Kristina Seiffert-Sinha

Investigation of human keratinocyte cell adhesion using atomic force microscopy

UNLABELLED Desmosomal junctions are specialized structures critical to cellular adhesion within epithelial tissues. Disassembly of these junctions is seen consequent to the development of autoantibodies directed at specific desmosomal proteins in blistering skin diseases such as pemphigus. However, many details regarding cell junction activity under normal physiological and disease conditions remain to be elucidated. Because of their complex structure, desmosomal junctions are not well suited to existing techniques for high-resolution three-dimensional structure-function analyses. Here, atomic force microscopy (AFM) is used for detailed characterization and visualization of the cell junctions of human epithelial cells. We demonstrate the ability to image the detailed three-dimensional structure of the cell junction at high magnification. In addition, the effect of specific antibody binding to desmosomal components of the cell junction is studied in longitudinal analyses before and after antibody treatment. We show that antibodies directed against desmoglein 3 (a major component of the desmosomal structural unit, and the major target of autoantibodies in patients with pemphigus vulgaris) are associated with changes at the cell surface of the human keratinocytes and alterations within keratinocyte intercellular adhesion structures, supporting the assertion that cell structures and junctions are modified by antibody binding. The present study indicates that the molecular structure of gap junctions can be more completely analyzed and characterized by AFM, offering a new technological approach to facilitate a better understanding of disease mechanisms and potentially monitor therapeutic strategies in blistering skin diseases. FROM THE CLINICAL EDITOR Disassembly of desmosomal junctions is seen in blistering skin diseases such as Pemphigus. This present study demonstrates that the molecular structure of gap junctions can be more completely analyzed and characterized by atomic force microscopy.

Quantitative Analysis of Human Keratinocyte Cell Elasticity Using Atomic Force Microscopy (AFM)

We present the use of atomic force microscopy (AFM) to visualize and quantify the dynamics of epithelial cell junction interactions under physiological and pathophysiological conditions at the nanoscale. Desmosomal junctions are critical cellular adhesion components within epithelial tissues and blistering skin diseases such as Pemphigus are the result in the disruption of these components. However, these structures are complex and mechanically inhomogeneous, making them difficult to study. The mechanisms of autoantibody mediated keratinocyte disassembly remain largely unknown. Here, we have used AFM technology to image and measure the mechanical properties of living skin epithelial cells in culture. We demonstrate that force measurement data can distinguish cells cultured with and without autoantibody treatment. Our demonstration of the use of AFM for in situ imaging and elasticity measurements at the local, or tissue level opens potential new avenues for the investigation of disease mechanisms and monitoring of therapeutic strategies in blistering skin diseases.

Bionanomanipulation Using Atomic Force Microscopy

This paper explains how an AFM-based nanorobot was developed to visualize and quantify the dynamics of cell proteins interactions under physiological and pathophysiological conditions at the nanoscale. As these events are directly related at the molecular level to the causes of many life-threatening or incurable diseases, the development of an AFM-based nanorobot, which can image and manipulate biological objects at the single molecule level, is a novel approach to reveal disease markers and elucidate the disease mechanisms.

A Globally Available Internet-Based Patient Survey of Pemphigus Vulgaris: Epidemiology and Disease Characteristics

The authors developed an anonymous, Web-based survey instrument available globally, and collected data from 171 pemphigus vulgaris (PV) patients to assemble epidemiologic data pertaining to an extensive set of clinical parameters in demographically diverse populations. The results showed female predominance, prevalent onset of disease in the fifth decade of life, and a strong correlation of PV with thyroid disease and type 1 diabetes in patients and family members. Most patients have a history of either mucosal-only or mucocutaneous lesions, but numerous patients self-report cutaneous lesions only, without previous or concurrent mucosal lesions, especially in the non-North American PV population.

Multiplexed autoantigen microarrays identify HLA as a key driver of anti-desmoglein and -non-desmoglein reactivities in pemphigus.

Significance Although the pathogenicity of autoantibodies (autoAbs) targeting the desmosomal cadherins desmoglein (Dsg) 3 and 1 is well established in pemphigus vulgaris (PV), the disease relevance of other autoAbs remains an area of intense research. By using disease-specific protein microarrays to characterize the autoAb response in PV, we identified Dsg and non-Dsg antigens as primary targets of patient autoAbs. Additionally, we found that healthy first- or second-degree relatives of patients with PV, specifically those expressing HLA DRB1*0402 or DQB1*0503 (PV susceptibility alleles) exhibit patterns of autoreactivity similar to that of patients with PV. These findings suggest a potential role for non-Dsg autoAbs in disease pathogenesis and implicate HLA as a primary driver of autoAb specificity in PV. Patients with pemphigus vulgaris (PV) harbor antibodies reactive against self-antigens expressed at the surface of keratinocytes, primarily desmoglein (Dsg) 3 and, to a lesser extent, Dsg1. Conventionally, only antibodies targeting these molecules have been thought to contribute to disease pathogenesis. This notion has been challenged by a growing pool of evidence that suggests that antibodies toward additional targets may play a role in disease. The aims of this study were to (i) establish high-throughput protein microarray technology as a method to investigate traditional and putative autoantibodies (autoAbs) in PV and (ii) use multiplexed protein array technology to define the scope and specificity of the autoAb response in PV. Our analysis demonstrated significant IgG reactivity in patients with PV toward the muscarinic acetylcholine receptor subtypes 3, 4, and 5 as well as thyroid peroxidase. Furthermore, we found that healthy first- and second-degree relatives of patients with PV express autoAbs toward desmoglein and non-Dsg targets. Our analysis also identified genetic elements, particularly HLA, as key drivers of autoAb expression. Finally, we show that patients with PV exhibit significantly reduced IgM reactivity toward disease-associated antigens relative to controls. The use of protein microarrays to profile the autoAb response in PV advanced the current understanding of disease and provided insight into the complex relationship between genetics and disease development.

Identification of a new disease cluster of pemphigus vulgaris with autoimmune thyroid disease, rheumatoid arthritis and type I diabetes

Pemphigus vulgaris (PV) is a potentially fatal autoimmune blistering skin disease. It is known that individuals with autoimmune diseases such as PV, as well as their family members, are at increased risk of developing other autoimmune diseases. However, it is unknown whether there are specific autoimmune diseases that cluster with PV.

Cellular level robotic surgery: Nanodissection of intermediate filaments in live keratinocytes.

We present the nanosurgery on the cytoskeleton of live cells using AFM based nanorobotics to achieve adhesiolysis and mimic the effect of pathophysiological modulation of intercellular adhesion. Nanosurgery successfully severs the intermediate filament bundles and disrupts cell-cell adhesion similar to the desmosomal protein disassembly in autoimmune disease, or the cationic modulation of desmosome formation. Our nanomechanical analysis revealed that adhesion loss results in a decrease in cellular stiffness in both cases of biochemical modulation of the desmosome junctions and mechanical disruption of intercellular adhesion, supporting the notion that intercellular adhesion through intermediate filaments anchors the cell structure as focal adhesion does and that intermediate filaments are integral components in cell mechanical integrity. The surgical process could potentially help reveal the mechanism of autoimmune pathology-induced cell-cell adhesion loss as well as its related pathways that lead to cell apoptosis.

Genome-wide expression analysis suggests unique disease-promoting and disease-preventing signatures in Pemphigus vulgaris.

To evaluate pathogenetic mechanisms underlying disease development and progression in the autoimmune skin disease Pemphigus vulgaris (PV), we examined global peripheral blood gene expression in patients and healthy controls. Our goals were to: (1) assign blood gene expression signatures to patients and controls; (2) identify differentially expressed genes (DEGs) and investigate functional pathways associated with these signatures; and (3) evaluate the distribution of DEGs across the genome to identify transcriptional ‘hot spots’. Unbiased hierarchical clustering clearly separated patients from human leukocyte antigen (HLA)-matched controls (MCRs; ‘disease’ signature), and active from remittent patients (‘activity’ signature). DEGs associated with these signatures are involved in immune response, cytoskeletal reorganization, mitogen-activated protein kinase (MAPK) signaling, oxidation-reduction and apoptosis. We further found that MCRs carrying the PV-associated HLA risk alleles cluster distinctly from unmatched controls (UMCR) revealing an HLA-associated ‘control’ signature. A subset of DEGs within the ‘control’ signature overlap with the ‘disease’ signature, but are inversely regulated in MCR when compared with either PV patients or UMCR, suggesting the existence of a ‘protection’ signature in healthy individuals carrying the PV HLA genetic risk elements. Finally, we identified 19 transcriptional ‘hot spots’ across the signatures, which may guide future studies aimed at pinpointing disease risk genes.

Peripheral blood gene expression in alopecia areata reveals molecular pathways distinguishing heritability, disease and severity

Alopecia areata (AA) is an autoimmune hair loss disorder in which systemic disturbances have been described, but are poorly understood. To evaluate disease mechanisms, we examined gene expression in the blood of defined clinical subgroups (patchy AA persistent type, AAP, n=5; alopecia universalis, AU, n=4) and healthy controls (unaffected relatives, UaR, n=5; unaffected non-relatives, UaNR, n=4) using microarrays. Unsupervised hierarchical clustering separates all four patient and control groups, producing three distinct expression patterns reflective of ‘inheritance’, ‘disease’ and ‘severity’ signatures. Functional classification of differentially expressed genes (DEGs) comparing disease (AAP, AU) vs normal (UaR) groups reveals upregulation in immune response, cytokine signaling, signal transduction, cell cycle, proteolysis and cell adhesion-related genes. Pathway analysis further reveals the activation of several genes related to natural killer-cell cytotoxicity, apoptosis, mitogen activated protein kinase, Wnt signaling and B- and T-cell receptor signaling in AA patients. Finally, 35 genes differentially expressed in AA blood overlap with DEGs previously identified in AA skin lesions. Our results implicate innate and adaptive immune processes while also revealing novel pathways, such as Wnt signaling and apoptosis, relevant to AA pathogenesis. Our data suggest that peripheral blood expression profiles of AA patients likely carry new biomarkers associated with disease susceptibility and expression.

Nanorobotic Investigation Identifies Novel Visual, Structural and Functional Correlates of Autoimmune Pathology in a Blistering Skin Disease Model

There remain major gaps in our knowledge regarding the detailed mechanisms by which autoantibodies mediate damage at the tissue level. We have undertaken novel strategies at the interface of engineering and clinical medicine to integrate nanoscale visual and structural data using nanorobotic atomic force microscopy with cell functional analyses to reveal previously unattainable details of autoimmune processes in real-time. Pemphigus vulgaris is a life-threatening autoimmune blistering skin condition in which there is disruption of desmosomal cell-cell adhesion structures that are associated with the presence of antibodies directed against specific epithelial proteins including Desmoglein (Dsg) 3. We demonstrate that pathogenic (blister-forming) anti-Dsg3 antibodies, distinct from non-pathogenic (non-blister forming) anti-Dsg3 antibodies, alter the structural and functional properties of keratinocytes in two sequential steps - an initial loss of cell adhesion and a later induction of apoptosis-related signaling pathways, but not full apoptotic cell death. We propose a “2-Hit” model for autoimmune disruption associated with skin-specific pathogenic autoantibodies. These data provide unprecedented details of autoimmune processes at the tissue level and offer a novel conceptual framework for understanding the action of self-reactive antibodies.