Cogan's syndrome with refractory abdominal aortitis and mesenteric vasculitis.

Cogan's syndrome is a rare multisystem disease characterized by ocular inflammation, vestibuloauditory dysfunction, and vasculitis. We report a 26-year-old Caucasian woman who died from Cogan's syndrome. Her case illustrates that patients with Cogan's syndrome can have abdominal aortitis and mesenteric vasculitis, and that the vasculitis can be refractory to methotrexate, cyclophosphamide, cyclosporine, and chlorambucil.

Human leukocyte antigen-class II-positive human corneal epithelial cells activate allogeneic T cells.

PURPOSE: To achieve a better understanding of the mechanism of corneal immune diseases, including corneal allograft rejection, the authors examined the potential of human corneal epithelial (HCE) cells to activate allogeneic T lymphocytes. METHODS: The mixed lymphocyte-HCE cell reaction (MLCER) was performed as follows: HCE cells from primary cultures, with or without treatment with interferon-gamma (IFN-gamma), were treated with mitomycin C and then mixed with peripheral blood lymphocytes (PBL) from normal volunteers. Triplicate cultures were incubated for 7 days. Interleukin-1-alpha (IL-1-alpha) was added to some cultures to examine its effect on MLCER: The lymphocyte responses were measured by 3H-thymidine uptake for the last 18 hours of incubation in MLCER: RESULTS: IFN-gamma-treated, HLA-class-II-bearing HCE cells stimulated allogenic lymphocytes, whereas IFN-gamma nontreated, class-II-negative HCE cells did not. The stimulation by IFN-gamma-treated HCE cells was blocked by anti-HLA class II monoclonal antibody. In addition, exogenous IL-1-alpha reduced the lymphocyte response in MLCER: This effort was inhibited by indomethacin. CONCLUSIONS: This study demonstrates that HLA-class-II-bearing HCE cells can activate allogeneic PBL by a major histocompatibility complex class II-dependent mechanism. In addition, HCE cells may regulate immune reactions, probably through prostaglandin production caused by IL-1.

Conjunctival epithelial cell hypermitosis and goblet cell hyperplasia in atopic keratoconjunctivitis.

Atopic diseases that include eczema (atopic dermatitis), asthma, and seasonal and perennial rhinoconjunctivitis are common manifestations of abnormal immediate hypersensitivity. Ocular involvement, such as atopic keratoconjunctivitis, characteristically includes conjunctival and corneal inflammation, and in a severe form, conjunctival scarring, symblepharon, corneal epitheliopathy, and visual loss. To examine the conjunctival cellular abnormalities in atopic keratoconjunctivitis, we studied the in vivo differentiation and tissue-culture growth characteristics of conjunctiva from normal subjects and patients with severe atopic keratoconjunctivitis. We examined conjunctival biopsy specimens to determine epithelial mitotic rate and goblet cell frequency, and we studied conjunctival explants to determine the latent period for fibroblast outgrowth and fibroblast doubling time. The mitotic rate for atopic keratoconjunctivitis, 6.7% +/- 2.1% (11 patients), was statistically significantly greater than for normal subjects, 2.0% +/- 0.63% (seven subjects) (P = .05). Also the goblet cell frequency for atopic keratoconjunctivitis, 14.6% +/- 3.4% (11 patients), was statistically significantly greater than for normal subjects, 4.8% +/- 0.92% (seven subjects) (P = .02). The latent period for fibroblast outgrowth and the fibroblast doubling time for atopic keratoconjunctivitis were not statistically significantly different from normal control subjects. Therefore, atopic keratoconjunctivitis was associated with conjunctival epithelial hypermitosis, goblet cell hyperplasia, and normal fibroblast tissue-culture growth. These characteristics may be useful in the diagnosis of atopic keratoconjunctivitis. We previously studied another disease characterized by chronic conjunctival inflammation and scarring, cicatricial pemphigoid, which also demonstrated conjunctival epithelial hypermitosis, but in contrast there was near absence of goblet cells, and the fibroblasts were hyperproliferative. These differences may be used to distinguish atopic keratoconjunctivitis from cicatricial pemphigoid.

Regulation of HLA class II antigen expression on cultured corneal epithelium by interferon-gamma.

The effect of recombinant human interferon gamma (IFN-gamma) on the induction of HLA class II (HLA-DR, -DP, -DQ) antigen expression on human corneal epithelial (HCE) cells was examined in different stages of culture. Primary cultures were established with limbal explants without endothelium. HCE cells in Stage 1 and Stage 2, with cells negative and positive for the 64K corneal keratin (the marker for advanced corneal epithelial differentiation), respectively, were prepared. HCE cells in both stages were treated with IFN-gamma at a concentration of 0 to 1000 U/ml for two to six days and were stained by the avidin-biotin peroxidase complex method. Class II antigens were not detected on HCE cells in either stage without IFN-gamma treatment. IFN-gamma induced three class II antigens on HCE cells in both stages in a dose- and time-dependent manner but at different levels for each antigen (DR greater than DP greater than DQ). In addition, DQ expression was related to cell differentiation, with DQ extremely rare at Stage 1 and more frequent at Stage 2 (5% vs. 20%). These findings indicate that the induction of class II antigens on HCE cells may be regulated by IFN-gamma independently for each of the antigens and that DQ induction may depend upon the differentiation of HCE cells in culture.

Growth of acanthamoeba on human corneal epithelial cells and keratocytes in vitro.

Acanthamoebic keratitis, a potentially devastating infection usually associated with contact lens wear, has been recognized with increasing frequency in recent years. Once the Acanthamoeba organisms gain access to the human cornea, it is not clear which constituents of the corneal milieu provide a substrate for their growth. The growth of Acanthamoeba polyphaga was investigated on cultured monolayers of human corneal epithelial cells, stromal keratocytes, and stromal homogenate suspensions. Growth was determined through organism counts and observation of cytopathic effects on tissue culture dishes. Compared with tissue culture media controls, acanthamoebic growth was supported by cultured epithelial cells and keratocytes but not stromal homogenates. These results suggest that in acanthamoebic keratitis the organisms depend on the cellular components of the cornea as substrates for growth. This in vitro model may also provide further information on the pathogenesis of keratitis and a system for drug sensitivity testing.

P-31 NMR analysis of phospholipids from cultured human corneal epithelial, fibroblast and endothelial cells.

Corneal epithelial, fibroblast and endothelial cells, cultured from human donors, were analyzed to determine their characteristic phospholipid profiles by 31P NMR. Tissue phospholipid profiles from epithelial, fibroblast and endothelial cell cultures were evaluated to differentiate the individual cell types and to identify resonances that typically appear in high-resolution phospholipid profiles of whole corneas. Phosphatidylcholine, phosphatidylethanolamine plasmalogen, an uncharacterized phospholipid at 0.13 delta, phosphatidylinositol, phosphatidylserine and sphingomyelin were determined to be, in decreasing order of concentration, the major phospholipids detected in these three cultured corneal cell types. Indices of phospholipid metabolism representing total plasmalogen content, total choline-containing lipids and the total choline-containing lipids less those synthesized through the plasmalogen pathway were found to differentiate the three cell types. Minor phospholipids cardiolipin, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine (LPC) and LPC plasmalogen not usually reported in studies of corneal phospholipids using other techniques, were useful in discriminating between cell types. Phospholipid profiles of the whole cornea provide important information concerning the biochemistry and pathology of the tissue, however, phospholipid analysis of individual components of the cornea, such as the epithelial, fibroblast and endothelial cells, makes it possible to understand the contribution of specific cellular constituents to the spectral information obtained from the whole cornea.

Survival of Acanthamoeba in contact lens rinse solutions.

Acanthamoeba may cause a severe keratitis in contact lens wearers. Since most sterilization techniques require rinsing the lenses prior to insertion, contaminated solutions represent a potential vector for transmission of Acanthamoeba. The ability of rinse solutions to sustain an inoculum of Acanthamoeba polyphaga was investigated. A. polyphaga was exposed to 0.1% benzalkonium chloride, 0.001% thimerosal/0.1% edetate disodium, 0.1% edetate disodium, saline, tap water, and distilled water. The status of the organism was evaluated with direct microscopic counts and cultures to confirm viability. Incubation with 0.1% edetate disodium, saline, tap water, and distilled water resulted in the maintenance of reduced populations of viable organisms for 7 days. Benzalkonium chloride preserved saline and solutions containing thimerosal with edetate rendered the Acanthamoeba nonviable.

Expression of two molecular forms of the complement decay-accelerating factor in the eye and lacrimal gland.

Complement is present in ocular fluids, but the molecular mechanism(s) restricting its activation to exogenous targets and not to autologous ocular cells are currently unknown. To clarify how this control is achieved, monoclonal antibody (mAb)-based techniques were used to examine the eye, the lacrimal gland, and ocular fluids for the decay-accelerating factor (DAF), a membrane regulatory protein which protects blood cells from autologous complement activation on their surfaces. Immunohistochemical staining of tissue sections revealed DAF antigen on corneal and conjunctival epithelia, corneal endothelium, trabecular meshwork, and retina, as well as on lacrimal gland acinar cells and in adjacent lumens. By flow cytometry, cultures of conjunctival epithelium exhibited the highest DAF levels and levels on corneal epithelium greater than corneal endothelium greater than conjunctival fibroblasts. Biosynthetic labeling of corneal endothelium yielded de novo DAF protein with an apparent molecular weight (Mr) of 75 kD, approximating that of blood cell DAF protein, and digestions of conjunctival epithelium with phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme which cleaves glycoinositolphospholipid membrane anchors, released approximately 70% of the ocular surface DAF protein similar to leukocyte surface DAF protein. Quantitations of DAF by radioimmunometric assay employing mAbs against two DAF epitopes revealed 325 ng/ml (n = 12), 4.8 ng/ml (n = 10), and 22.0 ng/ml (n = 8) of soluble DAF antigen in tears, aqueous humor, and vitreous humor, respectively. Western blot analyses of the tear DAF antigen revealed two DAF forms, one with an apparent Mr of 72 kD resembling membrane DAF forms in other sites, and a second with an apparent Mr of 100 kD, which is previously undescribed. Since DAF activity is essential physiologically in protecting blood cells from autologous complement attack, the identification of DAF on the ocular surface, intraocularly, in the lacrimal gland, and in tears suggests that DAF-mediated control of complement activation is also required in these locations.

Indications for keratoepithelioplasty.

Thirteen patients with ocular surface failure were treated by keratoepithelioplasty using allografts of corneal limbal epithelial cells from donor eyes. The ocular surface was stabilized with long-term healing of persistent epithelial defects in five of eight eyes followed up for 4 to 19 months. The procedure was performed on an additional 5 patients with superficial keratopathies. Three of those five procedures resulted in a stable and clear optical surface. These results suggest that epithelial transplantation may be a useful option in the care of chronic ocular surface failure unresponsive to conventional medical management.

Antibasement membrane antibody-mediated experimental conjunctivitis.

In ocular cicatricial pemphigoid, the binding of circulating antibodies to conjunctiva is believed to initiate an antibody-mediated cytotoxic response that results in inflammation and tissue damage. To develop a model of antibody-mediated conjunctival inflammation, we examined the effect on conjunctiva of local or systemic administration of a murine monoclonal antibody against basement membrane of stratified squamous epithelium. Neonatal rabbits were given either a single subconjunctival or intraperitoneal injection of the antibody. Eyes were graded clinically for inflammation and conjunctival biopsies were performed. After subconjunctival injection, clinical and histologic inflammation as well as murine antibody and rabbit complement binding to conjunctival basement membrane were detected. With systemic administration there was post-injection clinical inflammation, and conjunctival basement membrane-bound murine antibody was detected. There was no difference observed in conjunctival mitotic rate or goblet cell frequency between treatment groups and controls, following either route of administration. We have created, therefore, a model for antibody-mediated conjunctivitis in rabbits by local or systemic administration of a monoclonal antibody against a component of stratified squamous epithelial basement membrane.

Hyperproliferation of conjunctival fibroblasts from patients with cicatricial pemphigoid.

The characteristic conjunctival scarring in cicatricial pemphigoid is a consequence of subepithelial fibrosis. The fibroblast is the cellular element responsible for fibrosis. To increase the understanding of the pathogenesis of abnormal fibrosis in cicatricial pemphigoid, the growth characteristics of conjunctival fibroblasts, from untreated patients with cicatricial pemphigoid (n = 9) and normal controls (n = 6), were studied in tissue culture. The latent period until fibroblast outgrowth began from the conjunctival explant was determined, as were the plating efficiency and doubling time of cells from first-passage cultures. Outgrowths of fibroblasts from patients with cicatricial pemphigoid appeared significantly sooner than from controls, 8.1 +/- 3.8 vs 19.3 +/- 6.4 (mean +/- SD) days. While there was no significant difference in the plating efficiency between fibroblasts from cicatricial pemphigoid (mean +/- SD, 116.4% +/- 44.6%) and those from controls (71.0% +/- 39.4%), the doubling time was significantly faster for cicatricial pemphigoid than for controls, 26.5 +/- 8.5 vs 50.7 +/- 7.8 hours. Thus, conjunctival fibroblasts from patients with cicatricial pemphigoid are hyperproliferative in tissue culture when compared with normal controls. Therefore, scarring, which characterizes cicatricial pemphigoid, may be due, in part, to excessive fibroblast proliferation.

Diamond burring and surgical keratectomy. Morphologic comparison in the rabbit.

We compared the effects of diamond burring keratectomy (DBK) and surgical superficial lamellar keratectomy (SLK) on the rabbit corneal epithelial healing rate and corneal morphology 1, 4, 7, and 14 days after wounding. Epithelial defects healed significantly more slowly following 10-mm-diameter SLK than following DBK. Restoration of normal epithelial morphology occurred within two weeks in the DBK corneas. During the same interval, SLK corneas showed poor transition of basal to superficial cell morphology. Four days after surgery, the DBK corneas all exhibited marked keratocyte depletion in the anterior stroma but fibrocytes uniformly repopulated this area by 14 days after wounding. Conversely, the SLK corneas all had a hypercellular band of fibrocytes in the anterior stroma by four days following surgery and continued to increase in cellularity through 14 days after wounding. These data indicate that DBK is the preferred procedure for creation of a recipient bed for epithelial lenticles in keratoepithelioplasty and conjunctival transplantation.

Rapid diagnostic test for ocular adenovirus.

A new, direct, enzyme immunoassay test (Adenoclone, Cambridge BioScience, Hopkinton, MA) was evaluated for the rapid diagnosis of ocular adenovirus infections. In 36 culture-proven cases of adenovirus ocular infection, direct Adenoclone testing of conjunctival swabs was positive in 24 of 31 patients (77%) tested within 1 week of onset of symptoms, and in one of five patients (20%) who presented after 1 week (P less than 0.02). Overall sensitivity was 69%, whereas specificity was 100%. The authors conclude that a positive Adenoclone test is reliable in the rapid diagnosis of early adenovirus ocular infections.

The antiviral effects of rose bengal and fluorescein.

We evaluated the antiviral effects of rose bengal and fluorescein sodium. The direct antiviral activity was determined by an in vitro direct neutralization assay. The 50% inhibitory dose was 16 micrograms/mL for rose bengal and 460 micrograms/mL for fluorescein. The in vivo antiviral effects of these drugs were determined in the mouse herpetic keratitis model. Following topical application, rose bengal reduced surface virus titers (swabs) 1 million-fold, and residual ocular virus (eye homogenates) 32-fold, compared with controls. No infectious virus was recovered by swabbing after topical application of rose bengal. Fluorescein had no significant effect on virus replication. Thus, rose bengal, unlike fluorescein, has significant antiviral activity, and the diagnostic use of rose bengal prior to viral culture may preclude a positive result. Also, the use of rose bengal to grade keratitis in the study of new antiviral agents should be discouraged.

Epikeratophakia for control of pediatric bullous keratopathy.

A child who had had a congenital cataract phacoemulsified at age four months became contact lens intolerant, and at age two years had an implantation of a Worst metal-looped iris-clip intraocular lens. He developed painful pseudophakic bullous keratopathy three years later. Since the child remained with dense deprivation amblyopia and contact lens intolerance, when the IOL was removed an epikeratophakia graft was applied. The bullous keratopathy resolved and the patient has remained asymptomatic for 22 postoperative months. The proposed mechanism for relief of the bullous keratopathy is to increase tissue thickness and resistance, thus reducing fluid volume transferred to the subepithelial space.