isolated. Bacillus thuringiensis STB-1 produced bipyramidal inclusions and reacted with the H antiserum of B. thuringiensis ssp. kurstaki. The plasmid and protein profiles of B. thuringiensis STB-1 were compared with those of its reference strains, ssp. kurstaki and ssp. kenyae. To verify the gene type of B. thuringiensis STB-1, PCR analysis was performed with Spodoptera-specific cry gene primers. The result showed that B. thuringiensis STB-1, unlike its reference strains, had crylAa, crylAb, crylAc and crylE, suggesting that B. thuringiensis STB-1 was a unique strain with respect to gene type. In addition, B. thuringiensis STB-1 showed a high level of toxicity against both S. exigua and Bombyx mori, whereas B. thuringiensis ssp. kurstaki HD-1 or ssp. kenyae showed a high level of toxicity against only Bombyx mori or S. exigua, respectively.

Characterization of novel non-toxic Bacillus thuringiensis isolates from Korea.

Four Bacillus Thuringiensis isolates from soil samples produced parasporal inclusions which were non-toxic to insects. The isolates were named B. thuringiensis NTB-1, NTB-2, NTB-3 and NTB-4. The parasporal inclusions were shown to be ovoid by phase contrast and scanning electron microscopy. The serotypes of the four isolates were determined by agglutination using 33 antisera; NTB-1 and NTB-4 seemed to be subsp. israelensis, and NTB-2 seemed to be subsp. pondicheriensis. NTB-3 did not react with the 33 antisera. However, comparison of parasporal protein and plasmid DNA patterns of the four isolates with those of 15 known non-toxic B. thuringiensis strains demonstrated that the four isolates are novel.

Plakoglobin binding by human Dsg3 (pemphigus vulgaris antigen) in keratinocytes requires the cadherin-like intracytoplasmic segment.

Desmogleins are transmembrane desmosomal cadherins. Two desmogleins, Dsg3 and Dsg1, have been shown to bind plakoglobin, an intracytoplasmic (IC) desmosomal plaque protein. This binding may be critical for desmosome assembly or stability. The IC domain of desmogleins consists of subdomains that are either desmoglein specific or homologous with the IC region of classical cadherins. Here we identify the domains of human Dsg3 that are critical for plakoglobin binding in human keratinocytes. We constructed eukaryotic expression vectors containing chimeric cDNAs that encode the extracellular domain of mouse E-cadherin (Ecad) with the transmembrane and IC domains of Dsg3, with increasing truncations eliminating various IC subdomains from the carboxy-terminus. These constructs were used for transient transfection of HaCaT cells. Extracts were subjected to immunoprecipition with an anti-mouse Ecad antibody (that does not precipitate human Ecad), thus precipitating the chimeric protein and any tightly associated plakoglobin. Co-precipitation of plakoglobin was confirmed by immunoblotting. These data show that the desmoglein-specific IC subdomains are not necessary for plakoglobin binding, but the carboxy-terminal 87 amino acids of the IC-cadherin-like segment subdomain are critical. Finally, we confirmed these results outside cells with in vitro transcription and translation, which also demonstrates that the Dsg3-plakoglobin interaction is direct and does not depend on other cellular factors. These results underscore the importance of a region, highly conserved in all desmogleins, in the carboxy terminus of the IC-cadherin-like subdomain for the localization of plakoglobin to desmosomes.

Intracellular domain of desmoglein 3 (pemphigus vulgaris antigen) confers adhesive function on the extracellular domain of E-cadherin without binding catenins.

For the extracellular (EC) domain of E-cadherin to function in homophilic adhesion it is thought that its intracytoplasmic (IC) domain must bind alpha- and beta-catenins, which link it to the actin cytoskeleton. However, the IC domain of pemphigus vulgaris antigen (PVA or Dsg3), which is in the desmoglein subfamily of the cadherin gene superfamily, does not bind alpha- or beta-catenins. Because desmogleins have also been predicted to function in the cell adhesion of desmosomes, we speculated that the PVA IC domain might be able to act in a novel way in conferring adhesive function on the EC domain of cadherins. To test this hypothesis we studied aggregation of mouse fibroblast L cell clones that expressed chimeric cDNAs encoding the EC domain of E-cadherin with various IC domains. We show here that the full IC domain of PVA as well as an IC subdomain containing only 40 amino acids of the PVA intracellular anchor (IA) region confer adhesive function on the E-cadherin EC domain without catenin-like associations with cytoplasmic molecules or fractionation with the cell cytoskeleton. This IA region subdomain is evolutionarily conserved in desmogleins, but not classical cadherins. These findings suggest an important cell biologic function for the IA region of desmogleins and demonstrate that strong cytoplasmic interactions are not absolutely necessary for E-cadherin-mediated adhesion.