The aim of this study was to find out whether protease inhibitor 9 (PI-9) and granzyme B (GrB) molecules that contribute to immune response and the immunological privilege of various tissues are expressed in healthy and pathological human corneas. Using cryosections, cell imprints of control corneoscleral discs, we showed that PI-9 was expressed particularly in the endothelium, the superficial and suprabasal epithelium of healthy corneas, limbus, and conjunctiva. GrB was localized in healthy corneal and conjunctival epithelium, while the endothelium showed weak immunostaining. The expression of PI-6 and GrB was confirmed by qRT-PCR. Increased expression levels of the PI-9 and GrB genes were determined when the corneas were cultured with proinflammatory cytokines. Fluorescent and enzymatic immunohistochemistry of pathological corneal explants (corneal melting and herpes virus keratitis) showed pronounced PI-9, GrB, human leucocyte antigen (HLA)-DR, and leukocyte-common antigen (CD45) signals localized in multicellular stromal infiltrates and inflammatory cells scattered in the corneal stroma. We conclude that increased expression of the PI-9 and GrB proteins under pathological conditions and their upregulation in an inflammatory environment indicate their participation in immune response of the cornea during the inflammatory process.
- Publication type
- Journal Article MeSH
PURPOSE: To assess whether injured porcine endothelium of small and large corneoscleral disc differ in its reparative/regenerative capacity under various conditions of organ culture storage. MATERIAL AND METHODS: 166 paired porcine corneas were trephined to obtain tissues with diameter 12.0 mm and 17.5 mm (with area neighboring endothelial periphery). In tested discs, central endothelium was mechanically wounded. Density of live endothelial cells (LECD), percentage of dead cells (%DC), coefficient of variation and cell hexagonality were assessed in central and paracentral endothelium following 5- or 9-day incubation in medium with 2% or 10% fetal bovine serum. The parameters were assessed also in fresh and intact cultured discs. Dead endothelial cells (EC) were visualized by trypan blue, cell borders by Alizarin Red S dye. Endothelial imprints were immunoassayed for the proliferation marker Ki-67 and the nucleolar marker fibrillarin. RESULTS: In fresh corneas, the LECD/mm2 (mean ± standard deviation) were 3998.0 ± 215.4 (central area) and 3888.2 ± 363.1 (paracentral area). Only the length of storage had significant effect on wound repair. Lesion was repaired partially after 5-day and fully after 9-day cultivation. After 9-day storage in medium with 10% serum, the mean LECD detected in small discs were 2409.4 ± 881.8 (central area) and 3949.5 ± 275.5 (paracentral area) and in large discs the mean LECD were 2555.0 ± 347.0 (central area) and 4007.5 ± 261.2 (paracentral area). Ki-67 showed cell proliferation associated with healing of EC of both large and small corneas. CONCLUSIONS: The lesions were completely repaired within 9 days of storage. Presence of the area, where stem cells appear to be located, contributes to stimulation of endothelial reparation less than serum concentration and time of culture. Both cell migration and proliferation contribute to the wound repair.
- MeSH
- Wound Healing physiology MeSH
- Disease Models, Animal MeSH
- Organ Culture Techniques MeSH
- Cell Count MeSH
- Cell Movement MeSH
- Corneal Injuries pathology therapy MeSH
- Swine MeSH
- Endothelium, Corneal injuries pathology MeSH
- Wounds, Nonpenetrating pathology therapy MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Posterior polymorphous corneal dystrophy (PPCD) is a rare autosomal dominant genetically heterogeneous disorder. Nineteen Czech PPCD pedigrees with 113 affected family members were identified, and 17 of these kindreds were genotyped for markers on chromosome 20p12.1- 20q12. Comparison of haplotypes in 81 affected members, 20 unaffected first degree relatives and 13 spouses, as well as 55 unrelated controls, supported the hypothesis of a shared ancestor in 12 families originating from one geographic location. In 38 affected individuals from nine of these pedigrees, a common haplotype was observed between D20S48 and D20S107 spanning approximately 23 Mb, demonstrating segregation of disease with the PPCD1 locus. This haplotype was not detected in 110 ethnically matched control chromosomes. Within the common founder haplotype, a core mini-haplotype was detected for D20S605, D20S182 and M189K2 in all 67 affected members from families 1-12, however alleles representing the core mini-haplotype were also detected in population matched controls. The most likely location of the responsible gene within the disease interval, and estimated mutational age, were inferred by linkage disequilibrium mapping (DMLE+2.3). The appearance of a disease-causing mutation was dated between 64-133 generations. The inferred ancestral locus carrying a PPCD1 disease-causing variant within the disease interval spans 60 Kb on 20p11.23, which contains a single known protein coding gene, ZNF133. However, direct sequence analysis of coding and untranslated exons did not reveal a potential pathogenic mutation. Microdeletion or duplication was also excluded by comparative genomic hybridization using a dense chromosome 20 specific array. Geographical origin, haplotype and statistical analysis suggest that in 14 unrelated families an as yet undiscovered mutation on 20p11.23 was inherited from a common ancestor. Prevalence of PPCD in the Czech Republic appears to be the highest worldwide and our data suggests that at least one other novel locus for PPCD also exists.
- MeSH
- Corneal Dystrophies, Hereditary epidemiology genetics pathology MeSH
- Genes, Dominant MeSH
- Founder Effect * MeSH
- Exons MeSH
- Genetic Heterogeneity MeSH
- Genetic Loci MeSH
- Haplotypes MeSH
- Humans MeSH
- Chromosomes, Human, Pair 20 * MeSH
- Chromosome Mapping MeSH
- Mutation * MeSH
- Prevalence MeSH
- Repressor Proteins genetics MeSH
- Pedigree MeSH
- Cornea metabolism pathology MeSH
- Comparative Genomic Hybridization MeSH
- Case-Control Studies MeSH
- Linkage Disequilibrium * MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Czech Republic MeSH
The aim of this study was to determine if cytokeratins (CKs) 8 and 18--typical epithelial cell markers--are constitutively expressed in adult human corneal endothelium. Cryosections, paraffin-embedded sections and corneal endothelial imprints obtained from eleven adult human corneal discs not suitable for transplantation were used. Different fixative solutions were applied before indirect immunofluorescent or enzymatic staining was performed with antibodies against CK8 (Chemicon), CK18 (Dako and Sigma) and CK8/18 (Novocastra). Semi-quantitative RT-PCR and Western blotting (mRNA or proteins were isolated from Millicell membranes) were used to determine cytokeratin mRNA and protein levels. Approximately 50% of the corneal endothelial cells were positive for CK8 (Chemicon), CK18 (Sigma) and the CK pair 8/18 (Novocastra) in the endothelium when acetone was used for fixation. Four and 52% CK18-positive cells were observed using immunofluorescent and enzymatic immunohistochemistry, respectively, when the CK18 antibody from Dako was used. No signal was detected when 4% formalin or 10% paraformaldehyde was used as a fixative, irrespective of the antibody used. CK8 and CK18 proteins and mRNAs were detected in the endothelium of all tested corneas by Western blotting or semi-quantitative RT-PCR, respectively. We detected both CK8 and CK18 in the endothelium of all specimens at both the protein and mRNA levels. These results clearly demonstrate that cells of the corneal endothelium express CKs 8 and 18 and share some features with simple epithelia.
- MeSH
- Gene Expression MeSH
- Fluorescent Antibody Technique, Indirect methods MeSH
- Keratin-18 genetics metabolism MeSH
- Keratin-8 genetics metabolism MeSH
- Middle Aged MeSH
- Humans MeSH
- RNA, Messenger genetics MeSH
- Eye Proteins genetics metabolism MeSH
- Reverse Transcriptase Polymerase Chain Reaction methods MeSH
- Endothelium, Corneal metabolism MeSH
- Aged MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Aged MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Posterior polymorphous corneal dystrophy (PPCD) is a hereditary bilateral disorder affecting primarily the endothelium and Descemet's membrane (DM). The aim of this study was to determine the changes in the presence and localization of the alpha1-alpha6 collagen IV chains and alpha1, alpha2 collagen VIII chains in Czech patients with PPCD. Twelve corneal buttons from ten PPCD patients who underwent corneal grafting, as well as eight unaffected corneas, were used. Enzymatic indirect immunohistochemistry was performed on cryosections using antibodies against the alpha1-alpha6 collagen IV chains and alpha1, alpha2 collagen VIII chains. The intensity of the signal was examined separately in the basal membrane of the epithelium (BME), stroma and DM. More than 50% of PPCD specimens exhibited positivity for alpha1 and alpha2 collagen IV chains in the BME and in the posterior stroma, while no staining was detected in these areas in control specimens. The signal for the alpha1 and alpha2 collagen IV chains was more intense in DM of PPCD corneas compared to controls and it was shifted from the stromal side (in control tissue) to the endothelial side of DM (in the patients). A less intensive signal in PPCD corneas for the alpha3 and alpha5 chains in DM and an accumulation of alpha3-alpha5 in the posterior stroma in diseased corneas were the only differences in staining for the alpha3-alpha6 collagen IV chains. The alpha1 collagen VIII chain was detected on both the endothelial and the stromal sides of DM in 90% of patients with PPCD, compared with the prevailing localization on the stromal side of DM in control corneas. A change in the localization of the alpha2 collagen VIII chain in DM from vertically striated features in control specimens to double line positivity in the DM of PPCD corneas and positive staining in the posterior collagenous layer of four patients were also detected. In three PPCD patients a fibrous pannus located under the BME, positive for alpha1-alpha3, alpha5 collagen IV chains and alpha1 collagen VIII chain, was observed. The increased expression of the alpha1, alpha2 collagen IV and alpha1 collagen VIII chains and the change in their localization in DM may contribute to the increased endothelial proliferative capacity observed in PPCD patients.
- MeSH
- Basement Membrane metabolism pathology MeSH
- Bowman Membrane metabolism pathology MeSH
- Corneal Dystrophies, Hereditary metabolism pathology MeSH
- Adult MeSH
- Immunoenzyme Techniques MeSH
- Collagen Type VIII metabolism MeSH
- Collagen Type IV metabolism MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Cornea metabolism MeSH
- Aged MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Cieľ: Cieľom tejto práce bolo zistiť výskyt jednotlivých reťazcov kolagénu IV (?1, ?2, ?3, ?4, ?5 a ?6) v kontrolných rohovkách a v rohovkách pacientov so zadnou polymorfnou dystrofiou rohovky (ZPD). Metodika: Pre experimenty bolo použitých 7 kontrolných rohoviek a 7 rohovkových terčov získaných pri keratoplastike pacientov so ZPD. Jednotlivé reťazce kolagénu IV boli detekované v kryorezoch o hrúbke 7 Km pomocou nepriamej fluorescenčnej imunohistochémie. Výsledky: V kontrolných rohovkách sa v bazálnej membráne epitelu nachádzali reťazce ?3, ?5 a ?6, v strome reťazce ?3 a ?5. V Descemetovej membráne boli prítomné všetky reťazce kolagénu IV s výnimkou reťazca ?1, ktorý bol pozitívny len u jednej kontrolnej rohovky zo siedmych. V bunkách epitelu a endotelu kontrolných rohoviek nebol kolagén IV detekovaný. U rohoviek pacientov so ZPD sa v bazálnej membráne epitelu okrem reťazcov ?3, ?5 a ?6 vyskytoval aj reťazec ?2, v strome boli vedľa reťazcov ?3 a ?5 detekované i reťazce ?1, ?2 a ?4. V Descemetovej membráne rohoviek pacientov so ZPD boli vo zvýšenej (?1, ?2, ?3), zníženej (?4, ?6), alebo nezmenenej (?5) miere oproti kontrolám prítomné všetky reťazce kolagénu IV.V bunkách epitelu a endotelu rohoviek pacientov nebol kolagén IV detekovaný. Záver: Ľudská rohovka vykazuje značnú heterogenitu vo výskyte reťazcov ? kolagénu IV, a to predovšetkým v bazálnej membráne epitelu a v Descemetovej membráne, pre ktoré je prítomnosť kolagénu IV jednou z charakteristických čŕt. U pacientov so ZPD dochádza k zmenám expresie jednotlivých reťazcov kolagénu IV na úrovni Descemetovej membrány a stromy.
The expression of all six chains of collagen IV was studied using the indirect fluorescent immunohistochemistry in seven control corneas and seven corneas obtained from patients suffering from the posterior polymorphous corneal dystrophy. Heterogeneous staining, especially in the epithelial basement membrane and Descemet's membrane, was observed in the control corneas. An increase of the staining intensity for the ?1 and ?2 chains was observed, especially in the Descemet's membrane and the corneal stroma in samples obtained from the patients compared to the control tissues.
