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Recommended cytoskeletal antibodies for identification of epithelial subsets in ocular mucosal associated lymphoid tissues (MALT)
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The human lacrimal gland (LG) and ocular surface contain discrete regions of epithelial cells with specific functions and at different stages of cellular differentiation. Epithelial cells contain cytoskeletal antigens that show a differentiation-dependent pattern of expression. The objective of this study was to characterize the various epithelial...
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Context 1
... Our results indicate that these two acinar cell popula- tions have a similar pattern of immunoreactivity with cytoskeletal antibodies. Based on our findings, Table 3 presents our recommendations for choosing anti- bodies to stain the different epithelial subsets found in ocular MALT positively and negatively. ...
Citations
... Indeed, at the early stages of conjunctivalization, K3 is present in the conjunctival cells migrating over the cornea but is undetectable at later stages when the macroscopic signs of conjunctivalization occur [2,48]. Moreover, a weak K3 staining was detected in normal bovine, rabbit, and human conjunctiva [49][50][51]. ...
Regeneration of the corneal epithelium could be severely impaired in patients suffering from limbal stem cell deficiency. The purpose of this study was to evaluate the restoration of the corneal epithelium by grafting onto denuded corneas autologous limbal cells cultured on fibrin gels. The rabbit model was chosen to allow the microscopic evaluation over time after grafting.
Rabbit limbal epithelial cells (RLECs) were isolated and cultured from small limbal biopsies (3 mm2). The epithelium was separated from stroma after dispase digestion and put in culture on lethally irradiated fibroblasts used as a feeder layer. At the first passage, RLECs were cultured on a fibrin gel matrix. At confluence, the cultured epithelia were grafted in vivo on denuded autologous rabbit corneas. At different postoperative times, grafted and control (without graft or grafted with fibrin gels only) rabbit corneas were compared in vivo with a slit lamp microscope, and in situ by histological and immunohistological microscopy of harvested biopsies.
A small limbal biopsy was sufficient to generate enough RLECs to prepare several grafts and to perform cell analysis. Only two weeks were required to produce a cultured epithelium suitable for autologous transplantation. One month after grafting, a normal corneal phenotype was observed on the ocular surface of grafted rabbits in contrast to the control rabbits (ungrafted or grafted with fibrin gel only) where histological signs of conjunctivalization were found. The absence of goblet cells and negative staining for keratin 4 confirmed that the cultured cells persisted and that the epithelium regenerated after grafting was not from conjunctival origin.
Our results demonstrate that an autologous epithelium cultured on a physiologically biodegradable matrix can be prepared from a small biopsy and grafted on denuded cornea. The autologous graft allows epithelial regeneration from cultured cells and promotes corneal healing of unilateral total stem cell deficiency.
... Two other bands were variably present in the 42 and 62 kDa region. The latter most closely correlates with K3, a cornea-specific keratin that has been reported to be in the conjunctival epithelium, 15 whereas the former does not correlate with any reported cytokeratin molecular weight. ...
To document the cytokeratin expression patterns in the normal human conjunctival epithelium obtained directly from patients using impression cytology.
Impression cytology specimens were obtained from normal volunteers using pure nitrocellulose membranes rather than cellulose acetate. The 31 volunteers of both sexes ranged in age from 18 to 79 years. Impression cytology specimens were analyzed for individual cytokeratins by either immunocytochemistry or electrophoresis with immunoblotting using a defined panel of monoclonal antibodies.
Using the corroborative methods of immunocytochemistry and electrophoresis with immunoblotting, cytokeratins characteristic of nonkeratinized, stratified (K4 and K13), simple (K8 and K19), and glandular epithelia (K7) were present in the superficial layer (s) of normal human conjunctiva. Cytokeratins typical of keratinized epithelia (K1, K2, and K10) and the keratinization-related proteins filaggrin and involucrin were not expressed in normal conjunctival epithelium.
The normal human conjunctiva demonstrates a unique cytokeratin expression pattern containing cytokeratins characteristic of nonkeratinized, stratified epithelia, as well as others more typical of a simple differentiation pattern, a glandular differentiation pattern, or both. These findings provide a foundation for examining changes in the cytokeratin expression pattern in diseased human conjunctival epithelium using impression cytology.
... In the normal human LGs, the basal ductal epithelial cells stain intensely with an antibody specific for keratin no. 5 (AE-14), and the suprabasal ductal epithelia stain with an antibody specific for keratin no. 3 (AE-5). 22 In contrast, all layers of ductal epithelia in SS LGs react with both keratin no. 3-and no. ...
The lacrimal gland (LG) immunopathology of Sjögren's syndrome (SS) consists of a proliferation of B and CD4 lymphocytes surrounding epithelial structures (Pepose JS, et al: Ophthalmology 1990, 97:1599-1605). Based on the detection of EBV genomes in a greater percentage of SS than normal LG biopsies, we previously postulated that Epstein-Barr virus (EBV) is a risk factor for LG lymphoproliferation in SS (Pflugfelder SC, et al: Ophthalmology 1990, 97:976-984). The purpose of this study was to determine the cellular site(s) of infection, virus type, and antigen expression of EBV infecting normal and SS LGs. EBV DNA was detected by in situ hybridization in intraductal epithelia in 13-33% of lobules in 21% of normal LGs and in cells in areas of B lymphoproliferation as well as the majority of epithelia in 86% of SS LGs. EBV genomic sequences were amplified from 36% of normal and 88% of SS LG biopsies by polymerase chain reaction. Only type 1 EBV sequences were amplified in SS LGs; in contrast EBV nuclear antigen 2-deleted but not type 1 sequences were amplified in normal LGs. Immunohistochemistry with EBV-specific monoclonal antibodies was performed on normal and SS LGs. No EBV antigens were detected in normal LGs. In contrast, latent antigens (latent membrane protein, EBV nuclear antigen 2) were detected in lymphocytes in areas of B lymphoproliferation, and early and late lytic cycle antigens were observed in epithelia in SS LGs. These studies suggest that EBV may play a role in the LG B lymphoproliferation and epithelial pathologic changes observed in SS.
Previous studies have shown that activated CD4+ T cells infiltrate the conjunctival subepithelial layer during chronic allergic eye disease (CAED). The effectiveness of steroids and cyclosporin A (CsA) in treating the disease suggests an important immunological role for CD4+ T cells in CAED. There is some evidence supporting a role for epithelial cells in regulating T cell function in allergic disease, but little is known about the role of conjunctival epithelial cells. This study has investigated whether conjunctival epithelial cells are able to modulate T cell responses. Existing research with superficial conjunctival epithelial cells has been reported using impression cytology flow cytometry and immunocytochemistry. Biopsy derived conjunctival epithelial cells have only been studied using immunocytochemical methods. In this study, techniques for isolation, expansion and characterisation of conjunctival epithelial cells from biopsies has been estabilished and optimized for quantitative analysis by flow cytometry. The expression of HLA-DR and costimulatory molecules (ICAM-1, CD80, CD86, and CD40) on normal conjunctival epithelial cells and a human conjunctival epithelial cell line (ChWK) after treatment with various cytokines (IFN-γ, TNF-α, IL-4 and IL-13) were determined using flow cytometry and confocal microscopy. The antigen presentation function of conjunctival epithelial cells was assessed by their ability to support T lymphocyte proliferation using ChWK in mixed lymphocyte reactions (MLR). These studies suggest conjunctival epithelial cells can potentially act as antigen presenting cells (APC) and are involved in recruiting T cells to the subepithelial layer during CAED.
Reconstruction of the conjunctiva is an essential part of ocular surface regeneration, especially if an extensive area or the whole ocular surface is affected, such as in patients with ocular cicatricial pemphigoid, Stevens-Johnson syndrome, toxic epidermal necrolysis, or chemical/thermal burns. In these situations, corneal reconstruction almost inevitably fails unless the conjunctival surface is first repaired and a deep fornix is restored. The growing field of tissue engineering and advances in stem cell research offer promising new alternatives for these challenges. This article reviews the present approaches for reconstruction of the conjunctival surface, considering the established strategies and new potential methodologies.
Keratins are a group of water-insoluble proteins constituting paired acidic and basic keratin molecules that form 10-nm intermediate filaments in epithelial cells. Expression of the K3/K12 keratin pair characterizes the cornea-type differentiation. However, the mechanism that regulates this cornea-specific K12 expression remains unknown. To provide a better understanding of the cornea-specific expression, we have cloned the K12 cDNA (Liu, C.-Y., Zhu, G., Westerhausen-Larson, A., Converse, R., Kao, C. W.-C., Sun, T.-T., and Kao, W. W.-Y. (1993) Curr. Eye Res. 12, 963-974). In present studies, the murine K12 keratin gene (Krt1.12) was isolated and characterized. The murine Krt1.12 gene spans 6,567 base pairs of genomic DNA, and the mRNA encoding K12 keratin is distributed into eight exons. Chromosome mapping reveals that murine Krt1.12 is located within the Krt1 complex of mouse chromosome 11. In addition to the production of authentic K12 mRNA, the Krt1.12 gene gives rise to several alternate poly(A)+ RNAs by the use of alternative splicing in intron 2, an alternative promoter in intron 1, and/or both. Sequence analysis indicates that the transcripts derived from alternative splicing and/or the alternative promoter do not have a long open reading frame for keratin or keratin-like molecules. It is not known whether these alternate K12 poly(A)+ RNAs have any biological functions, e.g. regulation of K12 gene expression.
The conjunctival epithelium is intrinsically different from the corneal epithelium in vivo, but sometimes can transform into an epithelium morphologically indistinguishable from the latter after healing of a total corneal epithelial defect. It remains unclear whether this morphologic transformation represents a process of extrinsic modultation or transdifferentiation of intrinsically divergent epithelium. In air-lifted organotypic cultures, rabbit conjunctival epithelial cells lost goblet cell differentiation and were stratified to the same extent as corneal epithelial cells, resembling the above in vivo morphologic transformation. Paired expression of K3 (64 kD) and K12 (55 kD) keratins has been regarded as a marker for corneal-type differentiation. Immunoblot analysis by monoclonal antibody AE5 revealed that K3 keratin was expressed by both submerged or air-lifted corneal and conjunctival cultures with or without 3T3 fibroblasts in collagen gel. In contrast, K12 keratin was expressed only by air-lifted corneal cultures with 3T3 fibroblasts using monoclonal antibody AK2 and two epitope-specific antibodies to N- and C- terminal oligopeptides deduced from the mouse K12 gene. This finding was also confirmed by Northern hybridization with a rabbit K12 cDNA probe. The expression of K12 keratin was more delayed than that of K3 keratin in air-lifted corneal cultures. This dissociated expression of these two keratins resembles that noted in vivo in the stem cell-containing limbal region. These results suggest that morphologic transformation of the conjunctival epithelium represents extrinsic environment modulation, and that differential expression of K12 but not K3 keratin signifies corneal epithelial differentiation.
The presence of IgA autoantibodies against human eye muscle was investigated in 40 patients with Graves' disease (33 had ophthalmopathy). IgA anti-eye muscle antibodies could be demonstrated in sera of patients using western blotting and immunohistochemical methods. For the detection of sera possessing autoantibodies against eye muscle antigen the indirect immunosorbent assay had been used. IgA anti-eye muscle antibodies could be demonstrated in 25 cases and IgG types in 16 cases out of 40 patients. These anti-muscle autoantibodies were associated with eye muscle rather than skeletal muscle, the number of positive cases with the latter being 5/40 for IgG and 2/40 for IgA. Immunoreactive bands of IgA autoantibodies against eye cytosol were found at 84, 64, 45, 40 and 25-23 kDa in 22, 2, 16, 2 and 18 cases, respectively. A difference was observed in the staining of IgG and IgA types of autoantibodies by immunohistochemical analysis of eye muscle tissue. The IgA anti-eye muscle antibodies reacted with muscle fibers and the IgG types showed staining on endomysium. No sera of Graves' disease patients gave staining on skeletal muscle tissue. The results supported the presence of IgA anti-human eye muscle antibodies in patients with Graves' ophthalmopathy, which might play a relevant role in the development of eye disease.
Conjunctiva associated lymphoid tissue shows several similarities to mucosa associated lymphoid tissue of the gut and respiratory tract. These similarities have been described in relation to lymphocyte subpopulations and epithelial cell morphology. However, unlike the lymphoid tissue of the gut and respiratory tract, mucosa specific lymphocytes have not been described in the ocular mucosa. In this study we demonstrated the presence of mucosa specific lymphocytes bearing the Human Mucosal Lymphocyte-1 antigen (beta 7 integrin), in the human conjunctiva, limbus and lacrimal gland. The distribution of this subset of lymphocytes corresponded to the distribution of CD8+ T-cells and was found maximally in the epithelium of the epibulbar conjunctiva and in the lacrimal gland. The Human mucosal lymphocyte antigen may function to determine mucosal homing of this particular subset of CD8+ T-cells, which in turn, may have special function in immunological defense and tolerance mechanisms occurring at mucosal surfaces.
