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Zlatko Kopecki - One of the best experts on this subject based on the ideXlab platform.
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Skin barrier and autoimmunity mechanisms and novel therapeutic approaches for autoimmune Blistering diseases of the Skin
Frontiers in Immunology, 2019Co-Authors: Natalie E Stevens, Allison J Cowin, Zlatko KopeckiAbstract:One of the most important functions of the Skin besides regulating internal body temperature includes formation of the barrier between the organism and the external environment, hence protecting against pathogen invasion, chemical and physical assaults and unregulated loss of water and solutes. Disruption of the protective barrier is observed clinically in blisters and erosions of the Skin that form in autoimmune Blistering diseases where the body produces autoantibodies against structural proteins of the epidermis or the epidermal-dermal junction. Although there is no cure for autoimmune Skin Blistering diseases, immune suppressive therapies currently available offer opportunities for disease management. In cases where no treatment is sought, these disorders can lead to life threatening complications and current research efforts have focused on developing therapies that target autoantibodies which contribute to disease symptoms. This review will outline the involvement of the Skin barrier in main Skin-specific autoimmune Blistering diseases by describing the mechanisms underpinning Skin autoimmunity and review current progress in development of novel therapeutic approaches targeting the underlying causes of autoimmune Skin Blistering diseases.
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cytoskeletal regulation of inflammation and its impact on Skin Blistering disease epidermolysis bullosa acquisita
International Journal of Molecular Sciences, 2016Co-Authors: Zlatko Kopecki, Ralf Ludwig, Allison J CowinAbstract:Actin remodelling proteins regulate cytoskeletal cell responses and are important in both innate and adaptive immunity. These responses play a major role in providing a fine balance in a cascade of biological events that results in either protective acute inflammation or chronic inflammation that leads to a host of diseases including autoimmune inflammation mediated epidermolysis bullosa acquisita (EBA). This review describes the role of the actin cytoskeleton and in particular the actin remodelling protein called Flightless I (Flii) in regulating cellular inflammatory responses and its subsequent effect on the autoimmune Skin Blistering disease EBA. It also outlines the potential of an antibody based therapy for decreasing Flii expression in vivo to ameliorate the symptoms associated with EBA.
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flightless i over expression impairs Skin barrier development function and recovery following Skin Blistering
The Journal of Pathology, 2014Co-Authors: Zlatko Kopecki, Hiroaki Iwata, Detlef Zillikens, Ralf Ludwig, Gink N Yang, Ruth M Arkell, Jessica E Jackson, Elizabeth Melville, Dedee F Murrell, Allison J CowinAbstract:Development of an intact epidermis is critical for maintaining the integrity of the Skin. Patients with epidermolysis bullosa (EB) experience multiple erosions, which breach the epidermal barrier and lead to increased microbial colocalization of wounds, infections and sepsis. The cytoskeletal protein Flightless I (Flii) is a known regulator of both development and wound healing. Using Flii+/−, WT and FliiTg/Tg mice, we investigated the effect of altering Flii levels in embryos and adult mice on the development of the epidermal barrier and, consequently, how this affects the integrity of the Skin in EB. Flii over-expression resulted in delayed formation of the epidermal barrier in embryos and decreased expression of tight junction (TJ) proteins Claudin-1 and ZO-2. Increased intercellular space and transepidermal water loss was observed in FliiTg/Tg adult mouse Skin, while FliiTg/Tg keratinocytes showed altered TJ protein localization and reduced transepithelial resistance. Flii is increased in the blistered Skin of patients with EB, and over-expression of Flii in experimental EBA showed impaired Claudin-1 and -4 TJ protein expression and delayed recovery of functional barrier post-Blistering. Immunoprecipitation confirmed Flii associated with TJ proteins and in vivo actin assays showed that the effect of Flii on actin polymerization underpinned the impaired barrier function observed in FliiTg/Tg mice. These results therefore demonstrate an important role for Flii in the development and regulation of the epidermal barrier, which may contribute to the impaired healing and Skin fragility of EB patients. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd
Detlef Zillikens - One of the best experts on this subject based on the ideXlab platform.
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Assessment of Healthcare Costs for Patients With Pemphigus and Bullous Pemphigoid in an Academic Centre in Germany
British Journal of Dermatology, 2020Co-Authors: Sascha Ständer, Detlef Zillikens, B Färber, S Radeke, E. Schmidt, Ralf LudwigAbstract:: The autoimmune Skin Blistering diseases pemphigus (vulgaris and foliaceus) and bullous pemphigoid (BP) pose a high burden on affected patients. With current treatment options, induction of remission is achieved in most patients. However, prolonged immunosuppression is required to maintain remission, and treatment-related morbidity and mortality further adds to the patients' burden1,2 . Hence, development of novel therapeutic strategies that are effective and safe is highly warranted. Recently, insights into pemphigus and BP pathogenesis identified new therapeutic targets and drugs3 .
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flightless i over expression impairs Skin barrier development function and recovery following Skin Blistering
The Journal of Pathology, 2014Co-Authors: Zlatko Kopecki, Hiroaki Iwata, Detlef Zillikens, Ralf Ludwig, Gink N Yang, Ruth M Arkell, Jessica E Jackson, Elizabeth Melville, Dedee F Murrell, Allison J CowinAbstract:Development of an intact epidermis is critical for maintaining the integrity of the Skin. Patients with epidermolysis bullosa (EB) experience multiple erosions, which breach the epidermal barrier and lead to increased microbial colocalization of wounds, infections and sepsis. The cytoskeletal protein Flightless I (Flii) is a known regulator of both development and wound healing. Using Flii+/−, WT and FliiTg/Tg mice, we investigated the effect of altering Flii levels in embryos and adult mice on the development of the epidermal barrier and, consequently, how this affects the integrity of the Skin in EB. Flii over-expression resulted in delayed formation of the epidermal barrier in embryos and decreased expression of tight junction (TJ) proteins Claudin-1 and ZO-2. Increased intercellular space and transepidermal water loss was observed in FliiTg/Tg adult mouse Skin, while FliiTg/Tg keratinocytes showed altered TJ protein localization and reduced transepithelial resistance. Flii is increased in the blistered Skin of patients with EB, and over-expression of Flii in experimental EBA showed impaired Claudin-1 and -4 TJ protein expression and delayed recovery of functional barrier post-Blistering. Immunoprecipitation confirmed Flii associated with TJ proteins and in vivo actin assays showed that the effect of Flii on actin polymerization underpinned the impaired barrier function observed in FliiTg/Tg mice. These results therefore demonstrate an important role for Flii in the development and regulation of the epidermal barrier, which may contribute to the impaired healing and Skin fragility of EB patients. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd
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gm csf modulates autoantibody production and Skin Blistering in experimental epidermolysis bullosa acquisita
Journal of Immunology, 2014Co-Authors: Unni Krishna S R L Samavedam, Enno Schmidt, Hiroaki Iwata, Susen Muller, Franziska S Schulze, Andreas Recke, Detlef Zillikens, Ralf LudwigAbstract:GM-CSF activates hematopoietic cells and recruits neutrophils and macrophages to sites of inflammation. Inhibition of GM-CSF attenuates disease activity in models of chronic inflammatory disease. Effects of GM-CSF blockade were linked to modulation of the effector phase, whereas effects on early pathogenic events, for example, Ab production, have not been identified. To evaluate yet uncharacterized effects of GM-CSF on early pathogenic events in chronic inflammation, we employed immunization-induced epidermolysis bullosa acquisita (EBA), an autoimmune bullous disease caused by autoantibodies to type VII collagen. Compared to wild-type mice, upon immunization, GM-CSF−/− mice produced lower serum autoantibody titers, which were associated with reduced neutrophil numbers in draining lymph nodes. The same effect was observed in neutrophil-depleted wild-type mice. Neutrophil depletion in GM-CSF−/− mice led to a stronger inhibition, indicating that GM-CSF and neutrophils have additive functions. To characterize the contribution of GM-CSF specifically in the effector phase of EBA, disease was induced by transfer of anti–type VII collagen IgG into mice. We observed an increased GM-CSF expression, and GM-CSF blockade reduced Skin Blistering. Additionally, GM-CSF enhanced reactive oxygen species release and neutrophil migration in vitro. In immunization-induced murine EBA, treatment with anti–GM-CSF had a beneficial effect on established disease. We demonstrate that GM-CSF modulates both autoantibody production and Skin Blistering in a prototypical organ-specific autoimmune disease.
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Genome-wide mapping of gene–microbiota interactions in susceptibility to autoimmune Skin Blistering
Nature Communications, 2013Co-Authors: Girish Srinivas, Detlef Zillikens, Steffen Möller, Jun Wang, Sven Künzel, John F. Baines, Saleh M. IbrahimAbstract:Susceptibility to chronic inflammatory diseases is determined by immunogenetic and environmental risk factors. Resident microbial communities often differ between healthy and diseased states, but whether these differences are of primary aetiological importance or secondary to the altered inflammatory environment remains largely unknown. Here we provide evidence for host gene–microbiota interactions contributing to disease risk in a mouse model of epidermolysis bullosa acquisita, an autoantibody-induced inflammatory Skin disease. Using an advanced intercross, we identify genetic loci contributing to Skin microbiota variability, susceptibility to Skin Blistering and their overlap. Furthermore, by treating bacterial species abundances as covariates with disease we reveal a novel disease locus. The majority of the identified covariate taxa are characterized by reduced abundance being associated with increased disease risk, providing evidence of a primary role in protection from disease. Further characterization of these putative probiotic species or species assemblages offers promising potential for preventative and therapeutic treatment development.
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genome wide mapping of gene microbiota interactions in susceptibility to autoimmune Skin Blistering
Nature Communications, 2013Co-Authors: Girish Srinivas, Detlef Zillikens, Steffen Möller, Jun Wang, Sven Künzel, John F. Baines, Saleh M. IbrahimAbstract:Susceptibility to chronic inflammatory diseases is determined by immunogenetic and environmental risk factors. Resident microbial communities often differ between healthy and diseased states, but whether these differences are of primary aetiological importance or secondary to the altered inflammatory environment remains largely unknown. Here we provide evidence for host gene–microbiota interactions contributing to disease risk in a mouse model of epidermolysis bullosa acquisita, an autoantibody-induced inflammatory Skin disease. Using an advanced intercross, we identify genetic loci contributing to Skin microbiota variability, susceptibility to Skin Blistering and their overlap. Furthermore, by treating bacterial species abundances as covariates with disease we reveal a novel disease locus. The majority of the identified covariate taxa are characterized by reduced abundance being associated with increased disease risk, providing evidence of a primary role in protection from disease. Further characterization of these putative probiotic species or species assemblages offers promising potential for preventative and therapeutic treatment development.
Gerhard Wiche - One of the best experts on this subject based on the ideXlab platform.
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the many faces of plectin and plectinopathies pathology and mechanisms
Acta Neuropathologica, 2013Co-Authors: Lilli Winter, Gerhard WicheAbstract:Plectin, a giant multifunctional cytolinker protein, plays a crucial role in stabilizing and orchestrating intermediate filament networks in cells. Mutations in the human plectin gene result in multiple diseases manifesting with muscular dystrophy, Skin Blistering, and signs of neuropathy. The most common disease caused by plectin deficiency is epidermolysis bullosa simplex (EBS)-MD, a rare autosomal-recessive Skin Blistering disorder with late-onset muscular dystrophy. EBS-MD patients and plectin-deficient mice display pathologic desmin-positive protein aggregates, degenerated myofibrils, and mitochondrial abnormalities, the hallmarks of myofibrillar myopathies. In addition to EBS-MD, plectin mutations have been shown to cause EBS-MD with a myasthenic syndrome, limb-girdle muscular dystrophy type 2Q, EBS with pyloric atresia, and EBS-Ogna. This review focuses on clinical and pathological manifestations of these plectinopathies. It addresses especially plectin’s role in skeletal muscle, where a loss of muscle fiber integrity and profound changes of myofiber cytoarchitecture are observed in its absence. Furthermore, the highly complex genetic and molecular structure of plectin is discussed; a high number of differentially spliced exons give rise to a variety of different isoforms, which fulfill distinct functions in different cell types and tissues. Plectin’s abilities to act as a dynamic organizer of intermediate filament networks and to interact with a multitude of different interaction partners are the basis for its function as a scaffolding platform for proteins involved in signaling. Finally, the article addresses a series of genetically manipulated mouse lines that were generated to serve as powerful models to study functional and molecular consequences of plectin gene defects.
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plectin gene defects lead to various forms of epidermolysis bullosa simplex
Dermatologic Clinics, 2010Co-Authors: Gunther A Rezniczek, Gernot Walko, Gerhard WicheAbstract:Plectin is an important organizer of the keratin filament cytoskeleton in basal keratinocytes. It is essential for anchoring these filaments to the extracellular matrix via hemidesmosomal integrins. Loss of plectin or incorrect function of the protein due to mutations in its gene can lead to various forms of the Skin Blistering disease, epidermolysis bullosa simplex. Severity and subtype of the disease is dependent on the specific mutation and can be associated with (late-onset) muscular dystrophy or pyloric atresia. Mouse models mimicking the human phenotypes allow detailed study of plectin function.
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targeted ablation of plectin isoform 1 uncovers role of cytolinker proteins in leukocyte recruitment
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Christina Abrahamsberg, Peter Fuchs, Irmgard Fischer, Selma Osmanagicmyers, Friedrich Propst, Adelheid Elbeburger, Gerhard WicheAbstract:Plectin, a typical cytolinker protein, is essential for Skin and skeletal muscle integrity. It stabilizes cells mechanically, regulates cytoskeleton dynamics, and serves as a scaffolding platform for signaling molecules. A variety of isoforms expressed in different tissues and cell types account for this versatility. To uncover the role of plectin 1, the major isoform expressed in tissues of mesenchymal origin, against the background of all other variants, we raised plectin isoform 1-specific antibodies and generated isoform-deficient mice. In contrast to plectin-null mice (lacking all plectin isoforms), which die shortly after birth because of severe Skin Blistering, plectin isoform 1-deficient mice were viable at birth, had a normal lifespan, and did not display the Skin Blistering phenotype. However, dermal fibroblasts isolated from plectin 1-deficient mice exhibited abnormalities in their actin cytoskeleton and impaired migration potential. Similarly, plectin 1-deficient T cells isolated from nymph nodes showed diminished chemotactic migration in vitro. Most strikingly, in vivo we found that leukocyte infiltration during wound healing was reduced in the mutant mice. These data show a specific role of a cytolinker protein in immune cell motility. Single isoform-deficient mice thus represent a powerful tool to unravel highly specific functions of plectin variants.
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plectin isoform specific rescue of hemidesmosomal defects in plectin keratinocytes
Journal of Investigative Dermatology, 2003Co-Authors: Kerstin Andra, Peter Fuchs, Iris Kornacker, Almut Jorgl, Michael Zorer, Irmgard Fischer, Daniel Spazierer, Gerhard WicheAbstract:The various plectin isoforms are among the major crosslinking elements of the cytoskeleton. The importance of plectin in epithelia is convincingly supported by the severe Skin Blistering observed in plectin-deficient humans and mice. Here, we identified plectin 1a (> 500 kDa), a full length plectin variant containing the sequence encoded by the alternative first exon 1a, as the isoform most prominently expressed in human and mouse keratinocytes. In Skin sections and cultured keratinocytes, plectin 1a was shown to colocalize with hemidesmosomal structures. In contrast, a second isoform expressed in epithelia, plectin 1c, differing from 1a merely by a short N-terminal sequence, colocalized with microtubules. Expression of plectin 1a, but not of its N-terminal fragment alone, or of a third alternative full length isoform (plectin 1), restored the reduced number of hemidesmosome-like stable anchoring contacts in cultured plectin-null keratinocytes. Our results show for the first time that different isoforms of a cytolinker protein expressed in one cell type perform distinct functions. Moreover, the identification of plectin 1a as the isoform defects in which cause Skin Blistering in plectin-related genetic diseases, such as epidermolysis bullosa simplex MD and epidermolysis bullosa simplex Ogna, could have implications for the future development of clinical therapies for patients.
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Plectin-Isoform-Specific Rescue of Hemidesmosomal Defects in Plectin (–/–) Keratinocytes
Journal of Investigative Dermatology, 2003Co-Authors: Kerstin Andra, Peter Fuchs, Iris Kornacker, Almut Jorgl, Michael Zorer, Irmgard Fischer, Daniel Spazierer, Gerhard WicheAbstract:The various plectin isoforms are among the major crosslinking elements of the cytoskeleton. The importance of plectin in epithelia is convincingly supported by the severe Skin Blistering observed in plectin-deficient humans and mice. Here, we identified plectin 1a (> 500 kDa), a full length plectin variant containing the sequence encoded by the alternative first exon 1a, as the isoform most prominently expressed in human and mouse keratinocytes. In Skin sections and cultured keratinocytes, plectin 1a was shown to colocalize with hemidesmosomal structures. In contrast, a second isoform expressed in epithelia, plectin 1c, differing from 1a merely by a short N-terminal sequence, colocalized with microtubules. Expression of plectin 1a, but not of its N-terminal fragment alone, or of a third alternative full length isoform (plectin 1), restored the reduced number of hemidesmosome-like stable anchoring contacts in cultured plectin-null keratinocytes. Our results show for the first time that different isoforms of a cytolinker protein expressed in one cell type perform distinct functions. Moreover, the identification of plectin 1a as the isoform defects in which cause Skin Blistering in plectin-related genetic diseases, such as epidermolysis bullosa simplex MD and epidermolysis bullosa simplex Ogna, could have implications for the future development of clinical therapies for patients.
Allison J Cowin - One of the best experts on this subject based on the ideXlab platform.
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Skin barrier and autoimmunity mechanisms and novel therapeutic approaches for autoimmune Blistering diseases of the Skin
Frontiers in Immunology, 2019Co-Authors: Natalie E Stevens, Allison J Cowin, Zlatko KopeckiAbstract:One of the most important functions of the Skin besides regulating internal body temperature includes formation of the barrier between the organism and the external environment, hence protecting against pathogen invasion, chemical and physical assaults and unregulated loss of water and solutes. Disruption of the protective barrier is observed clinically in blisters and erosions of the Skin that form in autoimmune Blistering diseases where the body produces autoantibodies against structural proteins of the epidermis or the epidermal-dermal junction. Although there is no cure for autoimmune Skin Blistering diseases, immune suppressive therapies currently available offer opportunities for disease management. In cases where no treatment is sought, these disorders can lead to life threatening complications and current research efforts have focused on developing therapies that target autoantibodies which contribute to disease symptoms. This review will outline the involvement of the Skin barrier in main Skin-specific autoimmune Blistering diseases by describing the mechanisms underpinning Skin autoimmunity and review current progress in development of novel therapeutic approaches targeting the underlying causes of autoimmune Skin Blistering diseases.
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cytoskeletal regulation of inflammation and its impact on Skin Blistering disease epidermolysis bullosa acquisita
International Journal of Molecular Sciences, 2016Co-Authors: Zlatko Kopecki, Ralf Ludwig, Allison J CowinAbstract:Actin remodelling proteins regulate cytoskeletal cell responses and are important in both innate and adaptive immunity. These responses play a major role in providing a fine balance in a cascade of biological events that results in either protective acute inflammation or chronic inflammation that leads to a host of diseases including autoimmune inflammation mediated epidermolysis bullosa acquisita (EBA). This review describes the role of the actin cytoskeleton and in particular the actin remodelling protein called Flightless I (Flii) in regulating cellular inflammatory responses and its subsequent effect on the autoimmune Skin Blistering disease EBA. It also outlines the potential of an antibody based therapy for decreasing Flii expression in vivo to ameliorate the symptoms associated with EBA.
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flightless i over expression impairs Skin barrier development function and recovery following Skin Blistering
The Journal of Pathology, 2014Co-Authors: Zlatko Kopecki, Hiroaki Iwata, Detlef Zillikens, Ralf Ludwig, Gink N Yang, Ruth M Arkell, Jessica E Jackson, Elizabeth Melville, Dedee F Murrell, Allison J CowinAbstract:Development of an intact epidermis is critical for maintaining the integrity of the Skin. Patients with epidermolysis bullosa (EB) experience multiple erosions, which breach the epidermal barrier and lead to increased microbial colocalization of wounds, infections and sepsis. The cytoskeletal protein Flightless I (Flii) is a known regulator of both development and wound healing. Using Flii+/−, WT and FliiTg/Tg mice, we investigated the effect of altering Flii levels in embryos and adult mice on the development of the epidermal barrier and, consequently, how this affects the integrity of the Skin in EB. Flii over-expression resulted in delayed formation of the epidermal barrier in embryos and decreased expression of tight junction (TJ) proteins Claudin-1 and ZO-2. Increased intercellular space and transepidermal water loss was observed in FliiTg/Tg adult mouse Skin, while FliiTg/Tg keratinocytes showed altered TJ protein localization and reduced transepithelial resistance. Flii is increased in the blistered Skin of patients with EB, and over-expression of Flii in experimental EBA showed impaired Claudin-1 and -4 TJ protein expression and delayed recovery of functional barrier post-Blistering. Immunoprecipitation confirmed Flii associated with TJ proteins and in vivo actin assays showed that the effect of Flii on actin polymerization underpinned the impaired barrier function observed in FliiTg/Tg mice. These results therefore demonstrate an important role for Flii in the development and regulation of the epidermal barrier, which may contribute to the impaired healing and Skin fragility of EB patients. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd
Leena Brucknertuderman - One of the best experts on this subject based on the ideXlab platform.
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the flavonoid luteolin inhibits fcγ dependent respiratory burst in granulocytes but not Skin Blistering in a new model of pemphigoid in adult mice
PLOS ONE, 2012Co-Authors: Eva Oswald, Alina Sesarman, Clauswerner Franzke, Ute Wolfle, Leena Brucknertuderman, Thilo Jakob, Stefan F Martin, Cassian SitaruAbstract:Bullous pemphigoid is an autoimmune Blistering Skin disease associated with autoantibodies against the dermal-epidermal junction. Passive transfer of antibodies against BP180/collagen (C) XVII, a major hemidesmosomal pemphigoid antigen, into neonatal mice results in dermal-epidermal separation upon applying gentle pressure to their Skin, but not in spontaneous Skin Blistering. In addition, this neonatal mouse model precludes treatment and observation of diseased animals beyond 2–3 days. Therefore, in the present study we have developed a new disease model in mice reproducing the spontaneous Blistering and the chronic course characteristic of the human condition. Adult mice were pre-immunized with rabbit IgG followed by injection of BP180/CXVII rabbit IgG. Mice pre-immunized against rabbit IgG and injected 6 times every second day with the BP180/CXVII-specific antibodies (n = 35) developed spontaneous sustained Blistering of the Skin, while mice pre-immunized and then treated with normal rabbit IgG (n = 5) did not. Blistering was associated with IgG and complement C3 deposits at the epidermal basement membrane and recruitment of inflammatory cells, and was partly dependent on Ly-6G-positive cells. We further used this new experimental model to investigate the therapeutic potential of luteolin, a plant flavonoid with potent anti-inflammatory and anti-oxidative properties and good safety profile, in experimental BP. Luteolin inhibited the Fcγ-dependent respiratory burst in immune complex-stimulated granulocytes and the autoantibody-induced dermal-epidermal separation in Skin cryosections, but was not effective in suppressing the Skin Blistering in vivo. These studies establish a robust animal model that will be a useful tool for dissecting the mechanisms of blister formation and will facilitate the development of more effective therapeutic strategies for managing pemphigoid diseases.
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molecular basis of inherited Skin Blistering disorders and therapeutic implications
Expert Reviews in Molecular Medicine, 2006Co-Authors: Monique Aumailley, Lucy Tunggal, Leena BrucknertudermanAbstract:: Epidermolysis bullosa (EB) and associated Skin-fragility syndromes are a group of inherited Skin diseases characterised by trauma-induced Blistering of the Skin and mucous membranes. Mutations in at least 14 distinct genes encoding molecular components of the epidermis or the dermal-epidermal junction (DEJ) can cause Blistering Skin diseases that differ by clinical presentation and severity of the symptoms. Despite great advances in discerning the genetic basis of this group of diseases, the molecular pathways leading to symptoms are not yet fully understood. Unravelling these pathways by molecular analysis of the structure and in vitro assessment of functional properties of the human proteins involved, combined with genetic models in lower organisms, should pave the way for specific cures for inherited Skin fragility.
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some but not all glycine substitution mutations in col7a1 result in intracellular accumulation of collagen vii loss of anchoring fibrils and Skin Blistering
Journal of Biological Chemistry, 1998Co-Authors: Nadja Hammamihauasli, Hauke Schumann, Michael Raghunath, Oliver Kilgus, Ursula Luthi, Thomas A Luger, Leena BrucknertudermanAbstract:COL7A1 gene mutations cause dystrophic epidermolysis bullosa, a Skin Blistering disorder. The phenotypes result from defects of collagen VII, the major component of the anchoring fibrils at the dermo-epidermal junction; however, the molecular mechanisms underlying the phenotypes remain elusive. We investigated naturally occurring COL7A1 mutations and showed that some, but not all, glycine substitutions in collagen VII interfered with biosynthesis of the protein in a dominant-negative manner. Three point mutations in exon 73 caused glycine substitutions G2006D, G2034R, and G2015E in the triple helical domain of collagen VII and interfered with its folding and secretion. Confocal laser scanning studies and semiquantitative immunoblotting determined that dystrophic epidermolysis bullosa keratinocytes retained up to 2.5-fold more procollagen VII within the rough endoplasmic reticulum than controls. Limited proteolytic digestions of mutant procollagen VII produced aberrant fragments and revealed reduced stability of the triple helix. In contrast, the glycine substitution G1519D in another segment of the triple helix affected neither procollagen VII secretion nor anchoring fibril function and remained phenotypically silent. These data demonstrate that collagen VII presents a remarkable exception among collagens in that not all glycine substitutions within the triple helix exert dominant-negative interference and that the biological consequences of the substitutions probably depend on their position within the triple helix.
