bromochloroacetic-acid and Corneal-Opacity

To identify the stem-cell property of the ex vivo expansion of limbal stem cells (LSCs) on amniotic membrane (AM) in culture system and after clinical transplantation.. Four key factors have to be performed in the defined culture system: (1) the label-retaining cells have to be identified; (2) the cells can be serially expanded and passaged in vitro; (3) the expanded cells can be labeled by tissue-specific keratin or markers, and (3) their stem cells cannot be labeled by those keratin or markers.. The ex vivo-expanded LSCs on AM were positive for p63 and ABCG2 and BrdU label-retaining studies on flat mount preparation. When the ex vivo-expanded LSCs with AM were transplanted into a subcutaneous layer of nude mice, they formed multiple layers of cells. Only the basal layer of cells was positive for p63 and BrdU. The cells over the suprabasal layers were positive for K12/K3. The pathologic studies of corneal specimen of successful LSC transplantation after penetrating keratoplasty demonstrated that P63-positive cells were noticed all over the basal layer of central cornea and AM could be identified at 10 months after LSC transplantation.. These results indicate that the AM provided the niche function for cultured LSCs and maintained the limbal-like environment for the transplanted area of cornea. The survival of cases depends on the severity of the disease entity, culture technique, and maintenance of the niche environment for LSCs in the culture and after clinical transplantation.

Topics: Amnion; Animals; Biomarkers; Cell Culture Techniques; Cell Differentiation; Cell Division; Cells, Cultured; Cornea; Corneal Opacity; Humans; Keratins; Keratoplasty, Penetrating; Mice; Mice, Nude; Postoperative Period; Stem Cell Transplantation; Stem Cells

To assess whether Pax6 functions directly in the cornea, a corneal-preferred promoter was used to overexpress Pax6 specifically in the cornea.. Transgenic mice harboring a construct containing mouse Pax6 coding sequences fused downstream of the aldehyde dehydrogenase 3a1 (Aldh3a1) promoter were generated (Pax6 Tg). Pax6 expression was analyzed by Western blot and immunohistochemistry. Eye sections were stained with hematoxylin and eosin, Schiff reagent, and fluorescein, to assess morphologic changes, the presence of goblet cells, and barrier integrity, respectively. Gene expression changes in mildly affected Pax6 Tg corneas were compared to age-matched, wild-type (WT) corneas by microarray analysis and quantitative PCR. Promoter regulation of several differentially expressed genes was examined by monitoring luciferase activity of reporter constructs after cotransfection with Pax6 in COS7 cells.. Corneal overexpression of Pax6 produces an abnormal cornea with altered epithelial cell morphology, neovascularization, immune cell invasion, and a compromised barrier; the lens appeared normal. Major changes in expression of genes involved in immune function, vascularization, and epithelial differentiation occurred in corneas from Pax6 Tg versus WT mice. The keratin (K) profile was dramatically altered in the Pax6 Tg corneas, as were several components of the Wnt signaling pathway. In severely affected Pax6 Tg corneas, K12 was reduced, and Pax6 was redistributed into the cytoplasm. Promoters from the chitinase 3-like 3, Wnt inhibitory factor 1, and fms-related tyrosine kinase 1/soluble VEGF receptor genes were upregulated five-, seven-, and threefold, respectively, by Pax6 in transfected COS7 cells.. Pax6 functions directly to maintain normal, corneal epithelial cells.

Topics: Adaptor Proteins, Signal Transducing; Animals; Blotting, Western; Cell Differentiation; Chitinases; Chlorocebus aethiops; Cornea; Corneal Opacity; COS Cells; Epithelial Cells; Epithelium, Corneal; Extracellular Matrix Proteins; Eye Proteins; Gene Expression; Homeodomain Proteins; Immunohistochemistry; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Keratin-12; Keratins; Lens, Crystalline; Mice; Mice, Transgenic; Neovascularization, Pathologic; Paired Box Transcription Factors; PAX6 Transcription Factor; Phenotype; Promoter Regions, Genetic; Repressor Proteins; RNA, Messenger; Signal Transduction; Tissue Distribution; Up-Regulation; Vascular Endothelial Growth Factor Receptor-1; Wnt Proteins

The Pbx TALE (three-amino-acid loop extension) homeodomain proteins interact with class 1 Hox proteins, which are master regulators of cell fate decisions. This study was performed to elucidate the role of the Pbx1 TALE protein in the corneal epithelium of mice.. Pbx1(f/f) mice were crossed with mice containing Cre recombinase under the control of the K14 promoter. Subsequently, the eyes of these mice were dissected and prepared for histologic or molecular analysis.. Tissue-specific deletion of Pbx1 in the corneal epithelium of mice resulted in corneal dystrophy and clouding that was apparent in newborns and progressively worsened with age. Thickening of the cornea epithelium was accompanied by stromal infiltration with atypical basal cells, severe disorganization of stromal collagen matrix, and loss of corneal barrier function. High epithelial cell turnover was associated with perturbed expression of developmental regulators and aberrant differentiation, suggesting an important function for Pbx1 in determining corneal identity.. These studies establish an essential role of the Pbx1 proto-oncogene in corneal morphogenesis.

Topics: Animals; Animals, Newborn; Apoptosis; Bromodeoxyuridine; Cell Differentiation; Cell Division; Cornea; Corneal Opacity; Corneal Stroma; Epithelium, Corneal; Eye Proteins; Female; Genotype; Homeodomain Proteins; Immunoenzyme Techniques; In Situ Nick-End Labeling; Integrases; Keratins; Male; Mice; Mice, Knockout; Mice, Transgenic; Morphogenesis; Paired Box Transcription Factors; PAX6 Transcription Factor; Pre-B-Cell Leukemia Transcription Factor 1; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors

The arrangement of collagen fibrils and glycosaminoglycans (GAGs) in substantia propria are important for maintaining transparency of the cornea. Interferences in collagen fibrils and GAG production could be adversative to corneal integrity. In this study, six dogs consisting of four Beagles with normal cornea (normal), one Beagles with opaque cornea (sample No. 1) and one Shih Tzu with neovascularization opaque cornea (sample No.2) were used. All samples were observed morphologically by light and electron microscopes to obtain diameter and distribution of collagen fibrils in substantia propria and were performed biochemically to investigate into GAGs and collagen types. The average diameter of collagen fibrils in the intact cornea of normal, sample No.1 and No.2 was 33.2, 35.0 and 25.0 nm, respectively. The percentage of matrix per unit area was 67% in normal, 87% in sample No.1 and 28.3% in sample No.2. The type III collagen ratio was 25.3% in normal, 21.3% in sample No.1 and 35.8% in sample No.2. The relative amount of heparan sulfate, chondroitin sulfate, dermatan sulfate and keratin sulfate was 1.5, 9.7, 51.1 and 37.7% in normal, 3.3, 26.0, 45.7 and 23.7% in sample No.1 and 1.2, 18.0, 16.6 and 54.1% in sample No.2. Hyaluronic acid was found only in sample No.1 with a relative amount of 1.3%. Since there was some relationship between collagen formation and GAGs composition, it might be speculated that disturbance in arrangement of collagen fibrils and GAG metabolism especially in substantia propria would bring up opacity of the cornea.

Topics: Animals; Chondroitin Sulfates; Collagen Type III; Cornea; Corneal Opacity; Dermatan Sulfate; Dogs; Extracellular Matrix; Glycosaminoglycans; Heparitin Sulfate; Hyaluronic Acid; Keratins

To report an assessment of the two-step surgical combination of cultivated autologous oral mucosal epithelial transplantation (COMET) and penetrating keratoplasty (PKP) used to treat patients with severe limbal deficiency disorders, and to investigate the keratin expression patterns of transplanted surviving oral mucosal epithelium.. Observational case series.. Two patients with Stevens-Johnson syndrome and chemical eye injury were treated by COMET followed, approximately six months later, by a PKP triple procedure. In the course of a mean follow-up period of 22.5 months, their clinical outcomes and the efficacy of this two-step surgical procedure were assessed. In addition, the keratin expression in corneal buttons excised during PKP were immunohistochemically examined to characterize the oral mucosal epithelium that survived ectopically on the cornea. In vivo laser confocal microscopy was used to investigate the structure of the epithelium on the corneal grafts.. The ocular surfaces were successfully reconstructed with cultivated autologous oral mucosal epithelial sheets and PKP. No clinical complications, such as persistent epithelial defects, rejections, or recurrence of cicatrization, were encountered. Postoperative best-corrected visual acuity was 20/125 in one patient and 20/100 in the other. The surviving oral mucosal epithelium, distinguished by its fluorescence pattern, consisted of an irregular, nonkeratinized, stratified epithelium without goblet cells. Immunohistochemical study demonstrated that K3, but not K12, was expressed in the transplanted cultivated oral mucosal epithelium that was similar to oral mucosal tissue. In vivo, the epithelial structure and cell density in the basal cell layer of the corneal grafts were similar to normal cornea.. This study presents a two-step surgical approach to treat severely scarred ocular surfaces by means of a combination of COMET and PKP. Clinical outcomes suggest that this treatment may be beneficial for the maintenance of the reconstructed ocular surface by providing oral mucosal epithelium around the corneal graft.

Topics: Aged; Cataract Extraction; Cell Count; Cell Transplantation; Cells, Cultured; Corneal Diseases; Corneal Opacity; Epithelial Cells; Epithelium, Corneal; Fluorophotometry; Humans; Keratins; Keratoplasty, Penetrating; Lens Implantation, Intraocular; Male; Microscopy, Confocal; Mouth Mucosa; Stevens-Johnson Syndrome; Transplantation, Autologous; Visual Acuity

There is an ongoing discussion whether Lisch corneal dystrophy (band-shaped and whorled microcystic dystrophy of the corneal epithelium) represents a disorder that is different from Meesmann corneal dystrophy. The purpose of this study was to evaluate at the molecular level if Lisch and Meesmann corneal dystrophies are genetically distinct.. We examined at the slit lamp a total of 48 members of a family with an aggregation of Lisch corneal dystrophy. Genomic DNA was extracted from leukocytes of the peripheral blood of seven affected and six unaffected members of this family. Mutational hotspots in the cornea-specific keratin genes K3 and K12 were scanned for mutations by single-strand conformation analysis. To test for linkage to the keratin K3 or K12 loci or for X-chromosomal inheritance, six (K3) and four (K12) microsatellite markers each flanking the keratin loci as well as 22 microsatellite markers covering the X-chromosome were typed. Linkage was analyzed using the MLINK and FASTMAP procedures.. A total of 19 trait carriers were identified in six generations of the family. No hereditary transmission from father to son was observed. Linkage was excluded for the keratin K3 and K12 genes. Furthermore, single-strand conformation analysis detected no mutations in these genes. Multipoint linkage analysis revealed linkage with a maximum likelihood of the odds (LOD) score of 2.93 at Xp22.3. Linkage was excluded for Xp22.2 to Xqter.. Lisch corneal dystrophy is genetically different from Meesmann corneal dystrophy. Evidence was found for linkage of the gene for Lisch corneal dystrophy to Xp22.3.

Topics: Adolescent; Adult; Aged; Child; Chromosome Mapping; Corneal Dystrophies, Hereditary; Corneal Opacity; DNA Mutational Analysis; DNA Primers; Female; Genetic Linkage; Humans; Keratins; Lod Score; Male; Microsatellite Repeats; Middle Aged; Pedigree; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Sex Chromosome Aberrations; X Chromosome

To describe a case of posterior polymorphous dystrophy associated with posterior amyloid degeneration of the cornea confirmed histopathologically and immunohistochemically.. An 80-year-old woman with corneal opacities required penetrating keratoplasty. The keratectomy specimen was evaluated by light microscopy and immunohistochemistry.. Microscopic examination of the keratectomy specimen showed scattered fusiform deposits located in the deep corneal stroma. Congo red stains of the fusiform deposits confirmed the diagnosis of amyloidosis. Immunohistochemical stains for cytokeratin (AE1/AE3) showed that the endothelial cells were immunoreactive, confirming the diagnosis of posterior polymorphous dystrophy.. To our knowledge, the association between posterior polymorphous dystrophy and posterior amyloid degeneration has not been reported previously.

Topics: Aged; Aged, 80 and over; Amyloidosis; Corneal Dystrophies, Hereditary; Corneal Opacity; Corneal Stroma; Endothelium, Corneal; Epithelium, Corneal; Female; Humans; Immunoenzyme Techniques; Keratins; Keratoplasty, Penetrating

Bsk (bare skin) is an autosomal dominant mutation linked to the Krt 1 (type 1 keratin) locus of mouse chromosome 11. The adult Bsk mouse manifests hair loss and corneal opacity. To identify and characterize the keratin genes involved in this mutation, we examined the hypothesis proposing that the Bsk mutation might involve a recombination event between cornea-specific (K12) and hair-specific (mHa 1, 2, 3 and 4) type I keratin genes.. The Bsk phenotype was examined by histochemical analysis, using light and electron microscopy. RFLP was used for their genotyping, and possible keratin gene expression was examined by immunohistochemical staining, Western analysis, RT-PCR and Northern hybridization.. Northern hybridization, RT-PCR and Western blot analysis revealed that mHa 1, 2, 3 and 4 keratins are expressed in the skin, but not in cornea, whereas the expression of K12 is limited to the corneas of the Bsk mice. These data ruled out the hypothesis that Bsk phenotype results from a recombination event between K12 and mHa 1, 2, 3 and 4. Ultrastructural and biochemical analyses also indicated that Bsk does not involve negative dominant mutations of keratin 12, mHa 1, 2, 3 and 4, epidermal-specific keratin 10, or basal cell-specific keratin 14. Expression of an acidic 50 kD keratin, recognized by monoclonal antibody AK 2, was up-regulated in the injured corneas of normal mice as well as Bsk corneas.. The gene linked to the Bsk mutation remains unknown. The pathological changes in the skin and corneas may be secondary to the loss of protecting hairs and lashes by an unknown mechanism.

Topics: Animals; Blotting, Northern; Blotting, Western; Cornea; Corneal Opacity; DNA Primers; DNA, Complementary; Hair Diseases; Keratins; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mutation; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Recombination, Genetic; Skin

A Japanese girl, 2 years, 8 months of age, with palmoplantar keratosis and dendritic corneal opacities, showed increased tyrosine levels in the plasma, urine, and cerebrospinal fluid. The mental and physical growth was not retarded. The hepatorenal functions were within normal limits. Electron microscopically, the epidermal keratinocytes showed increased tonofibrils and no structures suggestive of tyrosine crystals. Cytosol and mitochondrial tyrosine aminotransferase (TAT) activities of the liver were greatly decreased, while p-hydroxyphenyl pyruvate oxidase (p-HPPO) activity was not decreased. The plasma tyrosine levels were controlled for 3 years with low phenylalanine-tyrosine diet.

Topics: Amino Acid Metabolism, Inborn Errors; Child, Preschool; Corneal Opacity; Female; Humans; Keratins; Keratoderma, Palmoplantar; Phenylacetates; Phenylalanine; Phenylpropionates; Phenylpyruvic Acids; Syndrome; Tyrosine; Tyrosine Transaminase

Topics: Aged; Climate; Cold Climate; Cornea; Corneal Dystrophies, Hereditary; Corneal Opacity; Environmental Exposure; Epithelium; Female; Histocytochemistry; Humans; Hyalin; Keratins; Male; Microscopy, Electron; Middle Aged; Newfoundland and Labrador; Staining and Labeling; Ultraviolet Rays

Morphologic observations on a peculiar type of corneal reaction with a predisposition for the superficial stroma of the interpalpebral portion of the cornea are reviewed. Histochemical evidence is provided which indicates that the corneal concretions, though not homogenous, are proteinaceous in nature and contain amino acids not normally detectable in the cornea. The corneal concretions were associated with conjunctival elastosis (pingueculae) in all 22 instances in which the eyes were sectioned in the horizontal plane. Identical concretions were identified within these associated pingueculae, as well as in a large percentage of other pingueculae and cutaneous lesions with actinic elastosis. The findings suggest that the abnormal material arises in the pericorneal conjunctival connective tissue from whence it diffuses into, and deposits in, the superficial corneal stroma. The data also raise the possibility that the concretions may be derived, at least in part, from altered elastic tissue. Morphologic and epidemiologic observations on the condition taken together strongly suggest that this unique reaction is a sequel to the cumulative effect of chronic actinic irradiation. Further observations on this keratopathy are needed to establish whether this unique response can be provoked by other noxious stimuli.

Topics: Adult; Aged; Biopsy; Chronic Disease; Conjunctiva; Cornea; Corneal Opacity; Disulfides; Elastic Tissue; Eye Diseases; Female; Histocytochemistry; Humans; Keratins; Male; Microscopy, Electron; Middle Aged; Radiation Injuries

Topics: Corneal Opacity; Humans; Keratins

Topics: Adult; Aged; Amino Acids; Arginine; Cornea; Corneal Dystrophies, Hereditary; Corneal Injuries; Corneal Opacity; Epithelium; Histocytochemistry; Humans; Hyalin; Keratins; Male; Middle Aged; Sulfur