Publications by authors named "Manuel Koch"

124 Publications

Evaluation of a New Spike (S)-Protein-Based Commercial Immunoassay for the Detection of Anti-SARS-CoV-2 IgG.

Microorganisms 2021 Mar 31;9(4). Epub 2021 Mar 31.

Institute of Virology, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50935 Cologne, Germany.

: The investigation of the antibody response to SARS-CoV-2 represents a key aspect in facing the COVID-19 pandemic. In the present study, we compared the new Immundiagnostik IDK® anti-SARS-CoV-2 S1 IgG assay with four widely-used commercial serological assays for the detection of antibodies targeting S (spike) and NC (nucleocapsid) proteins. Serum samples were taken from an unbiased group of convalescent patients and from a negative control group. Sample were simultaneously analyzed by the new Immundiagnostik IDK anti-SARS-CoV-2 S1 IgG assay, by the DiaSorin LIAISON SARS-CoV-2 S1/S2 IgG assay, and by the Euroimmun anti-SARS-CoV-2 S1 IgG ELISA. Antibodies binding NC were detected by the Abbott SARS-CoV-2 IgG assay and by the pan-immunoglobulin immunoassay Roche Elecsys anti-SARS-CoV-2. Moreover, we investigated samples of a group of COVID-19 convalescent subjects that were primarily tested S1 IgG non-reactive. Samples were also tested by live virus and pseudovirus neutralization tests. Overall, the IDK® anti-SARS-CoV-2 S1 IgG assay showed the highest sensitivity among the evaluated spike (S) protein-based assays. Additionally, the Immundiagnostik assay correlated well with serum-neutralizing activity. The novel IDK anti-SARS-CoV-2 S1 IgG assay showed high sensitivity and specificity, representing a valid option for use in the routine diagnostic.
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http://dx.doi.org/10.3390/microorganisms9040733DOI Listing
March 2021

A new MMP-mediated prodomain cleavage mechanism to activate bone morphogenetic proteins from the extracellular matrix.

FASEB J 2021 Mar;35(3):e21353

Center for Biochemistry, Faculty of Medicine, University Hospital Cologne, University of Cologne, Cologne, Germany.

Since their discovery as pluripotent cytokines extractable from bone matrix, it has been speculated how bone morphogenetic proteins (BMPs) become released and activated from the extracellular matrix (ECM). In contrast to TGF-βs, most investigated BMPs are secreted as bioactive prodomain (PD)-growth factor (GF) complexes (CPLXs). Recently, we demonstrated that PD-dependent targeting of BMP-7 CPLXs to the extracellular fibrillin microfibril (FMF) components fibrillin-1 and -2 represents a BMP sequestration mechanism by rendering the GF latent. Understanding how BMPs become activated from ECM scaffolds such as FMF is crucial to elucidate pathomechanisms characterized by aberrant BMP activation and ECM destruction. Here, we describe a new MMP-dependent BMP-7 activation mechanism from ECM-targeted pools via specific PD degradation. Using Edman sequencing and mutagenesis, we identified a new and conserved MMP-13 cleavage site within the BMP-7 PD. A degradation screen with different BMP family PDs and representative MMP family members suggested utilization of the identified site in a general MMP-driven BMP activation mechanism. Furthermore, sandwich ELISA and solid phase cleavage studies in combination with bioactivity assays, single particle TEM, and in silico molecular docking experiments provided evidence that PD cleavage by MMP-13 leads to BMP-7 CPLX disintegration and bioactive GF release.
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http://dx.doi.org/10.1096/fj.202001264RDOI Listing
March 2021

Pivotal Role of Tenascin-W (-N) in Postnatal Incisor Growth and Periodontal Ligament Remodeling.

Front Immunol 2020 22;11:608223. Epub 2021 Jan 22.

Faculty of Medicine and University Hospital Cologne, Institute for Dental Research and Oral Musculoskeletal Biology, University of Cologne, Cologne, Germany.

The continuously growing mouse incisor provides a fascinating model for studying stem cell regulation and organ renewal. In the incisor, epithelial and mesenchymal stem cells assure lifelong tooth growth. The epithelial stem cells reside in a niche known as the cervical loop. Mesenchymal stem cells are located in the nearby apical neurovascular bundle and in the neural plexus. So far, little is known about extracellular cues that are controlling incisor stem cell renewal and guidance. The extracellular matrix protein tenascin-W, also known as tenascin-N (TNN), is expressed in the mesenchyme of the pulp and of the periodontal ligament of the incisor, and is closely associated with collagen 3 fibers. Here, we report for the first time the phenotype of tenascin-W/TNN deficient mice, which in a C57BL/6N background exhibit a reduced body weight and lifespan. We found major defects in the alveolar bone and periodontal ligament of the growing rodent incisors, whereas molars were not affected. The alveolar bone around the incisor was replaced by a dense scar-like connective tissue, enriched with newly formed nerve fibers likely leading to periodontal pain, less food intake and reduced body weight. Using soft food to reduce mechanical load on the incisor partially rescued the phenotype. hybridization and Gli1 reporter mouse experiments revealed decreased hedgehog signaling in the incisor mesenchymal stem cell compartment, which coordinates the development of mesenchymal stem cell niche. These results indicate that TNN deficiency in mice affects periodontal remodeling and increases nerve fiber branching. Through periodontal pain the food intake is reduced and the incisor renewal and the neurovascular sonic hedgehog secretion rate are reduced. In conclusion, tenascin-W/TNN seems to have a primary function in rapid periodontal tissue remodeling and a secondary function in mechanosensation.
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http://dx.doi.org/10.3389/fimmu.2020.608223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862723PMC
January 2021

Basement membrane stiffness determines metastases formation.

Nat Mater 2021 Jan 25. Epub 2021 Jan 25.

Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark.

The basement membrane (BM) is a special type of extracellular matrix and presents the major barrier cancer cells have to overcome multiple times to form metastases. Here we show that BM stiffness is a major determinant of metastases formation in several tissues and identify netrin-4 (Net4) as a key regulator of BM stiffness. Mechanistically, our biophysical and functional analyses in combination with mathematical simulations show that Net4 softens the mechanical properties of native BMs by opening laminin node complexes, decreasing cancer cell potential to transmigrate this barrier despite creating bigger pores. Our results therefore reveal that BM stiffness is dominant over pore size, and that the mechanical properties of 'normal' BMs determine metastases formation and patient survival independent of cancer-mediated alterations. Thus, identifying individual Net4 protein levels within native BMs in major metastatic organs may have the potential to define patient survival even before tumour formation. The ratio of Net4 to laminin molecules determines BM stiffness, such that the more Net4, the softer the BM, thereby decreasing cancer cell invasion activity.
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http://dx.doi.org/10.1038/s41563-020-00894-0DOI Listing
January 2021

Epithelial loss of mitochondrial oxidative phosphorylation leads to disturbed enamel and impaired dentin matrix formation in postnatal developed mouse incisor.

Sci Rep 2020 12 16;10(1):22037. Epub 2020 Dec 16.

Department of Periodontology and Operative Dentistry, University Medical Center of the Johannes Gutenberg University, Augustusplatz 2, 55131, Mainz, Germany.

The formation of dentin and enamel matrix depends on reciprocal interactions between epithelial-mesenchymal cells. To assess the role of mitochondrial function in amelogenesis and dentinogenesis, we studied postnatal incisor development in K320E-Twinkle mice. In these mice, a loss of mitochondrial DNA (mtDNA), followed by a severe defect in the oxidative phosphorylation system is induced specifically in Keratin 14 (K14+) expressing epithelial cells. Histochemical staining showed severe reduction of cytochrome c oxidase activity only in K14+ epithelial cells. In mutant incisors, H&E staining showed severe defects in the ameloblasts, in the epithelial cells of the stratum intermedium and the papillary cell layer, but also a disturbed odontoblast layer. The lack of amelogenin in the enamel matrix of K320E-Twinkle mice indicated that defective ameloblasts are not able to form extracellular enamel matrix proteins. In comparison to control incisors, von Kossa staining showed enamel biomineralization defects and dentin matrix impairment. In mutant incisor, TUNEL staining and ultrastructural analyses revealed differentiation defects, while in hair follicle cells apoptosis is prevalent. We concluded that mitochondrial oxidative phosphorylation in epithelial cells of the developed incisor is required for Ca2+ homeostasis to regulate the formation of enamel matrix and induce the differentiation of ectomesenchymal cells into odontoblasts.
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http://dx.doi.org/10.1038/s41598-020-77954-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744519PMC
December 2020

Rapid SARS-CoV-2 testing in primary material based on a novel multiplex RT-LAMP assay.

PLoS One 2020 2;15(11):e0238612. Epub 2020 Nov 2.

Department II of Internal Medicine, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany.

Background: Rapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose.

Methods: To avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (RT-LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK).

Results: Whilst specificity of standard RT-LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed RT-LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel RT-LAMP assays with SHERLOCK.

Conclusion: In summary, this approach reveals one-step multiplexed RT-LAMP assays as a prime-option for the development of easy and cheap POC test kits.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0238612PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605681PMC
November 2020

Collagen XII mediated cellular and extracellular mechanisms regulate establishment of tendon structure and function.

Matrix Biol 2021 01 20;95:52-67. Epub 2020 Oct 20.

College of Medicine, University of South Florida, Morsani, Tampa, FL, United States.

Tendons have a uniaxially aligned structure with a hierarchical organization of collagen fibrils crucial for tendon function. Collagen XII is expressed in tendons and has been implicated in the regulation of fibrillogenesis. It is a non-fibrillar collagen belonging to the Fibril-Associated Collagens with Interrupted Triple Helices (FACIT) family. Mutations in COL12A1 cause myopathic Ehlers Danlos Syndrome with a clinical phenotype involving both joints and tendons supporting critical role(s) for collagen XII in tendon development and function. Here we demonstrate the molecular function of collagen XII during tendon development using a Col12a1 null mouse model. Col12a1 deficiency altered tenocyte shape, formation of interacting cell processes, and organization resulting in impaired cell-cell communication and disruption of hierarchal structure as well as decreased tissue stiffness. Immuno-localization revealed that collagen XII accumulated on the tenocyte surface and connected adjacent tenocytes by building matrix bridges between the cells, suggesting that collagen XII regulates intercellular communication. In addition, there was a decrease in fibrillar collagen I in collagen XII deficient tenocyte cultures compared with controls suggesting collagen XII signaling specifically alters tenocyte biosynthesis. This suggests that collagen XII provides feedback to tenocytes regulating extracellular collagen I. Together, the data indicate dual roles for collagen XII in determination of tendon structure and function. Through association with fibrils it functions in fibril packing, fiber assembly and stability. In addition, collagen XII influences tenocyte organization required for assembly of higher order structure; intercellular communication necessary to coordinate long range order and feedback on tenocytes influencing collagen synthesis. Integration of both regulatory roles is required for the acquisition of hierarchal structure and mechanical properties.
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http://dx.doi.org/10.1016/j.matbio.2020.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870578PMC
January 2021

EMILIN proteins are novel extracellular constituents of the dentin-pulp complex.

Sci Rep 2020 09 18;10(1):15320. Epub 2020 Sep 18.

Center for Biochemistry, Medical Faculty, University of Cologne, 50931, Cologne, Germany.

Odontoblasts and pulp stroma cells are embedded within supramolecular networks of extracellular matrix (ECM). Fibrillin microfibrils and associated proteins are crucial constituents of these networks, serving as contextual scaffolds to regulate tissue development and homeostasis by providing both structural and mechanical properties and sequestering growth factors of the TGF-β superfamily. EMILIN-1, -2, and -3 are microfibril-associated glycoproteins known to modulate cell behaviour, growth factor activity, and ECM assembly. So far their expression in the various cells of the dentin-pulp complex during development, in the adult stage, and during inflammation has not been investigated. Confocal immunofluorescence microscopy and western blot analysis of developing and adult mouse molars and incisors revealed an abundant presence of EMILINs in the entire dental papilla, at early developmental stages. Later in development the signal intensity for EMILIN-3 decreases, while EMILIN-1 and -2 staining appears to increase in the pre-dentin and in the ECM surrounding odontoblasts. Our data also demonstrate new specific interactions of EMILINs with fibulins in the dentin enamel junction. Interestingly, in dentin caries lesions the signal for EMILIN-3 was significantly increased in inflamed odontoblasts. Overall our findings point for the first time to a role of EMILINs in dentinogenesis, pulp biology, and inflammation.
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http://dx.doi.org/10.1038/s41598-020-72123-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501263PMC
September 2020

Role of collagen XII in skin homeostasis and repair.

Matrix Biol 2020 12 2;94:57-76. Epub 2020 Sep 2.

Translational Matrix Biology, University of Cologne, Medical Faculty, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany. Electronic address:

Skin integrity and function depends to a large extent on the composition of the extracellular matrix, which regulates tissue organization. Collagen XII is a homotrimer with short collagenous domains that confer binding to the surface of collagen I-containing fibrils and extended flexible arms, which bind to non-collagenous matrix components. Thereby, collagen XII helps to maintain collagen suprastructure and to absorb stress. Mutant or absent collagen XII leads to reduced muscle and bone strength and lax skin, whereas increased collagen XII amounts are observed in tumor stroma, scarring and fibrosis. This study aimed at uncovering in vivo mechanisms by which collagen XII may achieve these contrasting outcomes. We analyzed skin as a model tissue that contains abundant fibrils, composed of collagen I, III and V with collagen XII decorating their surface, and which is subject to mechanical stress. The impact of different collagen XII levels was investigated in collagen XII-deficient (Col12-KO) mice and in mice with collagen XII overexpression in the dermis (Col12-OE). Unchallenged skin of these mice was histologically inconspicuous, but at the ultrastructural level revealed distinct aberrations in collagen network suprastructure. Repair of excisional wounds deviated from controls in both models by delayed healing kinetics, which was, however, caused by completely different mechanisms in the two mouse lines. The disorganized matrix in Col12-KO wounds failed to properly sequester TGFβ, resulting in elevated numbers of myofibroblasts. These are, however, unable to contract and remodel the collagen XII-deficient matrix. Excess of collagen XII, in contrast, promotes persistence of M1-like macrophages in the wound bed, thereby stalling the wounds in an early inflammatory stage of the repair process and delaying healing. Taken together, we demonstrate that collagen XII is a key component that assists in orchestrating proper skin matrix structure, controls growth factor availability and regulates cellular composition and function. Together, these functions are pivotal for re-establishing homeostasis after injury.
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http://dx.doi.org/10.1016/j.matbio.2020.08.002DOI Listing
December 2020

New specific HSP47 functions in collagen subfamily chaperoning.

FASEB J 2020 09 27;34(9):12040-12052. Epub 2020 Jul 27.

Faculty of Medicine, Center for Biochemistry, University of Cologne, Cologne, Germany.

Although collagens are the most abundant proteins implicated in various disease pathways, essential mechanisms required for their proper folding and assembly are poorly understood. Heat-shock protein 47 (HSP47), an ER-resident chaperone, was mainly reported to fulfill key functions in folding and secretion of fibrillar collagens by stabilizing pro-collagen triple-helices. In this study, we demonstrate unique functions of HSP47 for different collagen subfamilies. Our results show that HSP47 binds to the N-terminal region of procollagen I and is essential for its secretion. However, HSP47 ablation does not majorly impact collagen VI secretion, but its lateral assembly. Moreover, specific ablation of Hsp47 in murine keratinocytes revealed a new role for the transmembrane collagen XVII triple-helix formation. Incompletely folded collagen XVII C-termini protruding from isolated HSP47 null keratinocyte membrane vesicles could be fully restored upon the application of recombinant HSP47. Thus, our study expands the current view regarding the client repertoire and function of HSP47, as well as emphasizes its importance for transmembrane collagen folding.
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http://dx.doi.org/10.1096/fj.202000570RDOI Listing
September 2020

Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients.

Cell 2020 08 13;182(4):843-854.e12. Epub 2020 Jul 13.

Laboratory of Experimental Immunology, Institute of Virology, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany; German Center for Infection Research, Partner Site Bonn-Cologne, 50931 Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50931 Cologne, Germany. Electronic address:

The SARS-CoV-2 pandemic has unprecedented implications for public health, social life, and the world economy. Because approved drugs and vaccines are limited or not available, new options for COVID-19 treatment and prevention are in high demand. To identify SARS-CoV-2-neutralizing antibodies, we analyzed the antibody response of 12 COVID-19 patients from 8 to 69 days after diagnosis. By screening 4,313 SARS-CoV-2-reactive B cells, we isolated 255 antibodies from different time points as early as 8 days after diagnosis. Of these, 28 potently neutralized authentic SARS-CoV-2 with IC as low as 0.04 μg/mL, showing a broad spectrum of variable (V) genes and low levels of somatic mutations. Interestingly, potential precursor sequences were identified in naive B cell repertoires from 48 healthy individuals who were sampled before the COVID-19 pandemic. Our results demonstrate that SARS-CoV-2-neutralizing antibodies are readily generated from a diverse pool of precursors, fostering hope for rapid induction of a protective immune response upon vaccination.
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http://dx.doi.org/10.1016/j.cell.2020.06.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355337PMC
August 2020

Tenascin-C Orchestrates an Immune-Suppressive Tumor Microenvironment in Oral Squamous Cell Carcinoma.

Cancer Immunol Res 2020 09 14;8(9):1122-1138. Epub 2020 Jul 14.

Université Strasbourg, INSERM U1109-MN3T, The Microenvironmental Niche in Tumorigenesis and Targeted Therapy, and The Tumor Microenvironment Laboratory, Hopital Civil, Institut d'Hématologie et d'Immunologie, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Strasbourg, France.

Inherent immune suppression represents a major challenge in the treatment of human cancer. The extracellular matrix molecule tenascin-C promotes cancer by multiple mechanisms, yet the roles of tenascin-C in tumor immunity are incompletely understood. Using a 4NQO-induced oral squamous cell carcinoma (OSCC) model with abundant and absent tenascin-C, we demonstrated that tenascin-C enforced an immune-suppressive lymphoid stroma via CCL21/CCR7 signaling, leading to increased metastatic tumors. Through TLR4, tenascin-C increased expression of CCR7 in CD11c myeloid cells. By inducing CCL21 in lymphatic endothelial cells via integrin α9β1 and binding to CCL21, tenascin-C immobilized CD11c cells in the stroma. Inversion of the lymph node-to-tumor CCL21 gradient, recruitment of T regulatory cells, high expression of anti-inflammatory cytokines, and matrisomal components were hallmarks of the tenascin-C-instructed lymphoid stroma. Ablation of tenascin-C or CCR7 blockade inhibited the lymphoid immune-suppressive stromal properties, reducing tumor growth, progression, and metastasis. Thus, targeting CCR7 could be relevant in human head and neck tumors, as high tenascin-C expression and an immune-suppressive stroma correlate to poor patient survival.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0074DOI Listing
September 2020

Affinity-Enhanced Multimeric VEGF (Vascular Endothelial Growth Factor) and PlGF (Placental Growth Factor) Variants for Specific Adsorption of sFlt-1 to Restore Angiogenic Balance in Preeclampsia.

Hypertension 2020 10 6;76(4):1176-1184. Epub 2020 Jul 6.

From the Department II of Internal Medicine (M. Matin, B.S., P.T.B., T.B., H.H.), University of Cologne, Faculty of Medicine, University Hospital Cologne, Germany.

Preeclampsia is a potentially life-threatening multisystem disease affecting 4% to 8% of pregnant women after the 20th week of gestation. An excess of placental expressed antiangiogenic soluble VEGF (vascular endothelial growth factor)-receptor 1 (soluble FMS-like tyrosine kinase 1) scavenges VEGF and PlGF (placental growth factor), causing generalized endothelial dysfunction. Interventions to restore the angiogenic balance in preeclamptic pregnancies are intensively studied and improve maternal and neonatal outcomes. Especially extracorporeal strategies to remove sFlt-1 are promising in human pregnancy. However, available apheresis systems adsorb sFlt-1 unspecifically and with low efficiency. Affinity-enhanced ligands are needed to improve performance and compatibility of apheresis treatments. Using computerized molecular modeling, we developed multimeric VEGF molecules comprised of single-chain VEGF dimers (scVEGF). A short peptide linker hampers intrachain dimerization to induce assembly preferably as tetrameric molecules as visualized in negative staining electron microscopy. scVEGF multimers possess 1.2-fold higher affinity for sFlt-1 as compared to the available antibodies or monomeric VEGF. Consequently, scVEGF multimers have the ability to competitively release sFlt-1 bound PlGF and, in particular, VEGF. In ex vivo adsorption experiments using serum samples from patients with preeclampsia, scVEGF multimers reduce sFlt-1 levels by 85% and increase PlGF and VEGF levels by 20- and 9-fold, respectively. Finally, performance and stability of sFlt-1 capturing scVEGF multimers were scrutinized on different matrices of which biocompatible agarose matrix yielded optimal results. We introduce the first VEGF-based highly efficient sFlt-1 apheresis system that is directly applicable in vivo due to utilization of inert agarose matrix, using a homomultimeric form of VEGF to restore the angiogenic balance in preeclampsia.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.120.14974DOI Listing
October 2020

Collagen XII Is a Regulator of Corneal Stroma Structure and Function.

Invest Ophthalmol Vis Sci 2020 05;61(5):61

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Purpose: The aim of this study was to determine the roles of collagen XII in the regulation of stromal hierarchical organization, keratocyte organization, and corneal mechanics.

Methods: The temporal and spatial expression of collagen XII at postnatal days 4, 10, 30, 90, and 150 were evaluated in wild-type (WT) mice. The role of collagen XII in hierarchical organization was analyzed by measuring fibril diameter and density, as well as stromal lamellar structure, within ultrastructural micrographs obtained from WT and collagen XII-deficient mice (Col12a1-/-). Keratocyte morphology and networks were assessed using actin staining with phalloidin and in vivo confocal microscopy. The effects of collagen XII on corneal biomechanics were evaluated with atomic force microscopy.

Results: Collagen XII was localized homogeneously in the stroma from postnatal day 4 to day 150, and protein accumulation was shown to increase during this period using semiquantitative immunoblots. Higher fibril density (P < 0.001) and disruption of lamellar organization were found in the collagen XII null mice stroma when compared to WT mice. Keratocyte networks and organization were altered in the absence of collagen XII, as demonstrated using fluorescent microscopy after phalloidin staining and in vivo confocal microscopy. Corneal stiffness was increased in the absence of collagen XII. Young's modulus was 16.2 ± 5.6 kPa in WT and 32.8 ± 6.4 kPa in Col12a1-/- corneas. The difference between these two groups was significant (P < 0.001, t-test).

Conclusions: Collagen XII plays a major role in establishing and maintaining stromal structure and function. In the absence of collagen XII, the corneal stroma showed significant abnormalities, including decreased interfibrillar space, disrupted lamellar organization, abnormal keratocyte organization, and increased corneal stiffness.
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http://dx.doi.org/10.1167/iovs.61.5.61DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405808PMC
May 2020

Topical VEGF-C/D Inhibition Prevents Lymphatic Vessel Ingrowth into Cornea but Does Not Improve Corneal Graft Survival.

J Clin Med 2020 Apr 28;9(5). Epub 2020 Apr 28.

Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, 50937 Cologne, Germany.

Vascular endothelial growth factor-C/D (VEGF-C/D) regulates lymphangiogenesis. Ingrowth of lymphatic vessels is negatively associated with corneal transplantation success. In this study, we therefore analyzed the effect local blockade of VEGF-C/D has on inflamed corneas. We used the murine model of suture-induced neovascularization and subsequent high-risk corneal transplantation. Mice were treated with a VEGF-C/D trap prior to transplantation. Topical inhibition of VEGF-C/D significantly reduced lymphatic vessel ingrowth, but increased Macrophage numbers in the cornea. Furthermore, corneal transplantation success was not improved by the topical application of the compound. This study demonstrates that local VEGF-C/D inhibition is insufficient to increases corneal transplantation success, likely due to interaction with immune cells.
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http://dx.doi.org/10.3390/jcm9051270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7287580PMC
April 2020

Smart Hydrogels for the Augmentation of Bone Regeneration by Endogenous Mesenchymal Progenitor Cell Recruitment.

Adv Sci (Weinh) 2020 Apr 5;7(7):1903395. Epub 2020 Feb 5.

Department of Obstetrics University Hospital Zurich University of Zurich Schmelzbergstr. 12 Zurich 8091 Switzerland.

The treatment of bone defects with recombinant bone morphogenetic protein-2 (BMP-2) requires high doses precluding broad clinical application. Here, a bioengineering approach is presented that strongly improves low-dose BMP-2-based bone regeneration by mobilizing healing-associated mesenchymal progenitor cells (MPCs). Smart synthetic hydrogels are used to trap and study endogenous MPCs trafficking to bone defects. Hydrogel-trapped and prospectively isolated MPCs differentiate into multiple lineages in vitro and form bone in vivo. In vitro screenings reveal that platelet-derived growth factor BB (PDGF-BB) strongly recruits prospective MPCs making it a promising candidate for the engineering of hydrogels that enrich endogenous MPCs in vivo. However, PDGF-BB inhibits BMP-2-mediated osteogenesis both in vitro and in vivo. In contrast, smart two-way dynamic release hydrogels with fast-release of PDGF-BB and sustained delivery of BMP-2 beneficially promote the healing of bone defects. Collectively, it is shown that modulating the dynamics of endogenous progenitor cells in vivo by smart synthetic hydrogels significantly improves bone healing and holds great potential for other advanced applications in regenerative medicine.
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http://dx.doi.org/10.1002/advs.201903395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141038PMC
April 2020

Netrin-1 promotes naive pluripotency through Neo1 and Unc5b co-regulation of Wnt and MAPK signalling.

Nat Cell Biol 2020 04 30;22(4):389-400. Epub 2020 Mar 30.

Cellular Reprogramming and Oncogenesis Laboratory, Equipe labellisée la Ligue contre le cancer, Labex DEVweCAN, Université de Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en cancérologie de Lyon, Lyon, France.

In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3α/β and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. Here we show that the axon guidance cue Netrin-1 promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, we demonstrate that Netrin-1 can substitute for blockade of Gsk3α/β and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, we reveal how Netrin-1 and the balance of its receptors Neo1 and Unc5B co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase to inactivate Gsk3α/β and stabilize β-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.
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http://dx.doi.org/10.1038/s41556-020-0483-2DOI Listing
April 2020

Author Correction: Decellularization and antibody staining of mouse tissues to map native extracellular matrix structures in 3D.

Nat Protoc 2020 06;15(6):2140

Biotech Research and Innovation Centre (BRIC), University of Copenhagen (UCPH), Copenhagen, Denmark.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41596-020-0315-7DOI Listing
June 2020

Effectiveness of AbobotulinumtoxinA in Post-stroke Upper Limb Spasticity in Relation to Timing of Treatment.

Front Neurol 2020 28;11:104. Epub 2020 Feb 28.

IPSEN PHARMA GmbH, Munich, Germany.

Recent studies of botulinum toxin for post-stroke spasticity indicate potential benefits of early treatment (i. e., first 6 months) in terms of developing hypertonicity, pain and passive function limitations. This non-interventional, longitudinal study aimed to assess the impact of disease duration on the effectiveness of abobotulinumtoxinA treatment for upper limb spasticity. The early-BIRD study (NCT01840475) was conducted between February 2013 and 2018 in 43 centers across Germany, France, Austria, Netherlands and Switzerland. Adult patients with post-stroke upper limb spasticity undergoing routine abobotulinumtoxinA treatment were followed for up to four treatment cycles. Patients were categorized by time from stroke event to first botulinum toxin-A treatment in the study (as defined by the 1st and 3rd quartiles time distribution) into early-, medium- and late- start groups. We hypothesized that the early-start group would show a larger benefit (decrease) as assessed by the modified Ashworth scale (MAS, primary endpoint) on elbow plus wrist flexors compared with the late-start group. Of the 303 patients enrolled, 292 (96.4%) received ≥1 treatment and 186 (61.4%) received 4 injection cycles and completed the study. Patients in all groups showed a reduction in MAS scores from baseline over the consecutive injection visits (i.e., at end of each cycle). Although reductions in MAS scores descriptively favored the early treatment group, the difference compared to the late group did not reach statistical significance at the last study visit (ANCOVA: difference in adjusted means of 0.15, = 0.546). In this observational, routine-practice study, patients in all groups displayed a benefit from abobotulinumtoxinA treatment, supporting the effectiveness of treatment for patients at various disease stages. Although the data revealed some trends in favor of early vs. late treatment, we did not find strong evidence for a significant benefit of early vs. late start of treatment in terms of reduction in MAS scores.
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http://dx.doi.org/10.3389/fneur.2020.00104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058702PMC
February 2020

AMD-Associated HTRA1 Variants Do Not Influence TGF-β Signaling in Microglia.

Adv Exp Med Biol 2019 ;1185:3-7

Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany.

Genetic variants of high-temperature requirement A serine peptidase 1 (HTRA1) and age-related maculopathy susceptibility 2 (ARMS2) are associated with age-related macular degeneration (AMD). One HTRA1 single nucleotide polymorphism (SNP) is situated in the promotor region (rs11200638) resulting in increased expression, while two synonymous SNPs are located in exon 1 (rs1049331:C > T, rs2293870:G > T). HtrA1 is known to inhibit transforming growth factor-β (TGF-β) signaling, a pathway regulating quiescence of microglia, the resident immune cells of the brain and retina. Microglia-mediated immune responses contribute to AMD pathogenesis. It is currently unclear whether AMD-associated HTRA1 variants influence TGF-β signaling and microglia phenotypes. Here, we show that an HtrA1 isoform carrying AMD-associated SNPs in exon 1 exhibits increased proteolytic activity. However, when incubating TGF-β-treated reactive microglia with HtrA1 protein variants, neither the wildtype nor the SNP-associated isoforms changed microglia activation in vitro.
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http://dx.doi.org/10.1007/978-3-030-27378-1_1DOI Listing
February 2020

Decellularization and antibody staining of mouse tissues to map native extracellular matrix structures in 3D.

Nat Protoc 2019 12 8;14(12):3395-3425. Epub 2019 Nov 8.

Biotech Research and Innovation Centre (BRIC), University of Copenhagen (UCPH), Copenhagen, Denmark.

The extracellular matrix (ECM) is a major regulator of homeostasis and disease, yet the 3D structure of the ECM remains poorly understood because of limitations in ECM visualization. We recently developed an ECM-specialized method termed in situ decellularization of tissues (ISDoT) to isolate native 3D ECM scaffolds from whole organs in which ECM structure and composition are preserved. Here, we present detailed surgical instructions to facilitate decellularization of 33 different mouse tissues and details of validated antibodies that enable the visualization of 35 mouse ECM proteins. Through mapping of these ECM proteins, the structure of the ECM can be determined and tissue structures visualized in detail. In this study, perfusion decellularization is presented for bones, skeletal muscle, tongue, salivary glands, stomach, duodenum, jejunum/ileum, large intestines, mesentery, liver, gallbladder, pancreas, trachea, bronchi, lungs, kidneys, urinary bladder, ovaries, uterine horn, cervix, adrenal gland, heart, arteries, veins, capillaries, lymph nodes, spleen, peripheral nerves, eye, outer ear, mammary glands, skin, and subcutaneous tissue. Decellularization, immunostaining, and imaging take 4-5 d.
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http://dx.doi.org/10.1038/s41596-019-0225-8DOI Listing
December 2019

Gene Expression Profiling of the Extracellular Matrix Signature in Macrophages of Different Activation Status: Relevance for Skin Wound Healing.

Int J Mol Sci 2019 Oct 14;20(20). Epub 2019 Oct 14.

Center for Biochemistry, Medical Faculty, University of Cologne, 50931 Cologne, Germany.

The extracellular matrix (ECM) provides structural support for tissue architecture and is a major effector of cell behavior during skin repair and inflammation. Macrophages are involved in all stages of skin repair but only limited knowledge exists about macrophage-specific expression and regulation of ECM components. In this study, we used transcriptome profiling and bioinformatic analysis to define the unique expression of ECM-associated genes in cultured macrophages. Characterization of the matrisome revealed that most genes were constitutively expressed and that several genes were uniquely regulated upon interferon gamma (IFNγ) and dexamethasone stimulation. Among those core matrisome and matrisome-associated components transforming growth factor beta (TGFβ)-induced, matrix metalloproteinase 9 (MMP9), elastin microfibril interfacer (EMILIN)-1, netrin-1 and gliomedin were also present within the wound bed at time points that are characterized by profound macrophage infiltration. Hence, macrophages are a source of ECM components in vitro as well as during skin wound healing, and identification of these matrisome components is a first step to understand the role and therapeutic value of ECM components in macrophages and during wound healing.
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http://dx.doi.org/10.3390/ijms20205086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829210PMC
October 2019

Lgr5 and Col22a1 Mark Progenitor Cells in the Lineage toward Juvenile Articular Chondrocytes.

Stem Cell Reports 2019 10 12;13(4):713-729. Epub 2019 Sep 12.

School of Biomedical Sciences, The University of Hong Kong, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong SAR, China; The University of Hong Kong - Shenzhen Institute of Research and Innovation (HKU- SIRI), Hi-Tech Industrial Park, Nanshan, Shenzhen, China. Electronic address:

The synovial joint forms from a pool of progenitor cells in the future region of the joint, the interzone. Expression of Gdf5 and Wnt9a has been used to mark the earliest cellular processes in the formation of the interzone and the progenitor cells. However, lineage specification and progression toward the different tissues of the joint are not well understood. Here, by lineage-tracing studies we identify a population of Lgr5 interzone cells that contribute to the formation of cruciate ligaments, synovial membrane, and articular chondrocytes of the joint. This finding is supported by single-cell transcriptome analyses. We show that Col22a1, a marker of early articular chondrocytes, is co-expressed with Lgr5 cells prior to cavitation as an important lineage marker specifying the progression toward articular chondrocytes. Lgr5 cells contribute to the repair of a joint defect with the re-establishment of a Col22a1-expressing superficial layer.
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http://dx.doi.org/10.1016/j.stemcr.2019.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829785PMC
October 2019

Dominant collagen XII mutations cause a distal myopathy.

Ann Clin Transl Neurol 2019 10 11;6(10):1980-1988. Epub 2019 Sep 11.

National Institutes of Health, NINDS, NNDCS, Bethesda, Maryland.

Objective: To characterize the natural history and clinical features of myopathies caused by mono-allelic, dominantly acting pathogenic variants in COL12A1.

Methods: Patients with dominant COL12A1-related myopathies were characterized by history and clinical examination, muscle imaging, and genetic analysis. Pathogenicity of the variants was assessed by immunostaining patient-derived dermal fibroblast cultures for collagen XII.

Results: Four independent families with childhood-onset weakness due to novel, dominantly acting pathogenic variants in COL12A1 were identified. Adult patients exhibited distal-predominant weakness. Three families carried dominantly acting glycine missense variants, and one family had a heterozygous, intragenic, in-frame deletion of exon 52 of COL12A1. All pathogenic variants resulted in increased intracellular retention of collagen XII in patient-derived fibroblasts as well as loss of extracellular, fibrillar collagen XII deposition. Since haploinsufficiency for COL12A1 is largely clinically asymptomatic, we designed and evaluated small interfering RNAs (siRNAs) that specifically target the mutant allele containing the exon 52 deletion. Immunostaining of the patient fibroblasts treated with the siRNA showed a near complete correction of collagen XII staining patterns.

Interpretation: This study characterizes a distal myopathy phenotype in adults with dominant COL12A1 pathogenic variants, further defining the phenotypic spectrum and natural history of COL12A1-related myopathies. This work also provides proof of concept of a precision medicine treatment approach by proposing and validating allele-specific knockdown using siRNAs specifically designed to target a patient's dominant COL12A1 disease allele.
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http://dx.doi.org/10.1002/acn3.50882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801183PMC
October 2019

Solution Structure of C. elegans UNC-6: A Nematode Paralogue of the Axon Guidance Protein Netrin-1.

Biophys J 2019 06 3;116(11):2121-2130. Epub 2019 May 3.

Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada; Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; DiscoveryLab and Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada. Electronic address:

UNCoordinated-6 (UNC-6) was the first member of the netrin family to be discovered in Caenorhabditis elegans. With homology to human netrin-1, it is a key signaling molecule involved in directing axon migration in nematodes. Similar to netrin-1, UNC-6 interacts with multiple receptors (UNC-5 and UNC-40, specifically) to guide axon migration in development. As a result of the distinct evolutionary path of UNC-6 compared to vertebrate netrins, we decided to employ an integrated approach to study its solution behavior and compare it to the high-resolution structure we previously published on vertebrate netrins. Dynamic light scattering and analytical ultracentrifugation on UNC-6 (with and without its C-domain) solubilized in a low-ionic strength buffer suggested that UNC-6 forms high-order oligomers. An increase in the buffer ionic strength resulted in a more homogeneous preparation of UNC-6, that was used for subsequent solution x-ray scattering experiments. Our biophysical analysis of UNC-6 ΔC solubilized in a high-ionic strength buffer suggested that it maintains a similar head-to-stalk arrangement as netrins -1 and -4. This phenomenon is thought to play a role in the signaling behavior of UNC-6 and its ability to move throughout the extracellular matrix.
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http://dx.doi.org/10.1016/j.bpj.2019.04.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6554493PMC
June 2019

Local VEGF-A blockade modulates the microenvironment of the corneal graft bed.

Am J Transplant 2019 09 3;19(9):2446-2456. Epub 2019 Apr 3.

Department of Ophthalmology, University Hospital of Cologne, Cologne, Germany.

The microenvironment plays an important role in several immunological processes. Vascular endothelial growth factor-A (VEGF-A) not only regulates angiogenesis, but is known as a modulator of the immune microenvironment. Modulating the site of transplantation might be beneficial for subsequent transplant survival. In this study, we therefore analyzed the effect that a local blockade of VEGF-A in the inflamed cornea as the graft receiving tissue has on the immune system. We used the murine model of suture-induced neovascularization and subsequent high-risk corneal transplantation, which is an optimal model for local drug application. Mice were treated with VEGFR1/R2 trap prior to transplantation. We analyzed corneal gene expression, as well as protein levels in the cornea and serum on the day of transplantation, 2 and 8 weeks later. Local VEGF depletion prior to transplantation increases the expression of pro-inflammatory as well as immune regulatory cytokines only in the corneal microenvironment, but not in the serum. Furthermore, local VEGFR1/R2 trap treatment significantly inhibits the infiltration of CD11c+ dendritic cells into the cornea. Subsequent increased corneal transplantation success was accompanied by a local upregulation of Foxp3 gene expression. This study demonstrates that locally restricted VEGF depletion increases transplantation success by modulating the receiving corneal microenvironment and inducing tolerogenic mechanisms.
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http://dx.doi.org/10.1111/ajt.15331DOI Listing
September 2019

COMP and TSP-4 interact specifically with the novel GXKGHR motif only found in fibrillar collagens.

Sci Rep 2018 11 21;8(1):17187. Epub 2018 Nov 21.

Institute of Biochemistry, University of Cologne, Cologne, Germany.

COMP (cartilage oligomeric matrix protein) is a member of the thrombospondin family and forms homopentamers as well as mixed heterooligomers with its closely related family member TSP-4. COMP is long known to bind to collagens and to influence collagen fibril formation. Recent work indicates that already intracellular interaction with collagen is important for collagen secretion. However, the exact binding site of COMP on the collagen triple helix has not been described up to now. In this study we have identified a GXKGHR motif on the collagen II helix to bind to COMP, using a recombinantly expressed collagen II peptide library. This binding sequence is conserved throughout evolution and we demonstrate that TSP-4 binds to the same sequence. The identified binding motif overlaps with the recognition sites of many other collagen-binding partners (e.g. PEDF, Heparin) and also spans the lysine residues, which form collagen cross-links. COMP might thereby protect collagen helices from premature modification and cross-linking. Interestingly, this motif is only found in classical fibrillar collagens, although COMP is known to also bind other types. This might indicate that COMP has a unique interface for fibrillar collagens, thus making it an interesting target for the development of antifibrotic drugs.
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http://dx.doi.org/10.1038/s41598-018-35447-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249252PMC
November 2018

Genomic features of the Helicobacter pylori strain PMSS1 and its virulence attributes as deduced from its in vivo colonisation patterns.

Mol Microbiol 2018 12 25;110(5):761-776. Epub 2018 Oct 25.

Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, 10117, Germany.

The human gastric pathogen Helicobacter pylori occurs in two basic variants, either exhibiting a functional cagPAI-encoded type-4-secretion-system (T4SS) or not. Only a few cagPAI-positive strains have been successfully adapted for long-term infection of mice, including the pre-mouse Sydney strain 1 (PMSS1). Here we confirm that PMSS1 induces gastric inflammation and neutrophil infiltration in mice, progressing to intestinal metaplasia. Complete genome analysis of PMSS1 revealed 1,423 coding sequences, encompassing the cagPAI gene cluster and, unusually, the location of the cytotoxin-associated gene A (cagA) approximately 15 kb downstream of the island. PMSS1 harbours three genetically exchangeable loci that are occupied by the hopQ coding sequences. HopQ represents a critical co-factor required for the translocation of CagA into the host cell and activation of NF-κB via the T4SS. Long-term colonisation of mice led to an impairment of cagPAI functionality. One of the bacterial clones re-isolated at four months post-infection revealed a mutation in the cagPAI gene cagW, resulting in a frame shift mutation, which prevented CagA translocation, possibly due to an impairment of T4SS function. Rescue of the mutant cagW re-established CagA translocation. Our data reveal intriguing insights into the adaptive abilities of PMSS1, suggesting functional modulation of the H. pylori cagPAI virulence attribute.
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http://dx.doi.org/10.1111/mmi.14123DOI Listing
December 2018

Molecular diagnosis of anti-laminin 332 (epiligrin) mucous membrane pemphigoid.

Orphanet J Rare Dis 2018 07 6;13(1):111. Epub 2018 Jul 6.

Department of Dermatology, University Medical Center Freiburg, Hauptstrasse 7, 79104, Freiburg, Germany.

Background: Mucous 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.

Results: Using 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.

Conclusions: Our findings qualify the use of immunoassays with the laminin 332-rich preparations as an ancillary diagnostic tool in mucous membrane pemphigoid.
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http://dx.doi.org/10.1186/s13023-018-0855-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6035451PMC
July 2018