Patients suffering from dystrophic epidermolysis bullosa are prone to developing autoantibodies against skin proteins: A longitudinal confirmational study.

Epidermolysis bullosa (EB) is a heritable skin blistering disease caused by variants in genes coding for proteins that secure cell-cell adhesion and attachment of the epidermis to the dermis. Interestingly, several proteins involved in inherited EB are also associated with autoimmune blistering diseases (AIBD). In this study, we present a long-term follow-up of 15 patients suffering from recessive dystrophic or junctional EB. From these patients, 62 sera were analysed for the presence of autoantibodies associated with AIBD. We show that patients suffering from recessive dystrophic EB (RDEB) are more susceptible to developing autoantibodies against skin proteins than patients suffering from junctional EB (70% vs. 20%, respectively). Interestingly, no correlation with age was observed. Most patients showed reactivity to Type XVII collagen/linear IgA bullous dermatosis autoantigen (n = 5; 33%), followed by BP230 (n = 4; 27%), Type VII collagen (n = 4; 27%) and laminin-332 (n = 1; 7%). The pathogenicity of these autoantibodies remains a subject for future experiments.

Keratinocyte footprint assay discriminates antilaminin-332 pemphigoid from all other forms of pemphigoid diseases.

BACKGROUND: Antilaminin-332 mucous membrane pemphigoid is a chronic severe pemphigoid disease characterized by autoantibodies to laminin-332. At present no commercial assay is available to demonstrate antilaminin-332 antibodies, and diagnosis relies on in-house techniques with limited sensitivities. OBJECTIVES: In order to move, keratinocytes cultured in vitro secrete laminin-332 to attach to the culture dish. In that way, they leave behind a unique footprint trail of laminin-332. We aimed to develop a sensitive and specific laboratory assay to determine antilaminin-332 autoantibodies in patient serum based on binding of patient IgG to these unique footprints. METHODS: Normal human keratinocytes were grown on glass coverslips and incubated with patient or control serum for 1 h. The binding of IgG was then investigated by immunofluorescence. After validating the test for its ability to identify antilaminin-332 autoantibodies it was converted into a daily available test based on binding of IgG to dried coverslips that can be stored frozen. The staining patterns of sera from patients with antilaminin-332 pemphigoid were then compared with those of sera from patients with other autoimmune bullous diseases and normal human sera. RESULTS: IgG of all antilaminin-332 pemphigoid sera (n = 16) bound to laminin-332 footprints, while all normal human controls (n = 55) were negative. From the sera of patients with other diseases (n = 72) four sera tested positive. The footprint assay was also positive for sera that were negative by salt-split skin analysis, demonstrating that it is a very sensitive technique. CONCLUSIONS: The keratinocyte footprint assay is a fast and specific assay to confirm or rule out the presence of antilaminin-332 autoantibodies. What's already known about this topic? Antilaminin-332 mucous membrane pemphigoid is a severe form of pemphigoid, and patients may have an increased risk of malignancies. The diagnosis of antilaminin-332 mucous membrane pemphigoid is complicated by the lack of specific commercial tests for antilaminin-332 antibodies and can be confirmed only in specialized laboratories. Keratinocytes in culture need laminin-332 for adhesion and migration and therefore deposit it on the bottom of the culture dish. What does this study add? The keratinocyte footprint assay detects antilaminin-332 autoantibodies in patient serum using the native laminin-332 produced by cultured keratinocytes. What is the translational message? The keratinocyte footprint assay is a fast and specific assay to confirm or rule out the presence of antilaminin-332 autoantibodies.

Features of epidermolysis bullosa simplex due to mutations in the ectodomain of type XVII collagen.

BACKGROUND: Mutations in COL17A1, coding for type XVII collagen, cause junctional epidermolysis bullosa with an ultrastructural plane of cleavage through the lamina lucida of the epidermal basement membrane. OBJECTIVES: To identify the COL17A1 mutations in a child with reduced type XVII collagen expression and intraepidermal blister formation. PATIENT AND METHODS: Protein expression and level of tissue separation were studied by immunofluorescence and electron microscopy. The mutations were identified by analysing the patient's DNA and mRNA. RESULTS: Immunofluorescence microscopy performed on nonlesional skin demonstrated absence of the type XVII collagen endodomain and presence, although reduced, of the shed ectodomain. Electron microscopy showed that the plane of cleavage was through the basal cells, not through the lamina lucida. Two heterozygous mutations were identified in COL17A1: a new 3'-acceptor splice-site mutation in intron 21 (1877-2A-->C), and a deletion in exon 48 (3432delT). The splice-site mutation in intron 21 results in alternative transcripts of which two are in-frame, with deletions of the first nine codons of exon 22 and the entire exon 22, respectively. By Western blot analysis, a type XVII collagen molecule was detected that was slightly smaller than normal. CONCLUSIONS: Occasionally mutations in the COL17A1 gene may result in split levels suggesting epidermolysis bullosa simplex rather than junctional epidermolysis bullosa.

A very mild form of non-Herlitz junctional epidermolysis bullosa: BP180 rescue by outsplicing of mutated exon 30 coding for the COL15 domain.

Mutations in the gene COL17A1 cause non-Herlitz junctional epidermolysis bullosa. Here, we describe a patient who, despite two heterozygous mutations in COL17A1, has an extremely mild form of the disease missing most of the characteristic clinical features. DNA analysis revealed a frame-shift mutation 3432delT and a nonsense mutation 2356C-->T (Q751X). cDNA analysis showed that the deleterious effect of the latter mutation was skirted by deleting the premature termination codon containing exon 30. In this way, the reading frame was restored, resulting in a 36 nucleotides shorter mRNA transcript. Immunoblot analysis showed expression of the 180-kDa bullous pemphigoid antigen (BP180) with a slightly higher SDS-PAGE mobility, in line with the deletion of 12 amino acids from the COL15 domain. Immunofluorescence of skin sections showed diminished, but correctly localised expression of BP180, and this, in concert with the mild clinical phenotype, suggests that this COL15 mutated BP180 is still partly functional.

Antitumor reactivity induced by liposomal MTP-PE in a liver metastasis model of colon cancer in the rat.

The antitumor effects of muramyl tripeptide phosphatidylethanolamine, incorporated within the lipophilic phase of liposomes (lipMTP-PE) were studied using a model of liver metastasis of colon cancer in the rat. Intravenous immunotherapy with lipMTP-PE, when started 2 days before the inoculation of tumor cells and given twice a week, significantly reduced subsequent tumor growth in the liver. The main effect of treatment appeared to be a substantial local increase in the number of tumoricidal macrophages and lymphocytes. Tumor cell lysis by isolated macrophages in vitro, however, appeared not to be elevated above the level triggered by tumor growth alone. Therefore, the observed therapeutic effect of lipMTP-PE probably results from a combination of (1) an increase in the number of cytotoxic macrophages at the onset of metastatic growth in the liver, thus increasing the probability of lethal contacts between tumoricidal effectors and tumor cells and (2) indirect effects of lipMTP-PE, via the induction of cytokine production by liver macrophages, leading to increased numbers and/or activity of cytotoxic lymphocytes and natural killer cells.

Tumoricidal response of liver macrophages isolated from rats bearing liver metastases of colon adenocarcinoma.

Intraportal inoculation of CC531 adenocarcinoma cells into syngeneic rats causes an increase of liver macrophage cell number but not of major histocompatibility complex class II antigen expression. On day 1 after inoculation of 10(5) CC531 cells, a fixed number of isolated liver macrophages lysed significantly more target cells in vitro than did control cells. This effect was still present after 4 weeks. A 10-fold higher initial tumor dose significantly suppressed the macrophage response during the first 2 weeks. In contrast to tumoricidal activity induced by lipopolysaccharide in vitro, the tumoricidal response following in vivo challenge with tumor cells appeared not closely related to the production of reactive nitrogen intermediates, as in the latter case it was not abrogated in the presence of nitric oxide synthase inhibitor. Furthermore, the liver macrophage population appeared not fully activated after tumor inoculation as lipopolysaccharide further increased tumoricidal activity in vitro. The observed numerical and functional response of liver macrophages to intraportally inoculated tumor cells points at an important role of these cells in aspecific immune reactivity aimed at the reduction of local tumor growth. Results suggest that mechanistic differences exist between macrophage tumoricidal activity induced by tumor cells as compared with lipopolysaccharide.

Liver metastasis model of colon cancer in the rat: immunohistochemical characterization.

Inoculation of 3 x 10(4) to 3 x 10(5) CC531 colon adenocarcinoma cells into the portal vein of syngeneic WAG/Rij rats provides a reproducible animal model of colon cancer liver metastasis with macroscopically visible tumor nodules at day 25 after inoculation. In this study, the inflammatory cell response in the liver sinusoids against locally induced metastases was also investigated using immunohistochemical methods. Host immune reactive cells accumulated in the liver sinusoids of tumor-bearing livers, especially resident Kupffer cells (KC; ED1+/ED2+), and to a lesser extent pit cells (OX8+/0X19-), T lymphocytes (OX8+/OX19+) and polymorphonuclear leukocytes (PMN; HIS48+). All these cell types may be involved in the lysis of CC531 cells in the liver, although only major histocompatibility complex class II+ monocytes (3D11+/ED1+/ED2-), but not resident KC, T lymphocytes nor PMN, accumulated at the peritumoral area between liver parenchyma and tumor foci. Especially monocytes but also PMN, lymphocytes and pit cells appeared to infiltrate the stromal tumor compartment. In view of the low cell dose needed, the rat liver metastasis model of colon cancer used in this study reflects well the human, weakly immunogenic, tumor-host relationships and provides an excellent opportunity for the study of cellular reactions related to chemo- and/or immunotherapy protocols.