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Biomedical subjects

D Meller

Publications and source records attributed to D Meller.

At least 19 recordsLinked to original sources
PubMed2006

[Adjuvant topical interferon-alpha-2b treatment in epithelial tumors of the ocular surface].

PURPOSE: The aim of this study was to evaluate the role of topical interferon-alpha-2b in the adjuvant treatment of corneal and conjunctival tumors. METHOD: In this noncomparative, prospective, interventional case series, five patients with histologically proven conjunctival intraepithelial neoplasia (CIN) after primary excision and amniotic membrane transplantation were treated with interferon (IFN)-alpha-2b eye drops five times daily over a period of 6 weeks (1 million IU/ml Intron A, Schering). An in situ hybridization technique was used to detect human papillomavirus (HPV) in all specimens. Frequent follow-up was undertaken clinically and photographically for evidence of tumor recurrence. RESULTS: In the follow-up period (13.2+/-4,97 months) no clinical evidence of recurrence with only limited treatment side effects such as mild conjunctival hyperemia were recorded after 6 weeks of interferon. In one CIN specimen HPV 16/18 could be detected. CONCLUSIONS: The combination of excisional biopsy and topical interferon-alpha-2b application seems to be an effective and safe treatment for conjunctival intraepithelial neoplasias. Therefore, we prefer this combined treatment to topical interferon-alpha-2b treatment alone, more destructive approaches such as radiation and cryotherapy, or treatment with antimetabolites such as 5-fluorouracil or mitomycin C.

Administration, Topical↗
PubMed2006

[Analysis of ocular surface alterations following proton beam radiation in eyes with conjunctival malignant melanoma].

BACKGROUND: In cases of large, diffuse or multilocular growth pattern of conjunctival melanoma, proton beam irradiation can serve as an alternative therapy to exenteration. In extended tumours, ocular surface problems can result after therapy. In this study we examined ocular surface integrity of ten patients who underwent proton beam radiation between 1996 and 2002. METHODS: The patients were examined during their follow-up. Eight of the ten cases who underwent proton radiotherapy were recurrent tumours, which were previously treated with other adjuvant therapies. We performed a standard ophthalmological examination and detailed tear film diagnostics. RESULTS: The follow-up was 17-87 months (mean: 40.9+/-20.1). In six cases more than 50% of the upper and lower eyelids were included in the radiation field. All of these cases showed moderate to severe sicca symptoms. The impression cytology revealed squamous metaplasia of conjunctival cells in nine of ten cases. CONCLUSIONS: Squamous metaplasia of conjunctival epithelia indicates a radiogenic, persisting disturbance of differentiation of the conjunctival epithelial cells. The tear film instability correlates with the loss of mucin-secreting goblet cells and meibomian gland dysfunction.

Adult↗
PubMed2005

[Neurotrophic keratopathy--a post-LASIK case report].

BACKGROUND: Neurotrophic keratopathy is a degenerative affection of the cornea caused by impairment of corneal sensitivity and represents a therapeutic challenge for ophthalmologists. The present article concentrates on the etiology, pathogenesis, diagnosis, therapeutic aspects and future therapeutic models for treating neurotrophic keratopathy and will present a case report: Also we evaluate the effects of autologous serum eye drops in bilateral LASIK-induced neurotrophic keratopathy with epithelial breakdown revealed by positive fluorescence and rose bengal staining and reduced tear film break-up time. METHODS: We treated a 42-year-old patient with post-LASIK neurotrophic keratopathy and tear film instability with autologous serum eye drops (5 x daily) and emulsion eye drops (Refresh Endura, Allergan, Irvine, CA, USA) after insertion of punctal plugs. RESULTS: Stabilization of vision, healing of the epithelium and reduction of the previously experienced symptoms like redness, itching and burning were achieved within 6 weeks. 10 months after changing therapy, the patient only complained about slight pain during lid movement. CONCLUSIONS: Severe denervation after bilateral LASIK disrupts ocular tear film dynamics and causes irritation symptoms of the ocular surface. Autologous serum eye drops may be an effective treatment of severe epithelial breakdown and be helpful to reestablish the disturbed ocular surface integrity, as shown by negative vital staining. Therefore, autologous serum eye drops represent a significant approach in the therapy of LASIK-induced severe dry eye and associated pain.

Adult↗
PubMed2004

[Corneal perforation associated with vitamin-A-deficiency].

PURPOSE: Vitamin A has been identified as a substantial substrate for ocular surface integrity. Vitamin A deficiency, which can be caused by different etiologies, may lead to severe complications including corneal perforation. PATIENTS: Case reports of three patients with vitamin A deficiency. The observed etiologies leading to vitamin A deficiency were: (1) primary biliary cirrhosis, (2) malnutrition induced by alcohol abuse, and (3) malabsorption syndrome caused by a graft-versus-host disease of the intestine after bone marrow transplantation. RESULTS: In all three cases vitamin A serum levels were below normal values. In two patients retinol binding protein was also reduced. Two of three had corneal perforation, which required penetrating keratoplasty (pKP). In one of these cases, pKP was performed three times on both eyes due to a rapid corneal stromal melting leading always to corneal perforation. The third case developed severe corneal thinning of both eyes with the formation of a descemetocele. Vitamin A substitution improved symptoms and ocular surface integrity. CONCLUSION: Vitamin A in combination with zinc is a crucial factor for maintaining conjunctival and corneal ocular surface integrity. Moreover, vitamin A regulates mucin production of the ocular surface epithelium, which is known to be an important part of the ocular surface defense. Several different primary diseases might cause vitamin A deficiency. Especially patients with rapid progressive corneal ulceration should be examined for vitamin A deficiency. Substitution of vitamin A should be initiated as early as possible to prevent serious disease progression.

Adult↗
PubMed2003

Management of acute ulcerative and necrotising herpes simplex and zoster keratitis with amniotic membrane transplantation.

AIM: To report promoted healing of acute ulcerative and necrotising herpetic keratitis after amniotic membrane transplantation (AMT). METHODS: Retrospective, non-comparative case series of seven patients with acute ulcerative and necrotising herpetic stromal keratitis. Single or multilayer AMT with epithelial side facing up was performed. The main outcome measures were wound healing of the corneal ulcers and decrease of stromal inflammation. RESULTS: The mean follow up was 10.7 (SEM 1.4) months (range 5-15 months). AMT was performed once in five cases, and twice in further two. Improvement of stromal inflammation was noted within 16.4 (2.5) days (range 7-28 days). Epithelial defects healed within a mean of 17 (2.7) days (range 7-28 days). Vision improved in all but two patients. No serious side effects occurred during the follow up. CONCLUSIONS: Although performed in an uncontrolled and non-randomised series of patients, these findings indicate that the AMT shows promise in selected cases for the restoration of ocular surface integrity, reduction of stromal inflammation, and improvement of vision in acute ulcerative and necrotising herpetic keratitis.

Acute Disease↗
PubMed2002

Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures.

BACKGROUND/AIM: Amniotic membrane (AM) transplantation effectively expands the remaining limbal epithelial stem cells in patients with partial limbal stem cell deficiency. The authors investigated whether this action could be produced ex vivo. METHODS: The outgrowth rate on AM was compared among explants derived from human limbus, peripheral cornea, and central cornea. For outgrowth of human limbal epithelial cells (HLEC), cell cycle kinetics were measured by BrdU labelling for 1 or 7 days, of which the latter was also chased in primary cultures, secondary 3T3 fibroblast cultures, and in athymic Balb/c mice following a brief treatment with a phorbol ester. Epithelial morphology was studied by histology and transmission electron microscopy, and phenotype was defined by immunostaining with monoclonal antibodies to keratins and mucins. RESULTS: Outgrowth rate was 0/22 (0%) and 2/24 (8.3%) for central and peripheral corneal explants, respectively, but was 77/80 (96.2%) for limbal explants (p <0.0001). 24 hour BrdU labelling showed a uniformly low (that is, less than 5%) labelling index in 65% of the limbal explants, but a mixed pattern with areas showing a high (that is, more than 40%) labelling index in 35% of limbal explants, and in all (100%) peripheral corneal explants. Continuous BrdU labelling for 7 days detected a high labelling index in 61.5% of the limbal explants with the remainder still retaining a low labelling index. A number of label retaining cells were noted after 7 day labelling followed by 14 days of chase in primary culture or by 21 days of chase after transplantation to 3T3 fibroblast feeder layers. After exposure to phorbol 12-myristate 13-acetate for 24 hours and 7 day labelling, HLEC transplanted in athymic mice still showed a number of label retaining basal cells after 9 days of chase. HLEC cultured on AM were strongly positive for K14 keratin and MUC4 and slightly positive in suprabasal cells for K3 keratin but negative for K12 keratin, AMEM2, and MUC5AC. After subcutaneous implantation in athymic mice, the resultant epithelium was markedly stratified and the basal epithelial cells were strongly positive for K14 keratin, while the suprabasal epithelial cells were strongly positive for K3 keratin and MUC4, and the entire epithelium was negative for K12 keratin and MUC5A/C. CONCLUSIONS: These data support the notion that AM cultures preferentially preserve and expand limbal epithelial stem cells that retain their in vivo properties of slow cycling, label retaining, and undifferentiation. This finding supports the feasibility of ex vivo expansion of limbal epithelial stem cells for treating patients with total limbal stem cell deficiency using a small amount of donor limbal tissue.

Adult↗
PubMed2001

[Amniotic membrane transplantation for reconstruction of the ocular surface].

Amniotic membrane transplantation has been recently gained wide-spread attention as a new method for reconstruction of the ocular surface. However long term prognosis of the surgical intervention as well as action mechanism are poorly defined. Therefore, we review the current literature concerning the application of amniotic membrane in ophthalmology and the outcome of the surgery. Amniotic membrane is used as graft or patch to promote epithelial healing of persistent epithelial defects, corneal ulcers and following penetrating keratoplasty. It is also used to seal corneal perforations and to treat bullous keratopathy. Amniotic membrane patches facilitate epithelial healing and reduce the number of complications after chemical burns. For partial limbal deficiency, amniotic membrane alone can be sufficient, while total limbal deficiency requires combination with stem-cell transplantation. Furthermore amniotic membrane grafts restore conjunctival surfaces following removal of lesions such as pterygium, tumor, scar, symblepharon, and conjunctivochalasis. The prognosis of amniotic membrane transplantation depends on the underlying disease and the quality of the ocular surface and is inversely related to the degree of inflammation. Recent investigations of the action mechanisms describe intrinsic antiinflammatory properties of the amniotic membrane.

Amnion↗
PubMed2001

[Ex-vivo expansion of cornea stem cells. Experimental principles and initial clinical results].

The ocular surface epithelium is composed of conjunctiva, limbus and cornea. These three compartments and an stable preocular tear film are crucial factors determining ocular surface health. Stem cells of the corneal epithelium are exclusively located at the limbus. Limbal stem cells are the ultimate source of regeneration of the entire corneal epithelium and in a variety of ocular surface diseases limbal stem cell deficiency has been observed to be a characteristic feature. Therefore renewal of the limbal stem cell population using different surgical techniques has been described as the only possible therapeutic strategy. Recent advancements in cell biology have enabled the development of new models of tissue engineering as a tool for tissue replacement. The aim of this review is to summarize recently reported clinical and experimental studies showing new developments in tissue engineering for the reconstitution of the ocular surface epithelium.

Animals↗
PubMed2001

Overexpression of collagenase (MMP-1) and stromelysin (MMP-3) by pterygium head fibroblasts.

BACKGROUND: The balance between matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) determines the extent of connective tissue degradation and remodeling. OBJECTIVE: To determine whether pterygium, characterized by fibrovascular invasion into the cornea, may in part be mediated by an increased activity of MMPs. MATERIALS AND METHODS: Expression of transcripts and proteins of MMPs, TIMPs, and urokinase plasminogen activator (uPA) by cultured human pterygium head, body, and subconjunctival fibroblasts, and normal corneal and conjunctival fibroblasts were determined by Northern hybridization, enzyme-linked immunosorbent assay, Western blotting, zymography, and quantitative collagenase assay, respectively. RESULTS: Compared with normal conjunctival fibroblasts from 6 subjects, the expression of MMP-1 and MMP-3 transcripts was dramatically increased in pterygium head fibroblasts of 8 patients, but not in pterygium body fibroblasts of 6 patients. The protein levels and collagenolytic and caseinolytic activities of MMP-1 and/or MMP-3 were also markedly increased in pterygium head fibroblasts. The MMP-1 and MMP-3 proteins and activity decreased in order from pterygium head to body to subconjunctival fibroblasts. There was no difference in the transcript and protein expression of MMP-2, TIMP-1, TIMP-2, and uPA among these groups. CONCLUSION: Pterygium head fibroblasts express increased mRNA, protein, and activities of MMP-1 and MMP-3. CLINICAL RELEVANCE: Overexpression of MMP-1 and MMP-3, a phenotype previously linked with UV exposure in dermal fibroblasts to explain the pathologic finding of elastotic degeneration, suggests that pterygium head fibroblasts might be intrinsically altered by UV, which might be responsible for corneal invasion.

Adult↗
PubMed2001

Improvement of HSV-1 necrotizing keratitis with amniotic membrane transplantation.

PURPOSE: Stromal herpes simplex virus keratitis (HSK) is an immune-mediated disease. Previous studies have indicated that T cells, neutrophils, and macrophages contribute to the tissue damage in HSK. It has been shown that human amniotic membrane promotes epithelial wound healing and has diverse anti-inflammatory effects. In this study, the effect of amniotic membrane transplantation (AMT) on corneal wound healing and on inflammation in mice with necrotizing HSK was examined. METHODS: BALB/c mice were corneally infected with 10(5) plaque-forming units (PFU) of HSV-1 (KOS strain). In 16 mice that exhibited severe ulcerating HSK, the cornea was covered with a preserved human amniotic membrane as a patch. Corneas in 16 infected mice remained uncovered and served as a control. On days 2 and 7 after surgery, the amniotic membrane was removed (eight mice in each group), the HSV-1-infected cornea was evaluated clinically, and the eye was enucleated. Tissue sections were analyzed histologically for epithelialization and cellular infiltration and immunohistochemically with anti-CD3 mAb to T cells, anti-CD11b mAb to both macrophages and neutrophils, or anti-F4/80 mAb to macrophages. RESULTS: Profound regression of corneal inflammation and rapid closure of epithelial defects were observed clinically within 2 days in the amniotic membrane-covered eyes, whereas HSV-1 keratitis and ulceration progressed in all mice in the control group (P < 0.001). Histologically, corneal edema and inflammatory infiltration, and immunohistochemically the number of CD3(+), CD11b(+), and F4/80(+) cells in the cornea were markedly decreased at 2 and 7 days after amniotic membrane application, compared with the uncovered control corneas (P < 0.001). CONCLUSIONS: AMT promotes rapid epithelialization and reduces stromal inflammation and ulceration in HSV-1 keratitis. AMT in mice with HSV necrotizing stromal keratitis appears to be a useful model for investigating the effect and the action mechanism of human amniotic membrane.

Amnion↗
PubMed2000

[Amniotic membrane transplantation with or without limbal allografts in corneal surface reconstruction in limbal deficiency].

PURPOSE: We examined whether amniotic membrane transplantation (AMT) is useful in preparing the perilimbal stroma to enhance the success of allograft limbal transplantation (ALT). METHODS: Forty-seven eyes of 42 consecutive patients with cytologically proven limbal deficiency (LD) were included in this prospective study. Based on the severity of LD, group A (mild) with 18 eyes received AMT alone, group B (moderate) with 13 eyes received AMT and ALT, and group C (severe) with 16 eyes received AMT, ALT and penetrating keratoplasty. All except for group A received continuous systemic cyclosporin A. RESULTS: Except for the two eyes with atopy, all amniotic membrane-covered surfaces showed rapid epithelialization in 2-4 weeks, reduced inflammation, vascularization and scarring, and became smooth. For the mean follow-up period of 23 months, 38 eyes (82.6%) showed visual improvement, consisting of > or = 6 lines (15 eyes), 4-5 lines (10 eyes), 1-3 lines (13 eyes). Visual improvement was noted in 16/18 eyes (88.9%) in group A, in 10/13 eyes (77%) in group B, and in 12/16 eyes (75%) in group C. In group C corneal graft rejection occurred in 12 of 16 eyes (75%). In group B and C, early reversible limbal allograft rejection was noted in 3 of 29 eyes (10.3%) and a recurrent limbal deficiency was observed in 8 of 29 eyes (27.6%). CONCLUSION: For partial LD with superficial involvement, AMT alone is sufficient and hence superior to ALT because of no need for using cyclosporin A. For total LD, additional ALT is needed and AMT helps reconstruct the perilimbal stroma with reduced inflammation and vascularization, which collectively may enhance ALT success.

Amnion↗
PubMed2000

Amniotic membrane transplantation for acute chemical or thermal burns.

PURPOSE: To determine whether preserved human amniotic membrane (AM) can be used to treat ocular burns in the acute stage. DESIGN: Prospective, noncomparative, interventional case series. PARTICIPANTS: Thirteen eyes from 11 patients with acute burns, 10 eyes with chemical burns and 3 with thermal burns of grades II-III (7 eyes) and grade IV (6 eyes), treated at 7 different facilities. METHODS: Patients received amniotic membrane transplantation (AMT) within 2 weeks after the injury. MAIN OUTCOME MEASURES: Integrity of ocular surface epithelium and visual acuity during 9 months of follow-up. RESULTS: Ten patients were male and one patient was female; most were young (38.2 +/- 10.6 years). For a follow-up of 8.8 + 4.7 months, 11 of 13 eyes (84.63%) showed epithelialization within 2 to 5 weeks (23.7 +/- 9.8 days), and final visual acuity improved > or = 6 lines (6 eyes), 4 to 5 lines (2 eyes), and 1 to 3 lines (2 eyes); only one eye experienced a symblepharon. Eyes with burns of grade II to III showed more visual improvement (7.3 +/- 3 lines) than those with burns of grade IV (2.3 +/- 3.0 lines; P < 0.05, unpaired t test). In the group with grade II or III burns, none had limbal stem cell deficiency. All eyes in the group with grade IV burns did experience limbal stem cell deficiency. CONCLUSIONS: Amniotic membrane transplantation is effective in promoting re-epithelialization and reducing inflammation, thus preventing scarring sequelae in the late stage. In mild to moderate burns, AMT alone rapidly restores both corneal and conjunctival surfaces. In severe burns, however, it restores the conjunctival ocular surface without debilitating symblepharon and reduces limbal stromal inflammation, but does not prevent limbal stem cell deficiency, which requires further limbal stem cell transplantation. These results underscore the importance of immediate intervention in the acute stage of eyes with severely damaged ocular surface. Further prospective randomized studies including a control group are required to determine the effectiveness of AMT in acute chemical and thermal burns of the eye.

Acids↗
PubMed2000

Amniotic membrane transplantation for symptomatic conjunctivochalasis refractory to medical treatments.

UNLABELLED: PURPOSE. To determine whether preserved human amniotic membrane can restore the large conjunctival defect created during surgical removal of conjunctivochalasis. METHODS: Amniotic membrane transplantation was performed at two facilities in 40 consecutive patients (47 eyes) with symptomatic conjunctivochalasis refractory to conventional treatments. RESULTS: The majority of patients were elderly (73.1 +/- 9.7 years) and women (75%). Over a follow-up period of 6.9 +/- 4.3 months, 46 (97.8%) eyes recovered smooth, quiet, and stable conjunctival surfaces. Epithelial defects healed in 16.5 +/- 7.3 days. Episodic epiphora was resolved in 24 of 30 (83.3%) eyes and improved in five other eyes. Notable relief was also noted for such symptoms as fullness or heaviness (19/19, 100%), sharp pain (6/6, 100%), redness (14/17, 88.2%), tiredness (17/20, 80.9%), itching (11/13, 78.6%), blurry or decreased vision (6/8, 75%), burning (8/13, 61.5%), foreign body sensation (8/13, 61.5%), and crust formation (1/2, 50%). Complications included focal inflammation of the host conjunctiva adjacent to the graft (six eyes), scar formation (five eyes), and suture-induced granuloma (one eye). CONCLUSION: Amniotic membrane transplantation can be considered as an effective means for conjunctival surface reconstruction during removal of conjunctivochalasis.

Aged↗
PubMed2000

Overexpression of MMP-1 and MMP-3 by cultured conjunctivochalasis fibroblasts.

PURPOSE: To determine whether conjunctivochalasis, denoting redundant, loose, nonedematous inferior bulbar conjunctiva, is associated with increased expression and activity of matrix metalloproteinases (MMPS) over their tissue inhibitors (TIMPs). METHODS: Expression of transcripts and proteins of MMPs, TIMPs, and urokinase plasminogen activator (uPA) by cultured normal human conjunctival and conjunctivochalasis fibroblasts was determined by Northern hybridization, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis, respectively. Gelatin and casein zymography and quantitative collagenase activity assay were performed in the serum-free conditioned media. RESULTS: Compared with normal conjunctival fibroblasts from six subjects, conjunctivochalasis fibroblasts from eight patients showed markedly increased transcript expression of MMP-1 (5- to 32-fold) and MMP-3 (4 to 30-fold), whereas that of MMP-2, TIMP-1, TIMP-2, and uPA was similar between the two groups. Protein levels were increased in the serum-free conditioned media of conjunctivochalasis fibroblasts for MMP-1 (3.5- to 7.6-fold) and MMP-3 (2.3- to 13-fold), determined by ELISA and Western blot analysis. There was increased caseinolytic activity of MMP-3 and collagenolytic activity of MMP-1 (2.2-fold) by conjunctivochalasis fibroblasts, whereas no difference was noted between these two types of fibroblasts in the protein and gelatinolytic activity of MMP-2 or expression of TIMP-1 and TIMP-2 proteins, although that of TIMP-1 transcript was slightly higher in some conjunctivochalasis fibroblasts. No expression of MMP-9 was detected. CONCLUSIONS: Overexpression of MMP-1 and MMP-3 mRNA by conjunctivochalasis fibroblasts is correlated with their increased protein levels and proteolytic activities. Collectively, these data help explain how conjunctivochalasis manifests excessive degradation of the conjunctival matrix and Tenon's capsule.

Blotting, Northern↗
PubMed2000

Regulation of collagenase, stromelysin, and gelatinase B in human conjunctival and conjunctivochalasis fibroblasts by interleukin-1beta and tumor necrosis factor-alpha.

PURPOSE: Overexpression and increased activities of matrix metalloproteinases (MMPs) have recently been reported in cultured conjunctival fibroblasts from patients with conjunctivochalasis. The role of inflammatory cytokines in modulating expression of MMPs, their tissue inhibitors (TIMPs), and urokinase plasminogen activator (uPA) as potential contributors to the pathogenesis of conjunctivochalasis was investigated. METHODS: Interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha) was added at 10 ng/ml to a serum-free medium. Expression of transcripts and proteins of MMPs, TIMPs, and uPA by cultured normal human conjunctival and conjunctivochalasis fibroblasts was determined by Northern hybridization, enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, respectively. Gelatin and casein zymographies were performed in serum-free conditioned media with and without the respective enzyme inhibitors. RESULTS: Without challenging the cells, conjunctivochalasis fibroblasts showed mRNA and protein overexpression of MMP-1 and MMP-3 compared with normal conjunctival fibroblasts, which showed minor or no expression of these enzymes. IL-1beta markedly and TNF-alpha to lesser extent increased mRNA and protein expression of MMP-1 and MMP-3 in conjunctivochalasis fibroblasts from 2 subjects when compared with normal conjunctival fibroblasts from 2 subjects and with their nonstimulated counterparts. In conjunctivochalasis fibroblasts and normal conjunctival fibroblasts, TNF-alpha, but not IL-1beta, induced a gelatinolytic activity of MMP-9, which was further confirmed by Western blot analysis and ELISA. Expression of MMP-2, TIMP-1, and TIMP-2 mRNA and protein was not influenced by IL-1beta or TNF-alpha, and no difference was found in the gelatinolytic activity of MMP-2 between both cell types. CONCLUSIONS: Inflammatory cytokines such as IL-1beta and TNF-alpha, which can potentially be derived from the ocular surface and tears, may be responsible for increased expression of MMPs in cultured conjunctivochalasis fibroblasts. Ocular inflammation might be one important denominator in the pathogenesis of conjunctivochalasis.

Blotting, Northern↗
PubMed1999

The fine structure of chromatin alterations in conjunctival epithelial cells in keratoconjunctivitis sicca.

PURPOSE: To study the ultrastructure of nuclear chromatin changes of conjunctival epithelial cells in imprints of dry-eye patients. METHODS: Imprints with nuclear chromatin changes of the conjunctival epithelium from dry-eye patients were first selected by light microscopy (LM). The latter allowed processing material with special interest (chromatin changes) for electron microscopy (EM). Ultrathin sections were cut tangential to the filter surface to obtain an large overview of the collected imprints. For scanning electron microscopy (SEM), selected specimens were dehydrated in ethanol and dried by the critical-point method or in some cases only air-dried. As controls, imprints of healthy patients were used. RESULTS: EM of the control samples showed sheets of well-preserved conjunctival nongoblet epithelial cells and goblet cells filled with secretory granules. Intercellular processes contributed to the formation of a cohesive sheet of epithelial cells. In dry-eye patients, signs of squamous metaplasia such as enlargement of nongoblet epithelial cells, increased keratinization, and nuclear chromatin changes were disclosed. The goblet cell density was drastically reduced or lost. Nuclear chromatin changes showed a pathologic distribution of eu- and heterochromatin, resulting in an axial snake- or stick-like chromatin condensation. Intracellular filament-like structures were identified in severe cases to cause a segmentation of nuclei. CONCLUSION: The fine structure of cellular and nuclear changes occurring in KCS are shown in detail. The data are in line with EM observations of impression cytology of contact lens wearers, thus confirming the ubiquitous origin of squamous metaplasia and authenticity of the snake phenomenon in squamous metaplasia. Potential pathomechanisms that might be involved in the development of chromatin alterations in conjunctival epithelial cells undergoing squamous metaplasia are discussed.

Chromatin↗
PubMed1999

Conjunctival epithelial cell differentiation on amniotic membrane.

PURPOSE: Amniotic membrane (AM)-reconstructed conjunctival surfaces recover the normal epithelial phenotype with a significantly higher cell density than the control. The present study was undertaken to examine how AM modulates rabbit conjunctival epithelial cell differentiation. METHODS: Rabbit conjunctival epithelial cells (RCEs) were cultured on the basement membrane side of dispase-pretreated AM, with or without seeding rabbit conjunctival fibroblasts (RCFs) on the stromal side. After 7 to 12 days, half of the cultures were raised to the air-liquid interface, and the remainder stayed submerged. A small group of air-lifted cultures containing RCFs was treated with retinoic acid. After 1, 2, and 4 weeks, cultures were terminated and processed for immunostaining with antibodies directed against distinct types of mucins (SMC and AM3), glycocalyx (AMEM2), keratin K3 (AE5), and K12 (AK2). Additionally, western blot analysis was performed for K3 keratin expression. Ultrastructural changes were evaluated by transmission electron microscopy. RESULTS: In general, RCEs grown on AM were uniformly small, negative to AE5 and AK2 antibodies, and positive to AMEM2 and ASPG1 antibodies. Epithelial stratification and cell polarity with prominent microvilli, tight junctions, and hemidesmosomes were more pronounced in air-lifted cultures. RCEs cocultured with RCFs showed scattered AM3-positive goblet cells, which were not increased by retinoic acid. CONCLUSIONS: RCEs cultured on AM primarily exhibit a nongoblet conjunctival epithelial phenotype. Epithelial stratification and cell polarity, features essential for epithelial differentiation, are promoted by air-lifting. This culture model will be useful for studying how growth and differentiation of conjunctival epithelial cells can be modulated further.

Amnion↗