Large-Scale Electron Microscopy Maps of Patient Skin and Mucosa Provide Insight into Pathogenesis of Blistering Diseases

Sokol, Ena; Kramer, Duco; Diercks, Gilles F.H.; Kuipers, Jeroen; Jonkman, Marcel F.; Pas, Hendrikus; Giepmans, Ben N. G.

doi:10.1038/jid.2015.109

Cited by 77 publications

(104 citation statements)

References 29 publications

(32 reference statements)

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“…Serum IgG antibodies from patients with pemphigus vulgaris, as well as recombinant monovalent human anti-desmoglein 3 antibodies, interfere with desmosome assembly by causing internalization and degradation of desmoglein 3 proteins that are not yet integrated in desmosomes, leading to desmoglein 3-depleted desmosomes 62–64 . Interference with desmosome assembly by pathogenic pemphigus autoantibodies is supported by both immunofluorescence and electron microscopy findings in skin from patients with pemphigus foliaceus or pemphigus vulgaris, which show interdesmosomal intercellular widening in areas of desmoglein clustering 65,66 .…”

Section: Mechanisms/pathophysiologymentioning

confidence: 88%

Pemphigus

Kasperkiewicz

Ellebrecht

Takahashi

et al. 2017

Nat Rev Dis Primers

421

429

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Pemphigus is a group of IgG-mediated autoimmune diseases of stratified squamous epithelia, such as the skin and oral mucosa, in which acantholysis (the loss of cell adhesion) causes blisters and erosions. Pemphigus has three major subtypes: pemphigus vulgaris, pemphigus foliaceus and paraneoplastic pemphigus. IgG autoantibodies are characteristically raised against desmoglein 1 and desmoglein 3, which are cell–cell adhesion molecules found in desmosomes. The sites of blister formation can be physiologically explained by the anti-desmoglein autoantibody profile and tissue-specific expression pattern of desmoglein isoforms. The pathophysiological roles of T cells and B cells have been characterized in mouse models of pemphigus and patients, revealing insights into the mechanisms of autoimmunity. Diagnosis is based on clinical manifestations and confirmed with histological and immunochemical testing. The current first-line treatment is systemic corticosteroids and adjuvant therapies, including immunosuppressive agents, intravenous immunoglobulin and plasmapheresis. Rituximab, a monoclonal antibody against CD20+ B cells, is a promising therapeutic option that may soon become first-line therapy. Pemphigus is one of the best-characterized human autoimmune diseases and provides an ideal paradigm for both basic and clinical research, especially towards the development of antigen-specific immune suppression treatments for autoimmune diseases.

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Section: Mechanisms/pathophysiologymentioning

confidence: 88%

Pemphigus

Kasperkiewicz

Ellebrecht

Takahashi

et al. 2017

Nat Rev Dis Primers

421

429

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“…With the increased possibilities and tools for correlative and large-scale EM we anticipate a revival of EM in basic and medical research. Our method represented here is applied to a zebrafish brain injury model 5 , but has been used on human tissue 7 , rat brain 3 , in a rat model for diabetes 4 and in cell culture 2 , and can also be used in combination with a TEM-based approach 1 showing the versatility of this method.…”

Section: Discussionmentioning

confidence: 99%

“…Although for sample preparation relatively standard EM processing (fixation, embedding and sectioning) is needed [5][6][7] , it is technically challenging to cut large ultrathin sections completely devoid of artifacts. Sections are very fragile, easily break, fold or are destroyed during imaging, which typically takes multiple hours per dataset.…”

Section: Discussionmentioning

confidence: 99%

“…By large-scale EM areas up to square millimeters can be visualized. We and others previously applied nanotomy to rat pancreatic islets 4 , cell culture 2 , rat brain 3 , skin and mucosa 7 and whole zebrafish larvae 1,5 (www.nanotomy.org). Zebrafish are highly suitable for in vivo imaging, in particularly to visualize cell types which are difficult to access in mammalian tissues including brain immune cells 11 .…”

Section: Introductionmentioning

confidence: 99%

“…Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae [1][2][3][4][5][6][7] . Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features.…”

mentioning

confidence: 99%

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Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Kuipers¹,

Kalicharan²,

Wolters³

et al. 2016

JoVE

Self Cite

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Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae [1][2][3][4][5][6][7] . Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on onehole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 -50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture [1][2][3][4][5] . Here, zebrafish brains were analyzed in a noninvasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM) 8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

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Using Advanced Correlative Microscopy to Study Complex Biological Samples

Meo

Liv

Hoogenboom

2016

Encyclopedia of Analytical Chemistry

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Correlation between light, mostly fluorescence, and electron microscopy (EM) is needed to identify biological molecules within their ultrastructural context and/or to relate the ultrastructure to preceding dynamics of biological molecules. Recent development of labels, sample preparation techniques, and microscopy tools allow researchers to bridge the gap between these two modalities, while dedicated, integrated microscopes merge the two techniques into one. This not only allows broader possibilities for implementation of CLEM (correlative light and electron microscopy) in analytical sciences but also enables novel applications crossing boundaries between the traditional microscopes. We provide an overview of the different CLEM approaches, including common labels and sample preparation techniques, and focus attention specifically on the advanced instrumentation and the novel opportunities and challenges these bring for the chemical and biological sciences.

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Large-Scale Electron Microscopy Maps of Patient Skin and Mucosa Provide Insight into Pathogenesis of Blistering Diseases

Cited by 77 publications

References 29 publications

Pemphigus

Pemphigus

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Using Advanced Correlative Microscopy to Study Complex Biological Samples

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