involucrin and Disease-Models--Animal

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.

Topics: Animals; Cell Cycle Proteins; Disease Models, Animal; Epidermis; Fluorescence Resonance Energy Transfer; Gene Expression Regulation; High-Throughput Screening Assays; Humans; Keratinocytes; Male; Mice; Mice, Inbred C57BL; Primary Cell Culture; Protein Isoforms; Protein Precursors; Re-Epithelialization; Skin Ulcer; Small Molecule Libraries; Structure-Activity Relationship; Transcription Factors; Transcription, Genetic; Wounds, Nonpenetrating

Topics: Animals; Anti-Inflammatory Agents; Cytokines; Dermatitis, Atopic; Dinitrochlorobenzene; Disease Models, Animal; Fermentation; Filaggrin Proteins; Fruit; Histamine; Immunoglobulin G; Intermediate Filament Proteins; Membrane Proteins; Mice; Morinda; Occludin; Plant Extracts; Protein Precursors; Pruritus; Skin; T-Lymphocytes, Helper-Inducer; Zonula Occludens-1 Protein

The mTOR (mechanistic target of rapamycin) inhibitor rapamycin has long been known for its immune suppressive properties, but it has shown limited therapeutic success when given systemically to patients with psoriasis. Recent data have shown that the mTOR pathway is hyperactivated in lesional psoriatic skin, which probably contributes to the disease by interfering with maturation of keratinocytes. This study investigated the effect of topical rapamycin treatment in an imiquimod-induced psoriatic mouse model. The disease was less severe if the mice had received rapamycin treatment. Immunohistological analysis revealed that rapamycin not only prevented the activation of mTOR signalling (P-mTOR and P-S6 levels), but almost normalized the expression of epidermal differentiation markers. In addition, the influx of innate immune cells into the draining lymph nodes was partially reduced by rapamycin treatment. These data emphasize the role of mTOR signalling in the pathogenesis of psoriasis, and support the investigation of topical mTOR inhibition as a novel anti-psoriatic strategy.

Topics: Administration, Topical; Aminoquinolines; Animals; Caspase 14; Dendritic Cells; Disease Models, Animal; Imiquimod; Immunosuppressive Agents; Keratin-10; Keratin-14; Ki-67 Antigen; Langerhans Cells; Lymph Nodes; Macrophages; Membrane Proteins; Mice, Inbred BALB C; Neovascularization, Physiologic; Protein Precursors; Psoriasis; Sirolimus; Skin; TOR Serine-Threonine Kinases

The presence of congenitally impaired skin barrier followed by atopic dermatitis (AD) is an initial step in the atopic march. The maintenance of acidic pH in the stratum corneum (SC) has been suggested as a therapeutic or preventive strategy for barrier impairment caused by skin inflammation. To determine whether an AD murine model, flaky tail mice, with inherited filaggrin deficiency could develop airway inflammation by repeated topical application followed by nasal inhalation of house dust mite (HDM) antigen (defined as a novel "atopic march animal model"), and whether maintenance of an acidic SC environment by continuous application of acidic cream could interrupt the following atopic march. During the course of HDM treatment, acidic cream (pH2.8) or neutral cream (pH7.4) was applied to flaky tail mice twice daily. Repeated applications and inhalations of HDM to flaky tail mice induced AD skin lesions followed by respiratory allergies. Maintenance of SC acidity inhibited the occurrence of respiratory allergic inflammation as well as AD-like skin lesions. Collectively, a novel atopic march model could be developed by repeated epicutaneous and nasal applications of HDM to flaky tail mice, and that the acidification of SC could prevent the atopic march from AD to respiratory allergy.

Topics: Administration, Cutaneous; Animals; Antigens, Dermatophagoides; Cytokines; Dermatitis, Atopic; Disease Models, Animal; Disease Progression; Epidermis; Female; Filaggrin Proteins; Hydrogen-Ion Concentration; Immunoglobulin E; Inhalation Exposure; Intermediate Filament Proteins; Membrane Proteins; Mice; Protein Precursors; Respiratory Hypersensitivity; Skin Cream; Thymic Stromal Lymphopoietin

Mice lacking three epidermal barrier proteins-envoplakin, periplakin, and involucrin (EPI-/- mice)-have a defective cornified layer, reduced epidermal γδ T cells, and increased dermal CD4(+) T cells. They are also resistant to developing skin tumors. The tumor-protective mechanism involves signaling between Rae-1 expressing keratinocytes and the natural killer group 2D receptor on immune cells, which also plays a role in host defenses against infection. Given the emerging link between bacteria and cancer, we investigated whether EPI-/- mice have an altered skin microbiota. The bacterial phyla were similar in wild-type and EPI-/- skin. However, bacteria were threefold more abundant in EPI-/- skin and penetrated deeper into the epidermis. The major epithelial defense mechanism against bacteria is production of antimicrobial proteins (AMPs). EPI-/- skin exhibited enhanced expression of antimicrobial peptides. However, reducing the bacterial load by antibiotic treatment or breeding mice under specific pathogen-free conditions did not reduce AMP expression or alleviate the abnormalities in T-cell populations. We conclude that the atopic characteristics of EPI-/- skin are a consequence of the defective barrier rather than a response to the increased bacterial load. It is therefore unlikely that the increase in skin microbiota contributes directly to the observed cancer resistance.

Topics: Analysis of Variance; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Load; Disease Models, Animal; Disease Susceptibility; Enzyme-Linked Immunosorbent Assay; Female; In Situ Hybridization, Fluorescence; Membrane Proteins; Mice; Mice, Inbred C57BL; Microbiota; Peroxidase; Protein Precursors; Real-Time Polymerase Chain Reaction; Skin; Skin Absorption; Skin Neoplasms; Statistics, Nonparametric

Skin barrier defects play an important role in atopic dermatitis (AD). Involucrin, an important barrier protein suppressed in human AD, is downregulated by interleukin-4 (IL-4). However, the molecular mechanism for IL-4 downregulation of involucrin has not been delineated, and especially how Stat6, a transcriptional activator, represses involucrin expression is unknown. Since Stats usually recruit p300/CBP in the general transcription machinery of their target genes and involucrin expression also involves p300/CBP, we hypothesize that Stat6 activated by IL-4 may sequestrate p300/CBP from the involucrin transcription complex, thus suppressing involucrin expression in keratinocytes. Using IL-4 transgenic mice, an AD mouse model, we find that involucrin expression is similarly downregulated as in human AD. In HaCat cells, the Jak inhibitor and dominant negative studies indicate that the Jaks-Stat6 pathway is involved in IL-4 downregulation of involucrin. Next, we transfected HaCat cells with an involucrin promoter-luciferase construct and then treated them with IL-4. IL-4 greatly suppresses the promoter activity, which is totally abolished by cotransfecting the CREB-binding protein (CBP) expression vector, indicating that IL-4 cannot downregulate involucrin in the presence of excess CBP. Finally, chromatin immunoprecipitation assay demonstrates that IL-4 decreases CBP binding to the involucrin transcription complex. For the first time, we defined a molecular mechanism for IL-4 downregulation of involucrin in keratinocytes, which may play an important role in the pathogenesis of AD.

Topics: Animals; Cell Line; CREB-Binding Protein; Dermatitis, Atopic; Dermis; Disease Models, Animal; Down-Regulation; Gene Expression Regulation; Humans; Interleukin-4; Keratinocytes; Mice; Mice, Transgenic; Multiprotein Complexes; Protein Binding; Protein Precursors; STAT6 Transcription Factor; Transcription, Genetic

Psoriasis is a complex inflammatory skin disease that presents a wide variety of clinical manifestations. Human β defensin-2 (hBD-2) is highly up-regulated in psoriatic lesions and has been defined as a biomarker for disease activity. We explored the potential benefits of targeting hBD-2 by topical application of DEFB4-siRNA-containing SECosomes in a bioengineered skin-humanized mouse model for psoriasis. A significant improvement in the psoriatic phenotype was observed by histological examination, with a normalization of the skin architecture and a reduction in the number and size of blood vessels in the dermal compartment. Treatment leads to the recovery of transglutaminase activity, filaggrin expression and stratum corneum appearance to the levels similar to those found in normal regenerated human skin. The availability of a reliable skin-humanized mouse model for psoriasis in conjunction with the use of the SECosome technology may provide a valuable preclinical tool for identifying potential therapeutic targets for this disease.

Topics: Administration, Cutaneous; Animals; beta-Defensins; Bioengineering; Cell Differentiation; Cell Proliferation; Dermis; Disease Models, Animal; Elafin; Epidermis; Filaggrin Proteins; Gene Expression; Gene Silencing; Humans; Intermediate Filament Proteins; Keratin-1; Keratin-17; Ki-67 Antigen; Leukocyte L1 Antigen Complex; Liposomes; Mice; Molecular Targeted Therapy; Nanoparticles; Protein Precursors; Psoriasis; RNA, Small Interfering; S100 Calcium Binding Protein A7; S100 Proteins

Atopic dermatitis can result from loss of structural proteins in the outermost epidermal layers, leading to a defective epidermal barrier. To test whether this influences tumour formation, we chemically induced tumours in EPI-/- mice, which lack three barrier proteins-Envoplakin, Periplakin, and Involucrin. EPI-/- mice were highly resistant to developing benign tumours when treated with 7,12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The DMBA response was normal, but EPI-/- skin exhibited an exaggerated atopic response to TPA, characterised by abnormal epidermal differentiation, a complex immune infiltrate and elevated serum thymic stromal lymphopoietin (TSLP). The exacerbated TPA response could be normalised by blocking TSLP or the immunoreceptor NKG2D but not CD4+ T cells. We conclude that atopy is protective against skin cancer in our experimental model and that the mechanism involves keratinocytes communicating with cells of the immune system via signalling elements that normally protect against environmental assaults.DOI: http://dx.doi.org/10.7554/eLife.01888.001.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma, Squamous Cell; Cell Communication; Cell Differentiation; Cell Transformation, Neoplastic; Cytokines; Dermatitis, Atopic; Disease Models, Animal; Epidermis; Keratinocytes; Membrane Proteins; Mice, 129 Strain; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Nude; NK Cell Lectin-Like Receptor Subfamily K; Papilloma; Permeability; Plakins; Protein Precursors; Signal Transduction; Skin Neoplasms; Tetradecanoylphorbol Acetate; Thymic Stromal Lymphopoietin; Time Factors

To determine whether eye drop instillation of the disaccharide trehalose (TT) alleviates ocular surface damage in a dry eye murine model.. Dry eye was induced in mice using an intelligently controlled environmental system (ICES). After 21 days housed in the ICES without topical treatment, the mice were randomly divided into three groups: no eye drops (ICES) for three weeks, four times a day with PBS 0.01 M 10 µl/eye bilaterally (ICES+PBS), or with TT 87.6 mM 10 µl/eye bilaterally (ICES+TT). Another mice group that was not exposed to the ICES and received no treatment served as a control group (UT). The ocular surface integrity, in each group, was evaluated using Oregon Green dextran (OGD) and fluorescein staining. The expression and distribution of occludin, involucrin, and small proline-rich protein 2 were determined with immunohistology analysis on whole mounted corneas. Heat shock protein 70 (HSP70) and matrix metalloproteinase 9 (MMP-9) expression was estimated with immunohistology. Ocular surface inflammation associated with each treatment was estimated with real time-PCR of interleukin-1β (IL-1β), IL-2, IL-6, IL-17, and tumor necrosis factor-alpha in the conjunctiva.. OGD staining in the cornea epithelium was lower in the ICES+TT group than in the ICES and ICES+PBS groups. Corneal epithelial occludin staining was markedly more homogenous in the ICES+TT group than in ICES and ICES+PBS groups, and there were no desquamating apical epithelial cells. Involucrin and small proline-rich protein 2 labeling of whole mounted corneas revealed upregulation of their expression in the groups, which received no treatment or PBS instillation compared to the ICES+TT group. HSP70 and MMP-9 immunolabeling revealed a marked increase in corneal epithelial expression in response to the ICES. The group treated with trehalose showed a similar profile expression of HSP70 and MMP-9 as the control group (UT). Conjunctival IL-1β, IL-2, IL-6, IL-17, tumor necrosis factor-alpha (TNF-α), and MMP-9 mRNA expression was lower in the ICES+TT group than in the ICES or ICES+PBS group.. Trehalose application restored ocular surface integrity, suppressed inflammatory and proteolytic MMP-9 and HSP70 expression, and keratinization in mice with dry eye damaged by a desiccative model.

Topics: Animals; Base Sequence; Cornea; Cornified Envelope Proline-Rich Proteins; Cytokines; Disease Models, Animal; DNA Primers; Dry Eye Syndromes; Female; Gene Expression Profiling; HSP70 Heat-Shock Proteins; Immunohistochemistry; Matrix Metalloproteinase 9; Membrane Proteins; Mice; Mice, Inbred C57BL; Occludin; Ophthalmic Solutions; Protein Precursors; RNA, Messenger; Trehalose

Transient knock-down of the gap junction protein Cx43 by antisense and siRNA, or gap junction block with mimetic peptides, have been shown to enhance epidermal wound healing. However, patients with oculodentodigital dysplasia (ODDD) express mutant Cx43 that leads to a chronic reduction in gap junctional intercellular communication. To determine whether mutant Cx43 in keratinocytes would impact upon the wound healing process, we localized Cx43 in human and mouse skin tissue expressing mutant Cx43 and assessed the ability of primary keratinocytes derived from a mouse model of ODDD to proliferate, migrate and differentiate. In the epidermis from an ODDD patient and in the epidermis of mice expressing the G60S mutant or in keratinocytes obtained from mutant mice, Cx43 was frequently found within intracellular compartments and rarely localized to punctate sites of cell-cell apposition. Primary keratinocytes derived from G60S mutant mice proliferated faster but migrated similarly to keratinocytes derived from wild-type control mice. Keratinocytes derived from mutant mice expressed abundant Cx43 and higher levels of involucrin and loricrin under low calcium conditions. However, after calcium-induced differentiation, similar levels of Cx43, involucrin and loricrin were observed. Thus, we conclude that during wound healing, mutant Cx43 may enhance keratinocyte proliferation and promote early differentiation of keratinocytes.

Topics: Abnormalities, Multiple; Animals; Biopsy; Calcium; Cell Differentiation; Cell Movement; Cell Proliferation; Cells, Cultured; Connexin 43; Dental Enamel Hypoplasia; Disease Models, Animal; Face; Facial Asymmetry; Humans; Intercellular Junctions; Keratinocytes; Membrane Proteins; Mice; Mice, Mutant Strains; Microphthalmos; Mutation; Protein Precursors; Skin; Syndactyly; Wound Healing

Fatty acid-binding proteins (FABPs) are postulated to serve as lipid shuttles that solubilize hydrophobic fatty acids and deliver them to appropriate intracellular sites. Epidermal FABP (E-FABP/FABP5) is predominantly expressed in keratinocytes and is overexpressed in the actively proliferating tissue characteristic of psoriasis and wound healing. In this study, we found decreased expression of the differentiation-specific proteins keratin 1, involucrin, and loricrin in E-FABP(-/-) keratinocytes relative to E-FABP(+/+) keratinocytes. We also determined that incorporation of linoleic acid was significantly reduced in E-FABP(-/-) keratinocytes. Although linoleic acid did not directly affect keratinocyte differentiation, keratin 1 expression was induced by the linoleic acid derivative 13(S)-hydroxyoctadecadienoic acid (13(S)-HODE), and this induction was concomitant with increased NF-κB activity. In E-FABP(-/-) keratinocytes, the expression of 13(S)-HODE and the subsequent induction of NF-κB activity was lower than in wild-type keratinocytes. The reduction of linoleic acid in E-FABP(-/-) keratinocytes led to decreased cellular 13(S)-HODE content, resulting in decreased keratin 1 expression through downregulation of NF-κB activity. The regulation of fatty acid metabolism by E-FABP during keratinocyte differentiation suggests that E-FABP may have a role in the pathogenesis of psoriasis.

Topics: Animals; Cell Differentiation; Cells, Cultured; Disease Models, Animal; Epidermis; Fatty Acid-Binding Proteins; Fatty Acids; Keratin-1; Keratinocytes; Linoleic Acid; Linoleic Acids; Membrane Proteins; Mice; Mice, Knockout; Neoplasm Proteins; NF-kappa B; Protein Precursors; Psoriasis; Signal Transduction

To evaluate the effects of desiccating stress on conjunctival goblet cell density and morphology and the expression of cornified envelope precursors by the ocular surface epithelia.. Experimental dry eye (EDE) was created in C57BL/6 mice. Real-time PCR evaluated the expression of cornified envelope (CE) precursor proteins (involucrin and small proline-rich [Sprr] -1a, -1b, -2a, -2b, -2f, and -2g proteins), the cross-linking transglutaminase 1 enzyme (Tg-1) and Muc5AC mRNA transcripts by the ocular surface epithelia. Laser scanning confocal microscopy evaluated the expression of the CE precursor proteins Tg-1 and Muc5AC in cryosections. Tg-1 activity was measured by a fluorescein cadaverine assay. Muc5AC concentration was measured by ELISA.. Levels of involucrin; Sprr-1a, -1b, -2a, -2b, -2f, and -2g; and Tg1-1 mRNA transcripts in ocular surface tissues increased in response to desiccating stress. Expression and activity of Tg in the conjunctiva markedly increased after EDE. Desiccating stress caused progressive loss of mucin-filled goblet cells. The apical portion of the remaining conjunctival goblet cells became entrapped by adjacent stratified apical epithelia expressing increased levels of cornified envelope precursors.. Exposure to desiccating stress stimulates ocular surface epithelia to produce cornified envelope precursors and the tissue transglutaminase enzyme that cross-links them. This effect is accompanied by loss of mucin-filled goblet cells and entrapment of mucin contents in the remaining ones by cornifying cells that block the egress of mucin contents to the ocular surface. This mechanism may contribute to the conjunctival mucin deficiency that develops in dry eye.

Topics: Animals; Cell Count; Conjunctiva; Cornified Envelope Proline-Rich Proteins; Desiccation; Disease Models, Animal; Dry Eye Syndromes; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation; Goblet Cells; Male; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Mucin 5AC; Peptide Fragments; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stress, Psychological

Elongase of very long chain fatty acids-4 (ELOVL4) is the only mammalian enzyme known to synthesize C28-C36 fatty acids. In humans, ELOVL4 mutations cause Stargardt disease-3 (STGD3), a juvenile dominant macular degeneration. Heterozygous Stgd3 mice that carry a pathogenic mutation in the mouse Elovl4 gene demonstrate reduced levels of retinal C28-C36 acyl phosphatidylcholines (PC) and epidermal C28-C36 acylceramides. Homozygous Stgd3 mice die shortly after birth with signs of disrupted skin barrier function. In this study, we report generation of transgenic (Tg) mice with targeted Elovl4 expression driven by an epidermal-specific involucrin promoter. In homozygous Stgd3 mice, this transgene reinstates both epidermal Elovl4 expression and synthesis of two missing epidermal lipid groups: C28-C36 acylceramides and (O-linoleoyl)-omega-hydroxy C28-C36 fatty acids. Transgene expression also restores skin barrier function and rescues the neonatal lethality of homozygous Stgd3 mice. These studies establish the critical requirement for epidermal C28-C36 fatty acid synthesis for animal viability. In addition to the skin, Elovl4 is also expressed in other tissues, including the retina, brain, and testes. Thus, these mice will facilitate future studies to define the roles of C28-C36 fatty acids in the Elovl4-expressing tissues.

Topics: Animals; Animals, Newborn; Ceramides; Chromosome Disorders; Chromosomes, Human, Pair 6; Disease Models, Animal; Epidermis; Eye Proteins; Fatty Acids; Founder Effect; Heterozygote; Homozygote; Humans; Macular Degeneration; Membrane Proteins; Mice; Mice, Transgenic; Mutation; Permeability; Phosphatidylcholines; Plasmids; Promoter Regions, Genetic; Protein Precursors; Retina; Tolonium Chloride; Transfection; Transgenes

In a multi-project research line, we are currently testing whether a morphologically and functionally near normal epidermis can be cultured from human sweat gland (SG) cells and be used as a skin substitute. The present study focuses on the stratum corneum of the epidermis that assumes a vital barrier function for the skin. The main process in the formation of the cornified cell envelope in human epidermis, i.e. crosslinking of proteins and lipids, is catalyzed by several transglutaminases (TG). Therefore, we compared the expression patterns of various TG and their substrates in SG-derived versus keratinocyte-derived epidermal substitutes.. Sweat gland cells, keratinocytes, and fibroblasts were isolated from human skin samples and cultivated separately to generate epidermal substitutes. These were transplanted onto the back of athymic rats. After 2 weeks, the transplants were excised and analyzed histologically as well as by indirect immunofluorescence. We looked at the expression of TG1, 3, 5, and their substrates involucrin and loricrin (=markers of epidermal differentiation) in SG-derived and keratinocyte-derived skin substitutes as well as in normal skin.. The SG cell-derived epidermis was near normal anatomically, formed a cornified cell envelope and demonstrated TG1, 3, and 5 as well as involucrin and loricrin expression patterns similar to those found in keratinocyte-derived epidermis and normal control skin.. These findings support the thesis that SG cells have the potential to form a near normal stratified epidermal analog that might be used as a skin substitute. The expression of TG1 and 3, not normally expressed in human SG, suggests the presence of re-programmed SG cells and/or stem cells capable of both de novo generating and maintaining an epidermis.

Topics: Adolescent; Animals; Biomarkers; Cell Differentiation; Cells, Cultured; Child; Child, Preschool; Disease Models, Animal; Epidermal Cells; Epidermis; Female; Fluoroimmunoassay; Humans; Infant; Keratinocytes; Membrane Proteins; Mice; Protein Precursors; Rats; Rats, Nude; Skin, Artificial; Sweat Glands; Transglutaminases; Wound Healing; Wounds and Injuries

To investigate the protective effects of c-Jun N-terminal kinase (JNK)-1 and -2 gene knockout (KO) on the corneal epithelial response to desiccating stress.. The C57BL/6, JNK1KO, and JNK2KO mice were subjected to desiccating stress (DS) for 5 days. The effects of DS on the corneal epithelium were evaluated by measuring corneal smoothness and permeability. Expression of matrix metalloproteinases (MMP)-1, MMP-9, and cornified envelope protein precursors (small proline-rich protein [SPRR]-1a, SPRR-2a, and involucrin) in the corneal epithelia was evaluated by immunostaining and real-time polymerase chain reaction. Collagenase and gelatinase activity in corneal sections as measured with in situ fluorescent assays.. The JNK2KO mice had smoother corneal surfaces and less corneal barrier disruption in response to DS than JNK1KO mice and C57BL/6 wild-type control mice. The DS increased levels of MMP-1, MMP-9, SPRR-1a, SPRR-2a, involucrin immunoreactivity, and mRNA transcripts in the corneal epithelium of JNK1KO and C57BL/6 mice, but not in JNK2KO mice. Knockout of JNK2 prevented DS-induced increase in gelatinase and collagenase activity in the cornea.. The JNK2 protein appears to have an essential role in desiccation-induced corneal epithelial disease by stimulating production of MMP-1, MMP-9, and cornified envelope precursors. Clinical Relevance The JNK2 protein could be a novel therapeutic target in dry eye disease.

Topics: Animals; Corneal Diseases; Cornified Envelope Proline-Rich Proteins; Desiccation; Disease Models, Animal; Dry Eye Syndromes; Epithelium, Corneal; Fluorescent Dyes; Fluorophotometry; Matrix Metalloproteinase 1; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinase 9; Organic Chemicals; Permeability; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stress, Physiological

To evaluate the mechanism of apical corneal epithelial barrier disruption in response to experimental ocular surface desiccation and the effects of two anti-inflammatory agents (methylprednisolone and doxycycline) on this process.. Experimental dry eye (EDE) was created in C57BL/6 mice, without or with topical therapy, 1% methylprednisolone, 0.025% doxycycline, or physiologic saline solution (PSS) four times per day. Corneal smoothness and Oregon green dextran (OGD) permeability were assessed. Desquamation of and cornified envelope protein (involucrin and small proline-rich protein [SPRR]-2) expression by the corneal epithelium was evaluated by laser scanning confocal microscopy. Levels of cornified envelope proteins mRNA were measured by real-time PCR.. Corneal OGD permeability, surface irregularity, and the number of desquamating apical corneal epithelia significantly increased in EDE. Desiccating stress significantly increased expression of involucrin and SPRR-2 in the corneal epithelia. Treatment of EDE with methylprednisolone or doxycycline reduced corneal permeability to OGD, improved corneal smoothness, and decreased involucrin and SPRR-2 immunoreactivity compared with EDE+PSS.. Disruption of apical corneal epithelial barrier function in dry eye is accompanied by increased apical desquamation and increased expression of cornified envelope proteins. Topical treatment of EDE with the anti-inflammatory agents methylprednisolone or doxycycline preserves apical corneal barrier function.

Topics: Animals; Anti-Inflammatory Agents; Cornified Envelope Proline-Rich Proteins; Disease Models, Animal; Doxycycline; Dry Eye Syndromes; Epithelium, Corneal; Fluorescent Antibody Technique, Indirect; Membrane Proteins; Methylprednisolone; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Occludin; Organic Chemicals; Permeability; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transglutaminases

Phenylketonuria (PKU) is a metabolic disease causing increased levels of phenylalanine in blood and body fluids. Circulating phenylalanine is normally cleared by phenylalanine hydroxylase (PAH) expressed in the liver. The aim of this study is to exploit the skin as a 'metabolic sink' removing phenylalanine from the blood. We have previously showed that the overexpression of PAH and GTP cyclohydrolase I (GTP-CH), the rate-limiting enzyme in the synthesis of the cofactor for PAH, leads to high levels of phenylalanine clearance in primary human keratinocytes. In this study, we have investigated the 'metabolic sink' strategy in an in vivo model by developing three lines of transgenic mice expressing PAH and GTP-CH in various layers of the skin. The promoters used were keratin 14 (K14), involucrin (INV) and a truncated variant of Keratin 1 (K1). The mice were crossbred to a mouse model of human PKU, the PAH(enu2) mouse, in order to obtain mice that do not express PAH in the liver and the kidney. Transgenic mice containing the INV and K14 promoters expressed PAH and GTP-CH in the epidermis. However, the K1 promoter did not lead to detectable gene expression. Analysis of the mice showed that no phenotypic effect was observed in mice expressing PAH and GTP-CH from the INV promoter. However, low level of phenylalanine clearance was observed in mice expressing PAH and GTP-CH from the K14 promoter, suggesting that the skin can be genetically engineered to function as a 'metabolic sink'.

Topics: Animals; Base Sequence; Disease Models, Animal; DNA, Complementary; Gene Expression; Genetic Engineering; Genetic Therapy; GTP Cyclohydrolase; Humans; Keratin-14; Keratins; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Mice, Transgenic; Phenotype; Phenylalanine; Phenylalanine Hydroxylase; Phenylketonurias; Promoter Regions, Genetic; Protein Precursors; Skin

To determine the role played by keratinocyte transglutaminase (TG1, TG(K)) in the abnormal keratinization of the cornea.. Vitamin A-deficient rats were produced as a model of severe dry eyes, and the expression of the mRNA and the enzyme activity of TG1 were examined in the corneas. The envelope proteins and keratins of cornified cells were also examined immunohistochemically.. The expression and enzyme activity of TG1 mRNA on the ocular surface were significantly upregulated as the vitamin A deficiency developed. As the TG1 expression was upregulated, involucrin, loricrin, and keratin 10 began to be expressed on the epithelial cells of the cornea.. Upregulation of TG1 expression followed by the appearance of the envelope proteins and keratin10 in cornified cells indicated that TG1 is involved in the abnormal keratinization of the cornea.

Topics: Animals; Blotting, Northern; Cornea; Disease Models, Animal; Dry Eye Syndromes; Epithelial Cells; Gene Expression Regulation, Enzymologic; Immunoenzyme Techniques; Keratin-10; Keratins; Membrane Proteins; Protein Precursors; Rats; Rats, Sprague-Dawley; Retinaldehyde; RNA, Messenger; Transglutaminases; Up-Regulation; Vitamin A; Vitamin A Deficiency

Considerable indirect evidence suggests that T lymphocytes have a role in the pathogenesis of psoriasis. The goal of this study was to directly test the ability of T cells to maintain psoriasis pathology. Psoriatic skin was transplanted onto SCID mice, which were then injected with autologous T cells. T cells were cultured from either psoriatic skin lesions or peripheral blood and injected intradermally or intravenously. Control SCID mice transplanted with psoriasis grafts were not injected with T cells. After 10 wk, control psoriatic skin grafts not injected with T cells lost many of the features of psoriasis. Injection of peripheral blood T cells was not able to maintain these psoriatic features. In contrast, the injection of T cells derived from psoriatic skin was able to maintain the psoriatic phenotype. Psoriatic features that were maintained included epidermal thickness and labeling index and expression of HLA-DR, involucrin, and ICAM-1, as well as loss of expression of filaggrin. Injection of skin infiltrating T cells into skin of normal donors on SCID mice did not induce changes of psoriasis. The ability of T cells from lesional skin, but not peripheral blood, to maintain psoriasis suggests that psoriasis is mediated by an autoantigen reactive T cell, which is present at a higher frequency in the psoriatic lesion.

Topics: Adoptive Transfer; Animals; Cell Transplantation; Disease Models, Animal; Filaggrin Proteins; HLA-DR Antigens; Humans; Intercellular Adhesion Molecule-1; Keratinocytes; Mice; Phenotype; Protein Precursors; Psoriasis; Skin Transplantation; Spleen; T-Lymphocytes; Transplantation, Heterologous

Acne vulgaris is the result of multifactorial disorders of the pilosebaceous duct. The initial lesion is believed to be hyper-keratinization of the infundibulum. The Rhino mouse has been used as an experimental acne model system for screening anti-keratinizing and comedolytic agents. Using this system we show that trypsin could induce desquamation and utriculi-epidermal differentiation in the absence of irritation. Following five daily trypsin treatments, the biomechanical properties of the mouse skin improved, as demonstrated by cutometer measurements and increased elastin expression. Extensive programmed cell death and apoptosis are demonstrated in the utriculi epithelium of the untreated animals. This cell death is eliminated by the trypsin treatment. We speculate that co-administration of trypsin might increase the therapeutic value of topical acne treatments and improve skin elasticity while reducing irritating effects.

Topics: Acne Vulgaris; Animals; Apoptosis; Cricetinae; Disease Models, Animal; Gene Expression; Glyceraldehyde-3-Phosphate Dehydrogenases; Hair Follicle; Keratolytic Agents; Male; Mesocricetus; Mice; Mice, Hairless; Polymerase Chain Reaction; Protein Precursors; RNA, Messenger; Skin; Skin Aging; Trypsin

Human papillomaviruses (HPVs) are important human pathogens associated with a range of epithelial neoplasia. The rising incidence of HPV infection and association of HPV with malignancy has led to increased interest in appropriate management of these infections. Development of new therapies for viral warts has been frustrated by the lack of availability of models permissive for viral replication. Here we describe the development of HPV-severe combined immunodeficient mouse model which reproduces mature HPV-infected epithelia. Grafting of anogenital and laryngeal papillomas harbouring either HPV-6 or HPV-11 resulted in the formation of a differentiated neo-epithelium exhibiting the hallmark features of HPV infection including basal hyperplasia, acanthosis and koilocytosis. The reformed warty epithelium contained amplified HPV DNA and expressed capsid protein in the differentiated layers. A striking feature is the production of macroscopic papillomata in an anatomically relevant and accessible site, providing a system of particular relevance for the temporal evaluation of developing lesions and selection of antiviral agents.

Topics: Animals; Capsid; Condylomata Acuminata; Disease Models, Animal; DNA Replication; Epithelium; Gene Expression; Humans; Keratins; Laryngeal Neoplasms; Mice; Mice, SCID; Neoplasm Transplantation; Papilloma; Papillomaviridae; Papillomavirus Infections; Proliferating Cell Nuclear Antigen; Protein Precursors; Skin; Skin Transplantation; Transplantation, Heterologous; Tumor Virus Infections; Virus Replication

Involucrin is a precursor of the keratinocyte cornified envelope that is specifically expressed in the suprabasal layers of the epidermis and other stratifying squamous epithelia. To study involucrin gene expression and the function of involucrin, we expressed a 6 kb DNA fragment of the human involucrin gene, containing approximately 2.5 kb of upstream sequence and 0.5 kb of downstream sequence, in transgenic mice. The transgene produces a 68 kDa protein that is detected by a human involucrin-specific antibody, and is expressed in a tissue-specific and differentiation-appropriate manner (i.e., expression is confined to the suprabasal layers of the epidermis, extocervix, trachea, esophagus and conjunctiva). Soluble involucrin levels are two to four times higher in transgenic epidermal keratinocytes compared to human foreskin keratinocytes. Newborn heterozygous animals have a normal birth weight and a normal appearing epidermis and hair growth begins at 4 to 5 days of age (i.e., the same time as hair growth in non-transgenic animals). In a subpopulation of the newborn homozygous animals birth weight is reduced, the epidermis is scaly and hair growth begins late, at around 9 to 10 days of age. In addition, the hair tends to stand erect on both heterozygous and homozygous adult animals giving the appearance of diffuse alopecia. Immunofluorescent and electron microscopy localize involucrin in the hair follicle and cornified envelope, respectively. These results suggest that overexpression of involucrin may cause abnormalities in hair follicle structure/function and cornified envelope structure. These animals provide a new model for the study of cornified envelope structure and function.

Topics: Alopecia; Amino Acid Sequence; Animals; Animals, Newborn; Cell Differentiation; Disease Models, Animal; DNA, Complementary; Epidermis; Female; Gene Expression Regulation; Genotype; Hair; Humans; Keratinocytes; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Sequence Data; Organ Specificity; Phenotype; Protein Precursors; Recombinant Proteins