Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche.

BACKGROUND/AIMS: There is substantial evidence that mammalian epithelial stem cells are located within well defined niches. Although the corneoscleral limbus is acknowledged as the site of corneal epithelial stem cells no anatomical niche for such cells has yet been described. The authors undertook to re-evaluate the microanatomy of the limbus in order to identify possible sites that may represent a stem cell niche. METHODS: Systematic serial 5-7 microm sections of human corneoscleral segments obtained from cadaver donors, were examined. The sections were stained with haematoxylin and eosin or toludine blue. Sections with specific areas of interest were further examined immunohistologically for the corneal epithelial marker cytokeratin 14 and the "stem cell" marker ABCG2 transporter protein. RESULTS: Distinct anatomical extensions from the peripheral aspect of the limbal palisades were identified. These consist of a solid cord of cells extending peripherally or circumferentially. The cells stained positive for CK14 and ABCG2. CONCLUSIONS: A novel anatomical structure has been identified at the human limbus, which demonstrates characteristics of being a stem cell niche. The authors have termed this structure the limbal epithelial crypt.

Sodium hyaluronate (hyaluronic acid) promotes migration of human corneal epithelial cells in vitro.

PURPOSE: Sodium hyaluronate (hyaluronic acid) is known to promote corneal epithelial wound healing in vivo and in vitro, in animal experiments. Sodium hyaluronate is the ligand for CD44, a cell surface adhesion molecule which has been found on normal human corneal epithelial cells. The purpose of this study was to investigate the effect of sodium hyaluronate on human corneal epithelial cell migration, proliferation, and CD44 receptor expression. METHODS: Human corneal epithelial cell cultures were established from 32 donor corneoscleral rims and maintained separately in three different culture conditions: (1) culture medium only, (2) sodium hyaluronate enriched (0.6 mg/ml) medium, and (3) hydroxypropylmethylcellulose enriched (2.5 mg/ml) medium. The total area of migrating epithelial cell sheets in each case was measured by planimetry on days 4, 8, 12, and 16. Cytospin preparations of cells cultured in the different culture conditions were examined immunohistochemically for proliferation and CD44 receptor expression using antibodies directed against Ki67 and CD44 respectively. RESULTS: Cells cultured in the presence of sodium hyaluronate showed significantly increased migration at days 12 and 16 (Friedmen test: p = 0.0012, day 16; p = <0.001, day 12) compared with cells cultured in the other media. There was no difference in cell proliferation (Ki67) or CD44 expression on cells cultured in the different culture conditions. CONCLUSIONS: Sodium hyaluronate promotes migration but not proliferation or CD44 expression on human corneal epithelial cells in vitro. The beneficial effect of sodium hyaluronate in corneal wound healing is likely to be related to rapid migration of cells leading to rapid wound closure. This may be facilitated by the adhesion between CD44 on the cells and hyaluronic acid, which coats the surface of the denuded cornea.

Epithelial cell characteristics of cultured human limbal explants.

AIM: To determine the immunohistochemical characteristics of putative corneal epithelial stem cells remaining on limbal explants maintained in culture. METHODS: Human limbal explant cultures were generated from 25 residual corneoscleral donor rims following penetrating keratoplasty. Serial sections of these explants were studied using immunohistochemical techniques with a panel of antibodies, on day 0 and 1, 2, and 3 weeks. RESULTS: The number of epithelial cells expressing cytokeratin 19 and vimentin increased with duration in culture, while the number of cells expressing cytokeratin 3 decreased. Connexin 43 expression was lost by 1 week in culture. p63 was expressed by cells that had migrated around the explant and the number of p63 positive cells decreased with longer duration in culture. The explants were initially negative for Ki67, but the epithelial cells were positive at 1 week, and expression of Ki67 was progressively lost with increasing duration in culture. The initial uniform staining of the epithelium for epidermal growth factor receptor and alpha enolase remained unchanged at 3 weeks. CONCLUSIONS: There is an expansion of less differentiated (cytokeratin 3 negative and CK19/vimentin positive) epithelial cells on corneoscleral explants maintained in culture for 3 weeks. The pattern of expression of p63 noted in this study does not support the suggestion that it is a marker of limbal stem cells. The decline in p63 and Ki67 expression among the epithelial cells of the cultured explant button implies that as the epithelial sheet outgrowing from the explant button reaches confluence, the proliferative status of the cells remaining on the explant button declines. These findings are of clinical relevance as explants of limbal tissue are used in limbal stem cell transplantation. There is no information available to date on the fate of epithelial cells on such explants. This study provides some insight into this and suggests that an expansion of the stem cell pool or its progeny may occur in limbal explants.

Effect of ABO blood group mismatching on corneal epithelial cells: an in vitro study.

AIM: To determine, in vitro, the effects of blood group ABO mismatching on corneal epithelial cells. METHODS: Corneal epithelial cell cultures were established from 32 human cadaver donor eyes. Epithelial cells (100 microl of 4 x 10(2) cells per microl) were incubated for 4 hours with antibodies against blood group antigens A, B, and AB, with and without complement. Cell lysis was assayed by a chemiluminescent assay using Cytolite reagent. Live cells, remaining after incubation, were counted in a scintillation counter. The blood group of the donors was determined retrospectively, in a blinded manner. RESULTS: Retrospective tracing of donor blood groups was possible for 20 donors. In all cases the blood group corresponded with that suggested by the cell lysis assay. Significant cell lysis was observed when known A group cells were incubated with anti-A and anti-AB antibody, B group cells were incubated with anti-B and AB antibody, and AB group cells were incubated with anti-AB antibody. Lysis occurred only in the presence of complement. No lysis of O group cells was observed with any of the antibodies. In all cases, lysis was observed only with neat (serum) antibody concentrations. CONCLUSIONS: Blood group ABO mismatching results in significant lysis of corneal epithelial cells. The antibody concentration required for lysis equals that found in serum. Such levels of antibody are unlikely to be achieved in tears and/or aqueous. This may offer an explanation for the conflicting reports of the studies on the effect of blood group matching on corneal grafts. The variability in the outcome may reflect the levels of antibodies gaining access to the corneal cells and not the mismatching alone.

Clinical and pathologic findings in human keratolimbal allograft rejection.

OBJECTIVE: To characterize the clinical and pathologic features of cadaveric keratolimbal allograft (KLAL) rejection. DESIGN: The study design is descriptive. PARTICIPANTS: Four patients (five eyes) with KLAL rejection are reported. INTERVENTION: All patients were subjected to slit-lamp biomicroscopy, treatment of rejection, and ultimately required repeat KLAL surgery. In three patients (four eyes), specimens obtained at the time of repeat surgery were subjected to immunohistochemical staining against the following immune and surface human antigens: CD4, CD8, CD19, CD3, DR, CK19, CK3, and vimentin. RESULTS: Signs of allograft rejection included intense sectoral injection, diffuse or perilimbal conjunctival injection, edema, and infiltration of the KLAL grafts, leading to punctate epithelial erosions, epithelial defects, and surface keratinization. Rejected specimens revealed T-lymphocyte infiltration (CD4:CD8, 2:1) with strong HLA-DR (MHC class II) expression. The epithelium stain results were positive for cytokeratin 19 and weakly positive to absent for cytokeratin 3. The epithelial stain results were weakly positive for vimentin in only one specimen. CONCLUSIONS: KLAL rejection is a newly recognized entity. Pathologic findings of rejected specimens indicate that this is a T-cell mediated rejection phenomenon. The pattern of cytokeratin staining provided little evidence that the epithelium covering KLALs had a corneal phenotype. The scarcity of vimentin-positive epithelial cells suggests that the stem-cell/transient-cell pool was probably depleted. Early recognition of clinical rejection is important, as treatment with immunosuppressive therapy may reverse the process.

Epitope discovery using bacteriophage display: the minimum epitope of an anti-IRBP antibody.

The purpose of this study was to determine, using random peptide library (RPL) technologies, the minimal epitope requirements of the mouse monoclonal anti-interphotoreceptor-retinoid-binding protein antibody, H3B5. This previously characterized antibody is used as an example to examine whether RPL's offer a relatively easy and rapid route to obtaining detailed epitope mapping data.A pentadecamer random peptide library (RPL) displayed on the major coat protein (gene 8) of filamentous bacteriophage (F88-4-15) was used as a target for selection by the anti-IRBP monoclonal antibody, H3B5. Three rounds of library selection were performed, and 90 of the resultant RPL clones were examined for affinity to H3B5 by enzyme-linked immunosorbent assay (ELISA). DNA sequencing of ELISA positive clones provided sequence of the region encoding the random peptide. After three rounds of selection of the RPL, 76.7% of clones examined interacted with H3B5, 17.7% did not show significant binding and 6.6% bound to control antibody also. The essential elements of the peptide epitope were determined by sequence comparison of 24 clones to be the four amino-acid sequence (Aspartic or glutamic acid)-Proline-Arginine-(Leucine, Isoleucine or Valine). This motif [(D/E) PR (L/I/V)] is in agreement, but at greater resolution, than previous synthetic peptide studies where the motif AASEDPRL was identified. Other motifs were found which bound to H3B5 but did not share primary structure similarities (peptidomimetics). Selection from a RPL has rapidly defined the minimal requirements for the H3B5 epitope in fine detail. Such a process offers great potential for investigating antibody-antigen interactions and core sequences of an epitope, and enables the identification of motifs in other proteins which may be recognized by the antibody, providing information on possible cross-reactivity.

Lymphocyte subsets in conjunctival mucosa-associated-lymphoid-tissue after exposure to retinal-S-antigen.

PURPOSE: To evaluate the immune cell subsets in conjunctival mucosa-associated-lymphoid-tissue (C-MALT) following challenge with antigen. METHODS: Ten adult female Lewis rats were studied. Five rats received one drop (5 microL) of retinal S-antigen (500 microg/mL in phosphate buffered saline, PBS) instilled into the lower fornix twice daily for 10 consecutive days. Five rats received PBS only and served as controls for the experiment. Two days after the last instillation the animals were sacrificed and the orbital contents prepared for immunohistological staining. A panel of monoclonal antibodies was used: CD5, CD4, CD8, CD25, and CD45RA. The number of positive cells were counted in sections of epibulbar, forniceal, and tarsal conjunctiva. RESULTS: There was a significant increase in the number of CD8+ T lymphocytes in the conjunctiva of animals receiving retinal S-antigen when compared to control animals. CONCLUSION: Conjunctival instillation of retinal S-antigen causes an immune response in the C-MALT with a significant increase in the CD8+ T lymphocyte subset in this tissue. This response may be involved in the induction of tolerance to the encountered antigen.

The detection of autoantibodies to IgE in plasma of individuals infected with hookworm (Necator americanus) and the demonstration of a predominant IgG1 anti-IgE autoantibody response.

In this study we have demonstrated significantly elevated levels of circulating IgG autoanti-IgE antibody in hookworm infected individuals from Kebasob village on Karkar Island, Papua New Guinea. Although anti-IgE activity was demonstrable in IgG1, IgG3 and IgG4, IgG1 was by far the most important subclass of IgG anti-IgE in terms of frequency of detection (34/39; 87.2%) and magnitude of increase (P = 0.0000); with IgG3 (16/39; 41.0%) and IgG4 (15/39; 38.5%) antibodies being considerably less prevalent. Plasma levels of IgG1 anti-IgE (P = 0.0019) and IgG3 anti-IgE (P = 0.0034) showed significant correlations with total IgE concentrations, but not with IgE specific to excretory-secretory worm products; thus suggesting that anti-IgE synthesis is more related to polyclonal hyper IgE production than to antigen-specific IgE stimulation. No correlation was seen between IgG subclass anti-IgE levels and faecal egg counts or worm burden. Given that our data failed to show a negative or a positive correlation between anti-IgE and the degree of infection with hookworm, it is tempting to speculate that the main role of autoanti-IgE is to provide the host with protection against immune complex- and IgE-mediated hypersensitivity reactions to parasitic antigens.

The recognition of a recombinant human Fc epsilon fragment by the subclasses of IgG autoanti-IgE: disproportional subclasses distribution of complexed autoantibody.

Circulating IgG autoanti-IgE is detectable in a large proportion of individuals with allergic asthma where it is suggested to be potentially involved in the removal of IgE-allergen complexes. Since such a putative role will largely be determined by the subclass profile of complexed (i.e. IgE-bound) IgG anti-IgE, a study was undertaken to determine the subclass distribution of complexed IgG anti-IgE antibody in the sera of asthmatic patients. The study exploits the heat-labile property of IgE by heating (30 min at 56 degrees C) serum to liberate bound anti-IgE, pre- and post-heated sera are then assayed for IgG subclass anti-recombinant human Fc epsilon (rFc epsilon) activities by ELISA and any heat-induced increase in antibody activity is taken as a measure of complexed anti-IgE. This has revealed a disproportionately high amount of IgG4 in complexed (but not free) IgG anti-IgE. The propensity of IgG4 to form complexes with IgE has important biological consequences, particularly with regard to the activation of C1q and Fc gamma R by other subclasses.

Elucidation of the epitope locations of human autoanti-IgE: recognition of two epitopes located within the C epsilon 2 and the C epsilon 4 domains.

IgG autoanti-IgE is detectable in a large proportion of individuals with allergic asthma, where it is suggested to be potentially involved in modulating IgE-mediated hypersensitivity. Using a series of overlapping recombinant human epsilon-chain peptides, we have shown that circulating IgG anti-IgE antibodies recognise at least 2 epitopes located within the C epsilon 2 and the C epsilon 4 domains, respectively. The C epsilon 2 recognition site is located within the C-terminal portion of the C epsilon 2 domain (i.e. aa301-339) which is thought to contribute residues to the Fc epsilon RI-binding site on IgE. The recognition by autoanti-IgE of an effector function site of such pivotal importance in IgE-mediated hypersensitivity suggests that it plays a possible modulatory role during mast cell and basophil activation.