Keratinocyte Cell Adhesion Research Articles

Integration of transcriptomics and spatial biology analyses reveals Galactomyces ferment filtrate promotes epidermal interconnectivity via induction of keratinocyte differentiation, proliferation and cellular bioenergetics.

Human skin is the first line of defence from environmental factors such as solar radiation and is susceptible to premature ageing, including a disruption in epidermal differentiation and homeostasis. We evaluated the impact of a Galactomyces Ferment Filtrate (GFF) on epidermal differentiation and response to oxidative stress. We used transcriptomics, both spatial and traditional, to assess the impact of GFF on epidermal biology and homeostasis in keratinocytes (primary or immortalized) and in exvivo skin explant tissue. The effect of GFF on cell adhesion rates, cellular ATP levels and proliferation rates were quantitated. Oxidative phosphorylation and glycolytic rates were measured under normal and stress-induced conditions. Transcriptomics from keratinocytes and exvivo skin explants from multiple donors show GFF induces keratinocyte differentiation, skin barrier development and cell adhesion while simultaneously repressing cellular stress and inflammatory related processes. Spatial transcriptomics profiling of exvivo skin indicated basal keratinocytes at the epidermal-dermal junction and cornifying keratinocytes in the top layer of the epidermis as the primary cell types influenced by GFF treatment. Additionally, GFF significantly increases crosstalk between suprabasal and basal keratinocytes. To support these findings, we show that GFF can significantly increase cell adhesion and proliferation in keratinocytes. GFF also protected overall cellular bioenergetics under metabolic or oxidative stress conditions. Our findings provide novel insights into cellular differences and epidermal spatial localization in response to GFF, supporting previous findings that this filtrate has a significant impact on epidermal biology and homeostasis, particularly on spatially defined crosstalk. We propose that GFF can help maintain epidermal health by enhancing keratinocyte crosstalk and differentiation/proliferation balance as well as promoting an enhanced response to stress.

The Impact of Ultrashort Pulse Laser Structuring of Metals on In-Vitro Cell Adhesion of Keratinocytes.

Besides the need for biomaterial surface modification to improve cellular attachment, laser-structuring is favorable for designing a new surface topography for external bone fixator pins or implants. The principle of this study was to observe how bioinspired (deer antler) laser-induced nano-microstructures influenced the adhesion and growth of skin cells. The goal was to create pins that allow the skin to attach to the biomaterial surface in a bacteria-proof manner. Therefore, typical fixator metals, steel, and titanium alloy were structured using ultrashort laser pulses, which resulted in periodical nano- and microstructures. Surface characteristics were investigated using a laser scanning microscope and static water contact angle measurements. In vitro studies with human HaCaT keratinocytes focused on cell adhesion, morphology, actin formation, and growth within 7 days. The study showed that surface functionalization influenced cell attachment, spreading, and proliferation. Micro-dimple clusters on polished bulk metals (DC20) will not hinder viability. Still, they will not promote the initial adhesion and spreading of HaCaTs. In contrast, additional nanostructuring with laser-induced periodic surface structures (LIPSS) promotes cell behavior. DC20 + LIPSS induced enhanced cell attachment with well-spread cell morphology. Thus, the bioinspired structures exhibited a benefit in initial cell adhesion. Laser surface functionalization opens up new possibilities for structuring, and is relevant to developing bioactive implants in regenerative medicine.

The Role of EAAT4 in Epidermal Differentiation and Calcium Homeostasis During Aging

The Role of EAAT4 in Epidermal Differentiation and Calcium Homeostasis During Aging

Effect of Poly(Oxanorbonene)- and Poly(Methacrylate)-Based Polyzwitterionic Surface Coatings on Cell Adhesion and Gene Expression of Human Keratinocytes.

Polyzwitterions are generally known for their anti-adhesive properties, including resistance to protein and cell adhesion, and overall high bio-inertness. Yet there are a few polyzwitterions to which mammalian cells do adhere. To understand the structural features of this behavior, a panel of polyzwitterions with different functional groups and overall degrees of hydrophobicity is analyzed here, and their physical and biological properties are correlated to these structural differences. Cell adhesion is focused on, which is the basic requirement for cell viability, proliferation, and growth. With the here presented polyzwitterion panel, three different types of cell-surface interactions are observed: adhesion, slight attachment, and cell repellency. Using immunofluorescence methods, it is found that human keratinocytes (HaCaT) form focal adhesions on the cell-adhesive polyzwitterions, but not on the sample that has only slight cell attachment. Gene expression analysis indicates that HaCaT cells cultivated in the presence of a non-adhesive polyzwitterion have up-regulated inflammatory and apoptosis-related cell signaling pathways, while the gene expression of HaCaT cells grown on a cell-adhesive polyzwitterion does not differ from the gene expression of the growth control, and thus can be defined as fully cell-compatible.

Therapeutic effect of chinese herbal medicine gu-ben-hua-shi (AESS) formula on atopic dermatitis through regulation of yes-associated protein

Background: Atopic dermatitis (AD) is a chronic and recurrent skin disease. At present, there is a lack of sufficiently effective and safe medicines that can be used for a prolonged time and reduce the recurrence of AD. The Gu-Ben-Hua-Shi (AESS) formula has been used for many years with a good clinical effect on AD but its specific treatment mechanism is unknown. Methods: The main components of AESS were analyzed using ultra-high performance liquid chromatography (UPLC). The composition of AESS compounds in the serum from rats was analyzed using ultra-high performance liquid chromatography-mass spectrometry. An AD mouse model was constructed using 2,4-dinitrofluorobenzene stimulation in Balb/C mice and the effect on the reduction of skin lesions and Th1/Th2/Th17/Treg balance after AESS administration were measured. The effects of AESS serum on the proliferation and apoptosis of keratinocyte cell line HaCaT and adhesion of HaCaT to human monocyte cell line THP-1 were detected in an IFN-γ/TNF-α stimulated AD-like inflammatory cell model. The effects of Yes-associated protein (YAP) expression on the therapeutic effect and a related signaling pathway were also investigated. Results: In total, 10 components were confirmed using UPLC, namely five organic acids, three flavonoids, and two chromogenic ketones. Additionally, the similarity of the three batches of samples (S1–3) was above 0.98, indicating that the formula samples have good uniformity. These 10 compounds were also detected in rat serum, suggesting that they are absorbed into rat blood as prototype components. Furthermore, AESS effectively reduced the skin lesions in the AD mouse model, regulated the Th1/Th2/Th17/Treg imbalance, improved the proliferation ability of the AD-like cell model, and inhibited HaCaT apoptosis and adhesion to THP-1 cells. It also reduced the expression of YAP in Th17 and Treg cells of the mouse spleen and increased YAP expression in the skin. The change in YAP expression in keratinocytes weakened the curative effect of AESS, and AESS exerted its effects through the NF-κB signaling pathway. Conclusion: AESS may play a role in the treatment of AD by affecting the expression of YAP. These findings can be used to promote its use as an alternative medication for prolonged use with fewer side effects.

Pitfalls in the Application of Dispase-Based Keratinocyte Dissociation Assay for In Vitro Analysis of Pemphigus Vulgaris.

Pemphigus vulgaris (PV) is a chronic, life-altering autoimmune disease due to the production of anti-desmoglein antibodies causing the loss of cell–cell adhesion in keratinocytes (acantholysis) and blister formation in both skin and mucous membranes. The dispase-based keratinocyte dissociation assay (DDA) is the method of choice to examine the pathogenic effect of antibodies and additional co-stimuli on cell adhesion in vitro. Despite its widespread use, there is a high variability of experimental conditions, leading to inconsistent results. In this paper, we identify and discuss pitfalls in the application of DDA, including generation of a monolayer with optimized density, appropriate culturing conditions to obtain said monolayer, application of mechanical stress in a standardized manner, and performing consistent data processing. Importantly, we describe a detailed protocol for a successful and reliable DDA and the respective ideal conditions for three different types of human keratinocytes: (1) primary keratinocytes, (2) the HaCaT spontaneously immortalized keratinocyte cell line, and (3) the recently characterized HaSKpw spontaneously immortalized keratinocyte cell line. Our study provides detailed protocols which guarantee intra- and inter-experimental comparability of DDA.

Histatin‐1 is a novel osteogenic factor that promotes bone cell adhesion, migration, and differentiation

Histatin-1 is a salivary antimicrobial peptide involved in the maintenance of enamel and oral mucosal homeostasis. Moreover, Histatin-1 has been shown to promote re-epithelialization in soft tissues, by stimulating cell adhesion and migration in oral and dermal keratinocytes, gingival and skin fibroblasts, endothelial cells and corneal epithelial cells. The broad-spectrum activity of Histatin-1 suggests that it behaves as a universal wound healing promoter, although this is far from being clear yet. Here, we report that Histatin-1 is a novel osteogenic factor that promotes bone cell adhesion, migration and differentiation. Specifically, Histatin-1 promoted cell adhesion, spreading and migration of SAOS-2 cells and MC3T3-E1 pre-osteoblasts in vitro, when placed on a fibronectin matrix. Besides, Histatin-1 induced the expression of osteogenic genes, including osteocalcin, osteopontin and Runx2, and increased both activity and protein levels of alkaline phosphatase. Furthermore, Histatin-1 promoted mineralization in vitro, as it augmented the formation of calcium deposits in both SAOS-2 and MC3T3-E1 cells. Mechanistically, although Histatin-1 failed to activate ERK1/2, FAK and Akt, which are signaling proteins associated with osteogenic differentiation or cell migration, it triggered nuclear re-localization of β-catenin. Strikingly, the effects of Histatin-1 were recapitulated in cells that are non-osteogenically committed, since it promoted surface adhesion, migration and the acquisition of osteogenic markers in primary mesenchymal cells derived from the apical papilla and dental pulp. Collectively, these observations indicate that Histatin-1 is a novel osteogenic factor that promotes bone cell differentiation, surface adhesion and migration, as crucial events required for bone tissue regeneration. This article is protected by copyright. All rights reserved.

Cell proliferation, adhesion, and differentiation of keratinocytes in the developing beak and egg-tooth of the turtle Emydura macquarii.

The development of the beak in turtles is poorly known. Beak development has been analyzed by immunofluorescent methods for studying cell proliferation and localization of specific proteins. The flat two-layered epidermis covering the turtle embryo at mid stage of development becomes columnar in the oral region and is associated with an increase of mesenchymal density as in placodes. Using 5BrdU, an intense cell proliferation is observed in the oral and epidermal cells covering the maxilla and mandibular bones, probably stimulated by the underlying mesenchyme in continuation with maxillary and mandibular bones. Expansion and fusion of these placodes give rise to the corneous beak. Beta catenin, mainly junctional but also sparsely detected in nuclei of the germinal layer of the beak epithelium, maintains united the differentiating keratinocytes that form a stratified corneous epithelium. This is initially composed of some layers of large cells that accumulate intermediate filament keratins (IFKs) and give rise to a keratinized embryonic epidermis destined to slough around hatching. The following corneocytes accumulate IFKs but mainly type I/II corneous beta proteins (CBPs) and form a corneous beak. These CBPs appear present with lower intensity in the beak than in the shell, but the higher intensity obtained with a general antibody against CBPs indicates that other CBPs contribute to the composition and stiffness of beak corneous material. The egg-tooth in continuation with the stratum corneum of the maxillary beak develops from a localized proliferation and comprises a thick embryonic epidermis accumulating IFKs under which large beta-cells connected by adhesion proteins accumulate CBPs contributing to hardening of this temporary organ.

Physicochemical and Biological Properties of Gelatin Extracted from Marine Snail Rapana venosa.

In this study, we aimed to obtain gelatin from the marine snail Rapana venosa using acidic and enzymatic extraction methods and to characterize these natural products for cosmetic and pharmaceutical applications. Marine gelatins presented protein values and hydroxyproline content similar to those of commercial mammalian gelatin, but with higher melting temperatures. Their electrophoretic profile and Fourier transform infrared (FTIR) spectra revealed protein and absorption bands situated in the amide region, specific for gelatin molecule. Scanning electron microscopy (SEM) analysis showed significant differences in the structure of the lyophilized samples, depending on the type of gelatin. In vitro studies performed on human keratinocytes showed no cytotoxic effect of acid-extracted gelatin at all tested concentrations and moderate cytotoxicity of enzymatic extracted gelatin at concentrations higher than 0.5 mg/mL. Also, both marine gelatins favored keratinocyte cell adhesion. No irritant potential was recorded as the level of IL-1α and IL-6 proinflammatory cytokines released by HaCaT cells cultivated in the presence of marine gelatins was significantly reduced. Together, these data suggest that marine snails are an alternative source of gelatins with potential use in pharmaceutical and skincare products.

Autoimmunity and immunological tolerance in autoimmune bullous diseases.

Autoimmune diseases are devastating conditions in which the immune system is directed against the host, leading to life-threatening destruction of organs. Although autoantigens are ill-defined in most autoimmune diseases, this is not the case in the skin. Autoimmune bullous diseases have been extensively studied with detailed characterization of autoantigens, the epitopes that are targeted, and the mechanisms of action that mediate autoimmune tissue destruction. Pemphigus is an autoimmune bullous disease caused by circulating IgG that targets two desmosomal proteins, desmoglein 1 and 3, which are crucial for cell-cell adhesion of keratinocytes. Binding of auto-antibodies to desmogleins impairs keratinocyte adhesion, leading to severe blistering disease. Mouse models that recapitulate the human disease have been instrumental in elucidating the detailed pathophysiology. Taking advantage of the fact that desmogleins are specifically targeted in pemphigus, studying humoral and cellular autoimmunity against these autoantigens provides us with an opportunity to understand not only the effector mechanisms of B and T cells in mediating pathology but also how autoreactive lymphocytes are regulated during development in the thymus and post-development in the periphery. This review introduces pemphigus and its subtypes as prototypic autoimmune diseases from which recent basic and translational developments should provide insight into how autoimmunity develops.

Effect of Silk Fibroin on Cell Viability in Electrospun Scaffolds of Polyethylene Oxide.

In this study, a coating from electrospun silk fibroin was performed with the aim to modify the surface of breast implants. We evaluated the effect of fibroin on polymeric matrices of poly (ethylene oxide) (PEO) to enhance cell viability, adhesion, and proliferation of HaCaT human keratinocytes to enhance the healing process on breast prosthesis implantation. We electrospun six blends of fibroin and PEO at different concentrations. These scaffolds were characterized by scanning electron microscopy, contact angle measurements, ATR-FTIR spectroscopy, and X-ray diffraction. We obtained diverse network conformations at different combinations to examine the regulation of cell adhesion and proliferation by modifying the microstructure of the matrix to be applied as a potential scaffold for coating breast implants. The key contribution of this work is the solution it provides to enhance the healing process on prosthesis implantation considering that the use of these PEO–fibroin scaffolds reduced (p < 0.05) the amount of pyknotic nuclei. Therefore, viability of HaCaT human keratinocytes on PEO–fibroin matrices was significantly improved (p < 0.001). These findings provide a rational strategy to coat breast implants improving biocompatibility.

A Homozygous Nonsense Mutation in the DSG3 Gene Causes Acantholytic Blisters in the Oral and Laryngeal Mucosa

A Homozygous Nonsense Mutation in the DSG3 Gene Causes Acantholytic Blisters in the Oral and Laryngeal Mucosa

Podoplanin suppresses the cell adhesion of epidermal keratinocytes via functional regulation of β1-integrin.

Epidermal stem cells adhere more efficiently to the extracellular matrix (ECM) than the less adhesive differentiating cells due to their high expression of cell adhesion molecules including β1-integrin. Podoplanin is majorly expressed in the markedly proliferative and differentiating basal cells of the wounded and psoriatic epidermis. This study was designed to reveal podoplanin's function in human epidermal keratinocytes (HEK) focusing on its interaction with β1-integrin. We analyzed the adhesion and differentiation of HEK in both podoplanin-overexpressing and -knock-down cells, considering their β1-integrin levels. The basal layer of IL-22-treated hyperproliferative reconstituted epidermis cells (simulating basal hyperproliferative psoriatic epidermal basal cells) expressed higher podoplanin levels than the untreated control cells. The adhesiveness of HaCaT cells, which do not express podoplanin, was reduced after the overexpression of podoplanin. HEK with podoplanin overexpression suppressed the cell adhesion to type I collagen (while downregulating β1-integrin functions) and podoplanin silencing augmented it (by increasing active ECM-bound β1-integrin). The increased cell adhesion to type I collagen induced by podoplanin silencing could be reversed by addition of P5D2, a neutralizing antibody against β1-integrin. In the psoriatic epidermis, podoplanin expression was especially upregulated on the rete ridges of the basal cell layer. This expression pattern was inversely correlated with the total/ECM-bound active β1-integrin-expression, which was stronger at the basal cell layer covering the dermal papillae. Our results indicate that podoplanin inhibits the cell ECM attachment by suppressing β1-integrin and initiating HEK differentiation. Podoplanin is presumably involved in the pathogenesis of psoriasis.

Non-Desmoglein Antibodies in Patients With Pemphigus Vulgaris.

Pemphigus vulgaris (PV) is a potentially life-threatening mucocutaneous autoimmune blistering disease. Patients develop non-healing erosions and blisters due to cell–cell detachment of keratinocytes (acantholysis), with subsequent suprabasal intraepidermal splitting. Identified almost 30 years ago, desmoglein-3 (Dsg3), a Ca2+-dependent cell adhesion molecule belonging to the cadherin family, has been considered the “primary” autoantigen in PV. Proteomic studies have identified numerous autoantibodies in patients with PV that have known roles in the physiology and cell adhesion of keratinocytes. Antibodies to these autoantibodies include desmocollins 1 and 3, several muscarinic and nicotinic acetylcholine receptor subtypes, mitochondrial proteins, human leukocyte antigen molecules, thyroid peroxidase, and hSPCA1—the Ca2+/Mn2+-ATPase encoded by ATP2C1, which is mutated in Hailey–Hailey disease. Several studies have identified direct pathogenic roles of these proteins, or synergistic roles when combined with Dsg3. We review the role of these direct and indirect mechanisms of non-desmoglein autoantibodies in the pathogenesis of PV.

Histatins, wound healing, and cell migration.

Wounds in the oral mucosa heal faster and more efficiently than those in the skin, although the mechanisms underlying these differences are not completely clear. In the last 10years, a group of salivary peptides, the histatins, has gained attention on behalf of their ability to improve several phases of the wound-healing process. In addition to their roles as anti-microbial agents and in enamel maintenance, histatins elicit other biological effects, namely by promoting the migration of different cell types contained in the oral mucosa and in non-oral tissues. Histatins, and specifically histatin-1, promote cell adhesion and migration in oral keratinocytes, gingival and dermal fibroblasts, non-oral epithelial cells, and endothelial cells. This is particularly relevant, as histatin-1 promotes the re-epithelialization phase and the angiogenic responses by increasing epithelial and endothelial cell migration. Although the molecular mechanisms associated with histatin-dependent cell migration remain poorly understood, recent studies have pointed to the control of signaling endosomes and the balance of small GTPases. This review aimed to update the literature on the effects of histatins in cell migration, with a focus on wound healing. We will also discuss the consequences that this increasing field will have in disease and therapy design.

Caspase cascade pathways of apoptosis in oral pemphigus: An immunohistochemical study.

Background:Pemphigus is a group of autoimmune blistering diseases characterized by loss of keratinocyte cell adhesion that leads to blister formation clinically. Induction of apoptosis or of proapoptotic proteins by pemphigus immunoglobulin G (IgG) may be part of the mechanism by which IgG induces acantholysis. Some of the current data suggest that activation of proapoptotic proteins such as bax and caspase cysteine proteinases may sensitize cells to the acantholytic effects of pemphigus IgG. Thus, a central role of apoptosis in the mechanisms of blister induction is well recognized.Aims:This study aims (a) To find which pathway of apoptosis is involved in the pathogenesis of pemphigus and (b) to evaluate the expression of bax and caspase-8 and its key role in pemphigus.Materials and Methods:The study was conducted on 21 samples of oral pemphigus. The presence of apoptosis was evaluated in the sections taken from histopathologically diagnosed oral pemphigus archival blocks using peroxidase-antiperoxidase immunohistochemical method.Results:The expression and staining intensity of pro-apoptotic marker bax and apoptotic marker caspase-8 were observed in the various areas with varying intensity in different samples. The result was subjected to statistical analysis.Conclusion:The results obtained in the present study suggest that the process of apoptosis occurs in PV. Hence, inhibition of apoptosis in the patients could reduce the severity of the lesions, and they could also represent new specific targets for pemphigus treatment.

RNA-Seq Analysis of Differentiated Keratinocytes Reveals a Massive Response to Late Events during Human Papillomavirus 16 Infection, Including Loss of Epithelial Barrier Function.

ABSTRACTThe human papillomavirus (HPV) replication cycle is tightly linked to epithelial cell differentiation. To examine HPV-associated changes in the keratinocyte transcriptome, RNAs isolated from undifferentiated and differentiated cell populations of normal, spontaneously immortalized keratinocytes (NIKS) and NIKS stably transfected with HPV16 episomal genomes (NIKS16) were compared using next-generation sequencing (RNA-Seq). HPV16 infection altered expression of 2,862 cellular genes. Next, to elucidate the role of keratinocyte gene expression in late events during the viral life cycle, RNA-Seq was carried out on triplicate differentiated populations of NIKS (uninfected) and NIKS16 (infected). Of the top 966 genes altered (>log2 = 1.8, 3.5-fold change), 670 genes were downregulated and 296 genes were upregulated. HPV downregulated many genes involved in epithelial barrier function, which involves structural resistance to the environment and immunity to infectious agents. For example, HPV infection repressed expression of the differentiated keratinocyte-specific pattern recognition receptor TLR7, the Langerhans cell chemoattractant CCL20, and proinflammatory cytokines interleukin 1α (IL-1α) and IL-1β. However, the type I interferon regulator IRF1, kappa interferon (IFN-κ), and viral restriction factors (IFIT1, -2, -3, and -5, OASL, CD74, and RTP4) were upregulated. HPV infection abrogated gene expression associated with the physical epithelial barrier, including keratinocyte cytoskeleton, intercellular junctions, and cell adhesion. Quantitative PCR (qRT-PCR) and Western blotting confirmed changes in expression of seven of the most significantly altered mRNAs. Expression of three genes showed statistically significant changes during cervical disease progression in clinical samples. Taken together, the data indicate that HPV infection manipulates the differentiating keratinocyte transcriptome to create an environment conducive to productive viral replication and egress.IMPORTANCE HPV genome amplification and capsid formation take place in differentiated keratinocytes. The viral life cycle is intimately associated with host cell differentiation. Deep sequencing (RNA-Seq) of RNA from undifferentiated and differentiated uninfected and HPV16-positive keratinocytes showed that almost 3,000 genes were differentially expressed in keratinocytes due to HPV16 infection. Strikingly, the epithelial barrier function of differentiated keratinocytes, comprising keratinocyte immune function and cellular structure, was found to be disrupted. These data provide new insights into the virus-host interaction that is crucial for the production of infectious virus and reveal that HPV infection remodels keratinocytes for completion of the virus replication cycle.

Vanillin increases stem cell signal and cell adhesion in keratinocytes

Vanillin increases stem cell signal and cell adhesion in keratinocytes

Spectraplakin family proteins - cytoskeletal crosslinkers with versatile roles.

The different cytoskeletal networks in a cell are responsible for many fundamental cellular processes. Current studies have shown that spectraplakins, cytoskeletal crosslinkers that combine features of both the spectrin and plakin families of crosslinkers, have a critical role in integrating these different cytoskeletal networks. Spectraplakin genes give rise to a variety of isoforms that have distinct functions. Importantly, all spectraplakin isoforms are uniquely able to associate with all three elements of the cytoskeleton, namely, F-actin, microtubules and intermediate filaments. In this Review, we will highlight recent studies that have unraveled their function in a wide range of different processes, from regulating cell adhesion in skin keratinocytes to neuronal cell migration. Taken together, this work has revealed a diverse and indispensable role for orchestrating the function of different cytoskeletal elements in vivo.

Current concepts of pemphigus with a deep insight into its molecular aspects.

Pemphigus vulgaris is an autoimmune bullous disease involving both the skin and mucosal areas, which is characterized by intraepithelial flaccid blisters and erosions. The pathogenesis of this disease is not yet completely established, but novel intuitions into its pathogenesis have recently been published. An unanswered question in its pathophysiology is the mechanism of acantholysis or loss of keratinocyte cell adhesion. Acantholysis seems to result from a communal action of autoantibodies against numerous keratinocyte self-antigens, of which desmogleins 1 and 3, desmocollins and nondesmosome components, such as the mitochondrion, might take part in the disease initiation. Lately, apoptosis was described as a possible underlying mechanism of acantholysis. Likewise, apoptolysis is assumed to be the association between suprabasal acantholytic and cell death pathways. Hence, the present review focuses on the current concepts in the pathogenesis of the pemphigus in a nutshell.