Dataset Information

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

ABSTRACT:

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.

SUBMITTER: Giurdanella F

PROVIDER: S-EPMC7027452 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Publications

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

Giurdanella F F Nijenhuis A M AM Diercks G F H GFH Jonkman M F MF Pas H H HH

The British journal of dermatology 20190809 2

<h4>Background</h4>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.<h4>Objectives</h4>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. W ...[more]

PMID: 31090065

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Project description:BackgroundMucous membrane pemphigoid is a group of chronic subepithelial autoimmune blistering diseases that mainly affect mucous membranes. Laminin 332-specific autoantibodies are present in approximately 1/3 of the patients, being associated with an increased risk of malignancy. Because of the severe complications, an early recognition of the disease allowing a timely therapy is essential. The gold standard methods for detection of laminin 332-specific autoantibodies, including the immunoprecipitation and immunoblotting are non-quantitative, laborious and restricted to a few specialized laboratories worldwide. In addition, the use of radioimmunoassays, although highly sensitive and specific, are laborious, expensive and tightly regulated. Therefore, there is a stringent need for a quantitative immunoassay for the routine detection of laminin 332-specific autoantibodies more broadly available to diagnostic laboratories. The aim of this study was to compare different antigenic substrates, including native, recombinant laminin 332 and laminin 332-rich keratinocyte extracellular matrix, for development of an ELISA to detect autoantibodies in mucous membrane pemphigoid.ResultsUsing a relatively large number of sera from MMP patients with well-characterized autoantibody reactivity we show the suitability of ELISA systems using laminin 332 preparations as adjunct diagnostic tools in MMP. While glycosylation of laminin 332 does not appear to influence its recognition by MMP autoantibodies, ELISA systems using both purified, native and recombinant laminin 332 demonstrated a high sensitivity and good correlation with the detection of autoantibodies by immunoblotting. ELISA systems using different laminin 332 preparations represent a feasible and more accessible alternative for a broad range of laboratories.ConclusionsOur findings qualify the use of immunoassays with the laminin 332-rich preparations as an ancillary diagnostic tool in mucous membrane pemphigoid.

Project description:Mucous membrane pemphigoid (MMP) is an autoimmune blistering disease characterized by autoantibodies against the basal membrane zone of skin and surface-close epithelia and predominant mucosal lesions. The oral cavity and conjunctivae are most frequently affected, albeit clinical manifestations can also occur on the skin. MMP-associated lesions outside the oral cavity typically lead to scarring. Mechanisms underlying scarring are largely unknown in MMP and effective treatment options are limited. Herein, we assessed the collagen architecture in tissue samples of an antibody-transfer mouse model of anti-laminin-332 MMP. In MMP mice, increased collagen fibril density was observed in skin and conjunctival lesions compared to mice injected with normal rabbit IgG. The extracellular matrix of MMP skin samples also showed altered post-translational collagen cross-linking with increased levels of both lysine- and hydroxylysine-derived collagen crosslinks supporting the fibrotic phenotype in experimental MMP compared to control animals. In addition, we evaluated a potential anti-fibrotic therapy in experimental anti-laminin-332 MMP using disulfiram, an inhibitor of the aldehyde dehydrogenase (ALDH), which has been implicated in immune-mediated mucosal scarring. In addition, disulfiram also acts as a copper chelator that was shown to block lysyl oxidase activity, an enzyme involved in formation of collagen crosslinks. Topical use of disulfiram (300 μM in 2% [w/v] methocel) did not improve ocular lesions in experimental MMP over the 12-day treatment period in disulfiram-treated mice compared to vehicle-treated mice (n=8/group). Furthermore, C57BL6/J mice (n=8/group) were treated prophylactically with 200 mg/kg p.o. disulfiram or the solvent once daily over a period of 12 days. Systemic treatment did not show any reduction in the severity of oral and ocular lesions in MMP mice, albeit some improvement in skin lesions was observed in disulfiram- vs. vehicle-treated mice (p=0.052). No reduction in fibrosis was seen, as assessed by immunohistochemistry. Whilst blocking of ALDH failed to significantly ameliorate disease activity, our data provide new insight into fibrotic processes highlighting changes in the collagenous matrix and cross-linking patterns in IgG-mediated MMP.

Dataset Information

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

Background

Objectives

Methods

Results

Conclusions

Publications

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

Similar Datasets

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