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BACKGROUND: Pemphigoid diseases are characterized by subepidermal blister formation accompanied by autoantibodies targeting skin component molecules, such as BP180. It is suggested that an epitope-phenotype correlation exists among autoantibodies recognizing BP180. However, it is unclear which regions of BP180 are likely targets for autoantibodies. OBJECTIVE: To elucidate the portions of BP180 where antibodies tend to react under the breakdown of immune tolerance. METHODS: We immunized mice with full-length mouse BP180 (mBP180) to produce anti-mBP180 antibodies. Using the immunized mice, hybridoma cells were established to produce anti-mBP180 antibodies. We analyzed the characteristics of the anti-mBP180 antibodies that were produced in terms of epitopes, immunoglobulin subclasses, and somatic hypermutations. RESULTS: Hybridoma cells derived from immunized mice with full-length mBP180 produced antibodies targeting the intracellular domain (IC) and the shed ectodomain (EC) of mBP180. Using the domain-deleted mBP180 recombinant protein, we revealed that monoclonal anti-mBP180 EC antibodies react to neoepitopes on the 13th collagenous region of cleaved mBP180, which corresponds to the epitopes of linear IgA bullous dermatosis antibodies in human BP180. Furthermore, the subclasses of these antibodies could be distinguished by epitope: The subclass of the anti-mBP180 IC monoclonal antibodies was IgG, whereas that of the anti-mBP180 EC antibodies was IgM. Of note, a clone of these IgM mBP180 EC antibodies was a germline antibody without somatic hypermutation, which is also known as a natural antibody. CONCLUSION: These data suggest that mice potentially have natural antibodies targeting the neoepitopes of cleaved mBP180 EC.
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Colágenos no Fibrilares , Penfigoide Ampolloso , Humanos , Animales , Ratones , Colágenos no Fibrilares/genética , Autoantígenos , Autoanticuerpos , Epítopos , Piel , Inmunoglobulina MRESUMEN
Niche for stem cells profoundly influences their maintenance and fate during tissue homeostasis and pathological disorders; however, the underlying mechanisms and tissue-specific features remain poorly understood. Here, it is reported that fatty acid desaturation catabolized by stearoyl-coenzyme A desaturase 1 (SCD1) regulates hair follicle stem cells (HFSCs) and hair growth by maintaining the bulge, niche for HFSCs. Scd1 deletion in mice results in abnormal hair growth, an effect exerted directly on keratin K14+ keratinocytes rather than on HFSCs. Mechanistically, Scd1 deficiency impairs the level of integrin α6ß4 complex and thus the assembly of hemidesmosomes (HDs). The disruption of HDs allows the aberrant activation of focal adhesion kinase and PI3K in K14+ keratinocytes and subsequently their differentiation and proliferation. The overgrowth of basal keratinocytes results in downward extension of the outer root sheath and interruption of bulge formation. Then, inhibition of PI3K signaling in Scd1-/- mice normalizes the bulge, HFSCs, and hair growth. Additionally, supplementation of oleic acid to Scd1-/- mice reestablishes HDs and the homeostasis of bulge niche, and restores hair growth. Thus, SCD1 is critical in regulating hair growth through stabilizing HDs in basal keratinocytes and thus sustaining bulge for HFSC residence and periodic activity.
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Hemidesmosomas , Fosfatidilinositol 3-Quinasas , Ratones , Animales , Queratinocitos , Homeostasis , Estearoil-CoA DesaturasaRESUMEN
Despite advancements in technology and increase in favorable outcomes associated with oral cancer, early detection remains the most significant factor in limiting mortality. The current study aimed to develop early diagnostic and prognostic markers for oral tumorigenesis. Protein and ultrastructural alterations at cell-extracellular matrix (ECM) adhesion junctions were examined concurrently using immunohistochemistry (IHC) and transmission electron microscopy (TEM) on progressive grade of oral carcinomas (n = 285). The expression of hemidesmosome (HD) proteins-integrin ß4, BP180, and laminin-5 increased in hyperplasia as compared to normal, and significantly increased further, as the disease progressed. TEM analysis in parallel tissues revealed a significant decrease in HD number and increase in the length of basal lamina (BL) in hyperplasia. With cancer progression, the severity of ultrastructural alterations increased gradually and significantly. Overexpression of HD proteins, decrease in HD number and increase in BL length significantly correlated with nodal metastasis, local recurrence, and recurrence-free survival of patients. Concurrent use of IHC and TEM can add value to early recognition of neoplastic changes in primary carcinomas of oral cavity. In this regard, altered expression of integrin ß4 and laminin-5, loss of HDs, and increased BL length could offer criteria for early diagnosis and prognosis of oral malignancy.
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Carcinoma , Neoplasias de la Boca , Carcinoma/patología , Matriz Extracelular/metabolismo , Hemidesmosomas/metabolismo , Hemidesmosomas/patología , Hemidesmosomas/ultraestructura , Humanos , Hiperplasia/metabolismo , Hiperplasia/patología , Integrina beta4/metabolismo , Neoplasias de la Boca/diagnóstico , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , PronósticoRESUMEN
Somatic stem cells are essential for the maintenance of tissue homeostasis. Despite its importance, how the esophageal stratified squamous epithelium executes its self-renewal and maintenance remains elusive. In this study, using 5-bromo-2'-deoxyuridine label-chase in rats in vivo and rat esophageal organoids in vitro together with genome-wide DNA methylation and single-cell RNA sequencing, we identified a slow-cycling/quiescent stem cell population that contained high levels of hemidesmosomes (HDs) and low levels of Wnt signaling localized spatially and randomly at the basal layer of the esophageal epithelium. Pseudotime cell trajectory analysis indicated that tissue cells originated from quiescent basal stem cells in the basal layer. Perturbations of HD component expression and/or Wnt signaling reduced the stem cell population in the basal layer of esophageal keratinocyte organoids, resulting in alterations in the organoid formation rate, size, morphogenesis, and proliferation-differentiation homeostasis. Furthermore, not only high levels of HDs and low levels of Wnt signaling but also an interplay between HD and Wnt signaling defined the stem cells of the basal layer. Hence, HDs and Wnt signaling are critical determinants for defining the stem cells of the basal layer required for tissue homeostasis in mammalian esophagi.
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Carcinoma de Células Escamosas , Células Madre , Ratas , Animales , Células Madre/metabolismo , Epitelio/metabolismo , Esófago/metabolismo , Diferenciación Celular , Carcinoma de Células Escamosas/metabolismo , Vía de Señalización Wnt , Proliferación Celular , MamíferosRESUMEN
Hemidesmosomes (HDs) are multiprotein complexes that firmly anchor epidermal cells to the basement membrane of skin through the interconnection of the cytoplasmic intermediate filaments with extracellular laminin 332 (Ln332). Considerably less attention has been paid to HDs compared to focal complexes/focal adhesions (FC/FAs) in mechanistic single-cell structures due to the lack of suitable in vitro model systems. Here nanopatterns of Ln332 (100-1000 nm) are created to direct and study the formation of HD in adherent HaCaT cells. It is observed that HaCaT cells at Ln 332 nanopatterns adhere via hemidesmosomes, in stark contrast to cells at homogeneous Ln332 surfaces that adhere via FC/FAs. Clustering of α6 integrin is observed at nanopatterned Ln332 of 300 nm patches and larger. Cells at 500 nm diameter patterns show strong colocalization of α6 integrin with ColXVII or pan-cytokeratin compared to 300 nm/1000 nm indicating a threshold for HD initiation >100 nm but a pattern size selection for maturation of HDs. It is demonstrated that the pattern of Ln332 can determine the cellular selection of adhesion types with a size-dependent initiation and maturation of HDs. The protein nanopatterning approach that is presented provides a new in vitro route to study the role of HDs in cell signaling and function.
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Adhesiones Focales , Hemidesmosomas , Adhesión Celular , Adhesiones Focales/metabolismo , Integrina alfa6/metabolismo , LigandosRESUMEN
Hemidesmosomes (HDs) are adhesive structures that ensure stable anchorage of cells to the basement membrane. They are formed by α6ß4-integrin heterodimers and linked to intermediate filaments via plectin. It has been reported that one of the most common events during the pathogenesis of prostate cancer (PCa) is the loss of HD organization. While the expression levels of ß4-integrins are strongly reduced, the expression levels of α6-integrins and plectin are maintained or even elevated, and seem to promote tumorigenic properties of PCa cells, such as proliferation, invasion, metastasis, apoptosis- and drug-resistance. In this review, we discuss the potential mechanisms of how HD components might contribute to various cellular signaling pathways to promote prostate carcinogenesis. Moreover, we summarize the current knowledge on the involvement of α6ß4-integrins and plectin in PCa initiation and progression.
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Plectin, a high-molecular-weight cytoskeletal linker protein, binds with high affinity to intermediate filaments of all types and connects them to junctional complexes, organelles, and inner membrane systems. In addition, it interacts with actomyosin structures and microtubules. As a multifunctional protein, plectin has been implicated in several multisystemic diseases, the most common of which is epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). A great part of our knowledge about plectin's functional diversity has been gained through the analysis of a unique collection of transgenic mice that includes a full (null) knockout (KO), several tissue-restricted and isoform-specific KOs, three double KOs, and two knock-in lines. The key molecular features and pathological phenotypes of these mice will be discussed in this review. In summary, the analysis of the different genetic models indicated that a functional plectin is required for the proper function of striated and simple epithelia, cardiac and skeletal muscle, the neuromuscular junction, and the vascular endothelium, recapitulating the symptoms of humans carrying plectin mutations. The plectin-null line showed severe skin and muscle phenotypes reflecting the importance of plectin for hemidesmosome and sarcomere integrity; whereas the ablation of individual isoforms caused a specific phenotype in myofibers, basal keratinocytes, or neurons. Tissue-restricted ablation of plectin rendered the targeted cells less resilient to mechanical stress. Studies based on animal models other than the mouse, such as zebrafish and C. elegans, will be discussed as well.
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Modelos Animales de Enfermedad , Epidermólisis Ampollosa Simple/patología , Distrofia Muscular de Cinturas/patología , Mutación , Plectina/metabolismo , Animales , Epidermólisis Ampollosa Simple/etiología , Epidermólisis Ampollosa Simple/metabolismo , Humanos , Distrofia Muscular de Cinturas/etiología , Distrofia Muscular de Cinturas/metabolismo , Plectina/genética , Isoformas de ProteínasRESUMEN
Many chemical routes have been proposed to immobilize peptides on biomedical device surfaces, and in particular, on dental implants to prevent peri-implantitis. While a number of factors affect peptide immobilization quality, an easily controllable factor is the chemistry used to immobilize peptides. These factors affect peptide chemoselectivity, orientation, etc., and ultimately control biological activity. Using many different physical and chemical routes for peptide coatings, previous research has intensely focused on immobilizing antimicrobial elements on dental implants to reduce infection rates. Alternatively, our strategy here is different and focused on promoting formation of a long-lasting biological seal between the soft tissue and the implant surface through transmembrane, cell adhesion structures called hemidesmosomes. For that purpose, we used a laminin-derived call adhesion peptide. However, the effect of different immobilization chemistries on cell adhesion peptide activity is vastly unexplored but likely critical. Here, we compared the physiochemical properties and biological responses of a hemidesmosome promoting peptide immobilized using silanization and copper-free click chemistry as a model system for cell adhesion peptides. Successful immobilization was confirmed with water contact angle and X-ray photoelectron spectroscopy. Peptide coatings were retained through 73 days of incubation in artificial saliva. Interestingly, the non-chemoselective immobilization route, silanization, resulted in significantly higher proliferation and hemidesmosome formation in oral keratinocytes compared to chemoselective click chemistry. Our results highlight that the most effective immobilization chemistry for optimal peptide activity is dependent on the specific system (substrate/peptide/cell/biological activity) under study. Overall, a better understanding of the effects immobilization chemistries have on cell adhesion peptide activity may lead to more efficacious coatings for biomedical devices.
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Abstract: Bullous pemphigoid is the most frequent autoimmune bullous disease and mainly affects elderly individuals. Increase in incidence rates in the past decades has been attributed to population aging, drug-induced cases and improvement in the diagnosis of the nonbullous presentations of the disease. A dysregulated T cell immune response and synthesis of IgG and IgE autoantibodies against hemidesmosomal proteins (BP180 and BP230) lead to neutrophil chemotaxis and degradation of the basement membrane zone. Bullous pemphigoid classically manifests with tense blisters over urticarial plaques on the trunk and extremities accompanied by intense pruritus. Mucosal involvement is rarely reported. Diagnosis relies on (1) the histopathological evaluation demonstrating eosinophilic spongiosis or a subepidermal detachment with eosinophils; (2) the detection of IgG and/or C3 deposition at the basement membrane zone using direct or indirect immunofluorescence assays; and (3) quantification of circulating autoantibodies against BP180 and/or BP230 using ELISA. Bullous pemphigoid is often associated with multiple comorbidities in elderly individuals, especially neurological disorders and increased thrombotic risk, reaching a 1-year mortality rate of 23%. Treatment has to be tailored according to the patient's clinical conditions and disease severity. High potency topical steroids and systemic steroids are the current mainstay of therapy. Recent randomized controlled studies have demonstrated the benefit and safety of adjuvant treatment with doxycycline, dapsone and immunosuppressants aiming a reduction in the cumulative steroid dose and mortality.
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Humanos , Anciano , Penfigoide Ampolloso/diagnóstico , Esteroides/uso terapéutico , Autoinmunidad/fisiología , Técnica del Anticuerpo Fluorescente/métodos , Penfigoide Ampolloso/clasificación , Penfigoide Ampolloso/etiología , Penfigoide Ampolloso/tratamiento farmacológico , Diagnóstico DiferencialRESUMEN
Amelogenesis imperfecta (AI) is a heterogeneous group of inherited disorders characterized by abnormal formation of dental enamel, either in isolation or as part of a syndrome. Heterozygous variants in laminin subunit beta 3 ( LAMB3) cause AI with dominant inheritance in the absence of other cosegregating clinical features. In contrast, biallelic loss-of-function variants in LAMB3 cause recessive junctional epidermolysis bullosa, characterized by life-threatening skin fragility. We identified 2 families segregating autosomal dominant AI with variable degrees of a distinctive hypoplastic phenotype due to pathogenic variants in LAMB3. Whole exome sequencing revealed a nonsense variant (c.3340G>T, p.E1114*) within the final exon in family 1, while Sanger sequencing in family 2 revealed a variant (c.3383-1G>A) in the canonical splice acceptor site of the final exon. Analysis of cDNA from family 2 revealed retention of the final intron leading to a premature termination codon. Two unerupted third molar teeth from individual IV:5 in family 2 were subject to computerized tomography and scanning electron microscopy. LAMB3 molar teeth have a multitude of cusps versus matched controls. LAMB3 enamel was well mineralized but pitted. The architecture of the initially secreted enamel was abnormal, with cervical enamel appearing much less severely affected than coronal enamel. This study further defines the variations in phenotype-genotype correlation for AI due to variants in LAMB3, underlines the clustering of nonsense and frameshift variants causing AI in the absence of junctional epidermolysis bullosa, and highlights the shared AI phenotype arising from variants in genes coding for hemidesmosome proteins.
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Amelogénesis Imperfecta/genética , Moléculas de Adhesión Celular/genética , Amelogénesis Imperfecta/clasificación , Codón sin Sentido , Femenino , Mutación del Sistema de Lectura , Heterocigoto , Humanos , Masculino , Linaje , Fenotipo , KalininaRESUMEN
The testes of sexually mature males of six mammalian species (men, bulls, boars, rats, mice, guinea pigs) have been studied using biochemical as well as light and electron microscopical techniques, in particular immunolocalizations. In these tissues, the peritubular walls represent lamellar encasement structures wrapped around the seminiferous tubules as a bandage system of extracellular matrix layers, alternating with monolayers of very flat polyhedral "lamellar smooth muscle cells" (LSMCs), the number of which varies in different species from 1 to 5 or 6. These LSMCs are complete SMCs containing smooth muscle α-actin (SMA), myosin light and heavy chains, α-actinin, tropomyosin, smoothelin, intermediate-sized filament proteins desmin and/or vimentin, filamin, talin, dystrophin, caldesmon, calponin, and protein SM22α, often also cytokeratins 8 and 18. In the monolayers, the LSMCs are connected by adherens junctions (AJs) based on cadherin-11, in some species also with P-cadherin and/or E-cadherin, which are anchored in cytoplasmic plaques containing ß-catenin and other armadillo proteins, in some species also striatin family proteins, protein myozap and/or LUMA. The LSMC cytoplasm is rich in myofilament bundles, which in many regions are packed in paracrystalline arrays, as well as in "dense bodies," "focal adhesions," and caveolae. In addition to some AJ-like end-on-end contacts, the LSMCs are laterally connected by numerous vertical AJ-like junctions located in variously sized and variously shaped, overlapping (alter super alterum) lamelliform cell protrusions. Consequently, the LSMCs of the peritubular wall monolayers are SMCs sensu stricto which are laterally connected by a novel architectonic system of arrays of vertical AJs located in overlapping cell protrusions.
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Uniones Adherentes/metabolismo , Mamíferos/metabolismo , Miocitos del Músculo Liso/citología , Testículo/citología , Uniones Adherentes/ultraestructura , Animales , Membrana Basal/metabolismo , Membrana Basal/ultraestructura , Extensiones de la Superficie Celular/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Humanos , Masculino , Miocitos del Músculo Liso/ultraestructura , Epitelio Seminífero/metabolismo , Túbulos Seminíferos/citología , Túbulos Seminíferos/ultraestructura , Testículo/ultraestructuraRESUMEN
A good biological sealing between soft tissues and an implant could minimize the risk of peri-implantitis. Like the junctional epithelium, the peri-implant epithelium attaches to the surface of the implant via hemidesmosomes (HDs) and internal basal lamina (extracellular matrix containing laminin332, IBL). A multilayer coating modified with the laminin332 gene (LAMA3) on the titanium implant surface is developed here via layer-by-layer assembly and antibody-antigen specific binding for substrate-mediated gene transduction. The results in vitro indicated that the LAMA3-modified coating on the titanium surface could improve HaCaT cell adhension in the early stage, and promote the expression of laminin α3 on both the protein and the gene levels. Moreover, the formation of hemidesmosomes at the interface became obvious. In vivo experiments demonstrated that the LAMA3 gene coating on the implant surface could enhance the expression of laminin α3 and improve the biological sealing between the implant and the epithelium. The success of the LAMA3 functionalized multilayer coating in improving biological sealing between titanium implant and gingival epithelium might provide a new approach and experimental evidence for research on the interface of the implant and soft tissues.
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Células Epiteliales/metabolismo , Epitelio/metabolismo , Laminina/metabolismo , Titanio/metabolismo , Adhesión Celular , Línea Celular , Supervivencia Celular/efectos de los fármacos , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/metabolismo , Materiales Biocompatibles Revestidos/farmacología , Implantes Dentales , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Técnicas de Transferencia de Gen , Encía/citología , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/ultraestructura , Laminina/química , Laminina/genética , Microscopía Electrónica , Propiedades de Superficie , Titanio/químicaRESUMEN
Osteoporosis (OP) is a global bone disease prevalent in aging in humans, especially in older women. Bisphosphonates (BPs) are commonly used as therapy for OP as it influences hard and soft tissues calcium metabolism. Mucosal and dermal ulceration with exposure of underlying bone arises from incomplete epithelial recovery due to reduced desmosome formation deriving from lack of available calcium. Pathological situations such as bisphosphonate-related osteonecrosis of the jaw have been described. This hypothesis states other situations which demand intact functional desmosomes such as healing skin over chronic pressure points leading to pressure ulcers (as well-known as bedsores, pressure sores, pressure injuries, decubitus ulcers), and hemidesmosomes such as epithelial seals in contact with titanium surfaces will have a higher prevalence of breakdown among patients being treated with BPs. This may be proven through the diminished modulation of calcium ions due to BPs, and its effect on the formation of intercellular gap junctions.
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Conservadores de la Densidad Ósea/efectos adversos , Calcio/deficiencia , Difosfonatos/efectos adversos , Oseointegración/efectos de los fármacos , Úlcera por Presión/etiología , Osteonecrosis de los Maxilares Asociada a Difosfonatos/etiología , Osteonecrosis de los Maxilares Asociada a Difosfonatos/metabolismo , Calcio/metabolismo , Desmosomas/efectos de los fármacos , Desmosomas/metabolismo , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Femenino , Humanos , Masculino , Modelos Biológicos , Osteoporosis/tratamiento farmacológicoRESUMEN
Oral epithelial adhesion to the lamina propria underlies the physiologic function of the oral mucosa and contributes to resisting bacterial invasion, preventing body fluid loss, and maintaining routine chewing; thus, understanding the factors that positively influence oral epithelial adhesion is a research topic of great interest. Rete pegs contribute to oral epithelial adhesion by enlarging the contact areas, whereas integrins are the major molecules that mediate epithelial cell adhesion to the basement membrane. Keratinocyte growth factor (KGF) can promote both rete peg elongation in the skin and the expression of integrins in various cell types. Herein, we tested the effects of submucosal injection of KGF in the ventral surfaces of rat tongues on oral epithelial adhesion. The data confirmed that topical injection of KGF elevated the adhesive forces, elongated the rete pegs, and increased the abundance of integrins, KGF, and KGF receptor on the rat tongue ventral surface. However, HYD-1 (Lys-Ile-Lys-Met-Val-Ile-Ser-Trp-Lys-Gly), an integrin antagonist, inhibited the KGF-enhanced epithelial adhesion and rete peg elongation. Moreover, KGF promoted the expression of integrin subunits α6, ß4, α3, and ß1 in human immortalized oral epithelial cells in 2- and 3-dimensional culture systems. In vitro cell attachment assays demonstrated that HYD-1 inhibited the adhesion of human immortalized oral epithelial cells on Matrigel. Strikingly, the expression of integrins, KGF, and KGFR in human masticatory mucosae with longer rete pegs was more abundant than that in the lining mucosae with shorter rete pegs. In addition, rete peg lengths were positively correlated with the expression levels of integrins, KGF, and KGF receptor. These findings indicate that KGF strengthens oral epithelial adhesion and rete peg elongation via integrins.
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Adhesión Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Factor 7 de Crecimiento de Fibroblastos/farmacología , Integrinas/metabolismo , Mucosa Bucal/citología , Animales , Western Blotting , Técnicas de Cultivo de Célula , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Masculino , Oligopéptidos/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Spectraplakins are evolutionary conserved cytolinkers with characteristics of both the spectrin and the plakin family proteins. Caenorhabditis elegans possesses two categories of spectraplakin isoforms encoded by a single locus termed vab-10. Here we summarize the structure, homology, expression and functions of these spectraplakin family proteins in the nematode. We particularly focus on the diverse roles of VAB-10 isoforms in a number of organs and tissue types, as well as the similarities and distinctions of the underlying mechanisms. We also discuss the functional partners of VAB-10 in different contexts, plus the involvement of VAB-10 isoforms in physiological processes beyond cytoskeletal integration. We emphasize the importance of spectraplakin-related studies using Caenorhabditis elegans as the model, and their contributions to our understanding of spectraplakins across species.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Citoesqueleto/metabolismo , Proteínas de Microfilamentos/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Genoma , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Modelos BiológicosRESUMEN
OBJECTIVE: To generate a nomogram for predicting the risk of neck node metastasis in pathologically node-negative patients using a combination of variables comprising of protein expression, ultrastructural alterations and clinicopathological parameters. MATERIALS AND METHODS: Surgically removed oral tumours (n = 103) were analysed for the expression of desmosomal and hemidesmosomal assembly proteins by immunohistochemistry and ultrastructural alterations by transmission electron microscopy (TEM). Protein expression, ultrastructural alterations and clinicopathological variables were used to construct nomogram from the training set in 75 patients. Clinical utility of the nomogram was validated in a discrete set of 28 patients. RESULTS: Univariate and multivariate analyses were performed on the training set, and obtained significant variables comprising of integrin ß4 expression (p = .027), number of hemidesmosomes (p = .027)/desmosomes (p = .046), tumour differentiation grade (p = .033) and tumour thickness (p = .024) were used for construction of the nomogram. The area under the curve was calculated for both training 0.821 (95% CI 0.725-0.918) and validation sets 0.880 (95% CI 0.743-1.000). The nomogram demonstrated a predictive accuracy of 73.3% and 78.6% with the sensitivity of 81.4% and 83.3% in the training and validation sets, respectively. CONCLUSIONS: The nomogram constructed on postsurgical tumour samples will be a value addition to histopathology for the detection of neck node metastasis in pathologically node-negative patients.
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Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/secundario , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Nomogramas , Área Bajo la Curva , Carcinoma de Células Escamosas/ultraestructura , Desmosomas/metabolismo , Desmosomas/ultraestructura , Femenino , Hemidesmosomas/metabolismo , Hemidesmosomas/ultraestructura , Humanos , Integrina beta4/metabolismo , Metástasis Linfática , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/ultraestructura , Cuello , Clasificación del Tumor , Valor Predictivo de las Pruebas , Curva ROC , Factores de RiesgoRESUMEN
BACKGROUND: Normal myoepithelial cells (MECs) play an important tumour-suppressor role in the breast but display an altered phenotype in ductal carcinoma in situ (DCIS), gaining tumour-promoter functions. Matrix metalloproteinase-8 (MMP-8) is expressed by normal MECs but is lost in DCIS. This study investigated the function of MMP-8 in MECs and the impact of its loss in DCIS. METHODS: Primary normal and DCIS-associated MECs, and normal (N-1089) and DCIS-modified myoepithelial (ß6-1089) cell lines, were used to assess MMP-8 expression and function. ß6-1089 lacking MMP-8 were transfected with MMP-8 WT and catalytically inactive MMP-8 EA, and MMP-8 in N-1089 MEC was knocked down with siRNA. The effect on adhesion and migration to extracellular matrix (ECM), localisation of α6ß4 integrin to hemidesmosomes (HD), TGF-ß signalling and gelatinase activity was measured. The effect of altering MEC MMP-8 expression on tumour cell invasion was investigated in 2D and 3D organotypic models. RESULTS: Assessment of primary cells and MEC lines confirmed expression of MMP-8 in normal MEC and its loss in DCIS-MEC. Over-expression of MMP-8 WT but not MMP-8 EA in ß6-1089 cells increased adhesion to ECM proteins and reduced migration. Conversely, knock-down of MMP-8 in N-1089 reduced adhesion and increased migration. Expression of MMP-8 WT in ß6-1089 led to greater localisation of α6ß4 to HD and reduced retraction fibre formation, this being reversed by MMP-8 knock-down in N-1089. Over-expression of MMP-8 WT reduced TGF-ß signalling and gelatinolytic activity. MMP-8 knock-down enhanced TGF-ß signalling and gelatinolytic activity, which was reversed by blocking MMP-9 by knock-down or an inhibitor. MMP-8 WT but not MMP-8 EA over-expression in ß6-1089 reduced breast cancer cell invasion in 2D and 3D invasion assays, while MMP-8 knock-down in N-1089 enhanced cancer cell invasion. Staining of breast cancer cases for MMP-8 revealed a statistically significant loss of MMP-8 expression in DCIS with invasion versus pure DCIS (p = 0.001). CONCLUSIONS: These data indicate MMP-8 is a vital component of the myoepithelial tumour-suppressor function. It restores MEC interaction with the matrix, opposes TGF-ß signalling and MMP-9 proteolysis, which contributes to inhibition of tumour cell invasion. Assessment of MMP-8 expression may help to determine risk of DCIS progression.
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Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Intraductal no Infiltrante/genética , Carcinoma Intraductal no Infiltrante/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Células Epiteliales/metabolismo , Metaloproteinasa 8 de la Matriz/deficiencia , Biomarcadores de Tumor , Carcinoma Ductal de Mama/patología , Carcinoma Intraductal no Infiltrante/patología , Adhesión Celular , Línea Celular Transformada , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Integrina alfa6beta4/metabolismo , Metaloproteinasa 8 de la Matriz/genética , Metaloproteinasa 8 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Comunicación Paracrina , Transporte de Proteínas , Proteolisis , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Intermediate filaments (IFs) are involved in multiple cellular processes that are essential for the maintenance of cell and tissue integrity. To achieve this crucial function, IFs have to be organized as long and resistant filaments across the cells and to be tightly anchored at the cell periphery. This anchoring takes place at the level desmosomes and hemidesmosomes through proteins belonging to the spectraplakin family. Here, we focus on the sole nematode Caenorhabditis elegans spectraplakin locus vab-10 that is essential to connect the epidermis to the cuticle apically and to the muscles basally. After briefly reviewing the structure of the gene, we first present the genetic tools available to study this gene as well as the reagents to examine the distribution of its translation products. We discuss the functional assays that enable examining their function. Finally, we detail a genetic method to identify spectraplakin functional partners through RNAi screens, and a biochemical method to examine the phosphorylation status of IFs.
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Proteínas de Caenorhabditis elegans/genética , Animales , Secuencia de Bases , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/fisiología , Exones , Mutación , Estructura Terciaria de ProteínaRESUMEN
Bullous autoimmune diseases are organ-specific disorders characterized by an autoantibody-mediated blistering of skin and mucous membranes. The detection of tissue-bound and serum autoantibodies is prerequisite for the diagnosis of autoimmune blistering diseases. The individual entities of this group may be difficult to differentiate on clinical grounds alone. An accurate diagnosis is however important for prognosis and therapy. A preliminary diagnostic step includes direct and indirect immunofluorescence microscopy, which provide information about the binding pattern and isotype of autoantibodies and allow the diagnosis of the autoimmune blistering disease. Subsequent characterization of the molecular specificity of autoantibodies is necessary for the exact classification of autoimmune bullous dermatoses. The quantitative measurement of autoantibodies against structural proteins of the skin may be often used to assess disease severity at follow-up.
Asunto(s)
Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/inmunología , Inmunoensayo/métodos , Técnicas de Diagnóstico Molecular/métodos , Enfermedades de la Piel/diagnóstico , Enfermedades de la Piel/inmunología , Autoanticuerpos/inmunología , HumanosRESUMEN
A three dimensional tissue-engineered human oral mucosal model (3D OMM) used in the investigation of implant-soft tissue interface was recently reported. The aim of this study was to examine the ultrastructural features of soft tissue attachment to various titanium (Ti) implant surfaces based on the 3D OMM. Two techniques, that is, focus ion beam (FIB) and electropolishing techniques were used to prepare specimens for transmission electron microscopic (TEM) analysis of the interface. The 3D OM consisting of both epithelial and connective tissue layers was constructed by co-culturing human oral keratinocytes and fibroblasts onto an acellular dermis scaffold. Four types of Ti surface topographies were tested: polished, machined (turned), sandblasted, and TiUnite. The specimens were then processed for TEM examination using FIB (Ti remained) and electropolishing (Ti removed) techniques. The FIB sections showed some artifact and lack of details of ultrastructural features. In contrast, the ultrathin sections prepared from the electropolishing technique showed a residual Ti oxide layer, which preserved the details for intact ultrastructural interface analysis. There was evidence of hemidesmosome-like structures at the interface on the four types of Ti surfaces, which suggests that the tissue-engineered oral mucosa formed epithelial attachments on the Ti surfaces.