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Comparison of Two Diagnostic Assays for Anti-Laminin 332 Mucous Membrane Pemphigoid.

ABSTRACT: Anti-laminin 332 mucous membrane pemphigoid (MMP) is an autoimmune blistering disease characterized by predominant mucosal lesions and autoantibodies against laminin 332. The exact diagnosis of anti-laminin 332 MMP is important since nearly 30% of patients develop solid cancers. This study compared two independently developed diagnostic indirect immunofluorescence (IF) tests based on recombinant laminin 332 expressed in HEK239 cells (biochip mosaic assay) and the migration trails of cultured keratinocytes rich in laminin 332 (footprint assay). The sera of 54 anti-laminin 332 MMP, 35 non-anti-laminin 332 MMP, and 30 pemphigus vulgaris patients as well as 20 healthy blood donors were analyzed blindly and independently. Fifty-two of 54 and 54/54 anti-laminin 332 MMP sera were positive in the biochip mosaic and the footprint assay, respectively. In the 35 non-anti-laminin 332 MMP sera, 3 were positive in both tests and 4 others showed weak reactivity in the footprint assay. In conclusion, both assays are easy to perform, highly sensitive, and specific, which will further facilitate the diagnosis of anti-laminin 332 MMP.

SUBMITTER: Goletz S

PROVIDER: S-EPMC8657402 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Comparison of Two Diagnostic Assays for Anti-Laminin 332 Mucous Membrane Pemphigoid.

Goletz Stephanie S Giurdanella Federica F Holtsche Maike M MM Nijenhuis Miranda M Horvath Barbara B Diercks Gilles F H GFH Zillikens Detlef D Hashimoto Takashi T Schmidt Enno E Pas Hendri H HH

Frontiers in immunology 20211125

Anti-laminin 332 mucous membrane pemphigoid (MMP) is an autoimmune blistering disease characterized by predominant mucosal lesions and autoantibodies against laminin 332. The exact diagnosis of anti-laminin 332 MMP is important since nearly 30% of patients develop solid cancers. This study compared two independently developed diagnostic indirect immunofluorescence (IF) tests based on recombinant laminin 332 expressed in HEK239 cells (biochip mosaic assay) and the migration trails of cultured ker ...[more]

PMID: 34899726

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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.

Project description:ImportanceAn accurate diagnosis of mucous membrane pemphigoid (MMP) is essential to reduce diagnostic and therapeutic delay.ObjectiveTo assess the diagnostic accuracy of direct immunofluorescence microscopy on mucosal biopsy specimens and immunoserology in a large cohort of patients with suspected MMP.Design, setting, and participantsThis retrospective cohort study was carried out in a single tertiary care center for blistering diseases between January 2002 and March 2019. Eligible participants were patients with suspected MMP and paired data on at least a mucosal biopsy specimen for direct immunofluorescence microscopy (DIF) and indirect immunofluorescence microscopy (IIF) on a human salt-split skin substrate (SSS). In addition, an optional DIF test on a skin biopsy specimen and one or more performed routine immunoserologic tests were analyzed. Data analysis was conducted from April 2019, to June 2020.Main outcomes and measuresDiagnostic accuracy of DIF, IIF SSS, and immunoblot for BP180 and BP230.ResultsOf the 787 participants, 121 (15.4%) received the diagnosis of MMP (50 men [41.3%], 71 women [58.7%]; mean [SD] age at diagnosis, 60.1 [17.7] years). Sixty-seven of the patients with MMP (55.4%) had monosite involvement, of which oral site was the most frequently affected (51 [42.1%]). No significant difference was found between the sensitivity of DIF on a perilesional buccal biopsy and a normal buccal biopsy (89.3% vs 76.7%). Three patients with solitary ocular involvement showed a positive DIF of only the oral mucosa. In 6 patients with a negative mucosal DIF, a skin biopsy confirmed diagnosis of MMP. Overall, IIF SSS was less sensitive (44.6%), but highly specific (98.9%). The sensitivity of immunoblot (66.1%) was higher compared to SSS, but with lower specificity (91.3%).Conclusions and relevanceThis comparative diagnostic accuracy study of a cohort of 787 patients found a high sensitivity of a mucosal DIF biopsy for diagnosis of MMP, and lower sensitivity of serologic analysis. A biopsy can be taken from either perilesional or normal buccal mucosa. An additional DIF biopsy of another mucosal site or of affected or unaffected skin may increase the diagnostic yield and is recommended in patients with negative DIF results and high clinical suspicion.

Project description:Mucous Membrane Pemphigoid is an orphan multi-system autoimmune scarring disease involving mucosal sites, including the ocular surface (OcMMP) and gut. Loss of tolerance to epithelial basement membrane proteins and generation of autoreactive T cell and/or autoantibodies are central to the disease process. The gut microbiome plays a critical role in the development of the immune system. Alteration in the gut microbiome (gut dysbiosis) affects the generation of autoreactive T cells and B cell autoantibody repertoire in several autoimmune conditions. This study examines the relationship between gut microbiome diversity and ocular inflammation in patients with OcMMP by comparing OcMMP gut microbiome profiles with healthy controls. DNA was extracted from faecal samples (49 OcMMP patients, 40 healthy controls), amplified for the V4 region of the 16S rRNA gene and sequenced using Illumina Miseq platform. Sequencing reads were processed using the bioinformatics pipeline available in the mothur v.1.44.1 software. After adjusting for participant factors in the multivariable model (age, gender, BMI, diet, proton pump inhibitor use), OcMMP cohort was found to be associated with lower number of operational taxonomic units (OTUs) and Shannon Diversity Index when compared to healthy controls. Within the OcMMP cohort, the number of OTUs were found to be significantly correlated with both the bulbar conjunctival inflammation score (p=0.03) and the current use of systemic immunotherapy (p=0.02). The linear discriminant analysis effect size scores indicated that Streptococcus and Lachnoclostridium were enriched in OcMMP patients whilst Oxalobacter, Clostridia uncultured genus-level group (UCG) 014, Christensenellaceae R-7 group and butyrate-producing bacteria such as Ruminococcus, Lachnospiraceae, Coprococcus, Roseburia, Oscillospiraceae UCG 003, 005, NK4A214 group were enriched in healthy controls (Log10 LDA score < 2, FDR-adjusted p <0.05). In conclusion, OcMMP patients have gut dysbiosis correlating with bulbar conjunctival inflammation and the use of systemic immunotherapies. This provides a framework for future longitudinal deep phenotyping studies on the role of the gut microbiome in the pathogenesis of OcMMP.

Dataset Information

Comparison of Two Diagnostic Assays for Anti-Laminin 332 Mucous Membrane Pemphigoid.

Publications

Comparison of Two Diagnostic Assays for Anti-Laminin 332 Mucous Membrane Pemphigoid.

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