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Report of the Corneal Diseases Panel

The cornea is the transparent convex tissue at the front of the eye (figure at right) that serves two specialized functions. First, it forms, with the sclera, a protective physical barrier that shields the inner eye from the external environment. Equally important is its ability to protect itself from various types of damage, ranging from physical trauma and biochemical injury to infections by myriad pathogenic organisms, to the deleterious effects of long-term exposure to light itself. In protecting itself, the cornea also safeguards many underlying ocular structures from similar damage. Second, the cornea serves as the main refractive element of the visual system, directing incoming light onto the crystalline lens, which focuses it onto the retina. Refraction depends on the cornea acquiring transparency during embryonic development and maintaining it throughout adult life.

Although the cornea, which continues laterally with the layers of the sclera, appears to be one clear membrane, it is composed of several discrete layers (figure above):

Conjunctiva—This is the thin, flexible layer of tissue covering both the inner surface of the eyelids and the sclera (the white of the eye). Its elasticity contributes to the ease of eye movements; its goblet cells contribute to the tear film by producing mucus; it heals rapidly with little formation of opaque scar tissue.

Tear film—The liquid tear layer bathing the cornea and conjunctiva performs optical, protective, and lubricative functions. It creates a perfectly smooth liquid outer layer that polishes the corneal surface, mechanically traps and flushes out foreign bodies and chemicals, contains bacteriostatic substances that inhibit the growth of microorganisms, and reduces the surface friction associated with eyelid blinking and eye movement.

Corneal epithelium—This is a renewable, transparent tissue that, along with the tear film, forms a refracting optical surface. It is the only corneal tissue that is innervated and can signal pain. Like all epithelia, it presents a barrier against the external environment, with intercellular junctions that prevent invasion by pathogens. The epithelium usually must be damaged before an infectious agent can become established.

Corneal stroma—This layer of connective tissue, composed of extracellular matrix molecules and collagen fibrils, is responsible for the strength and shape of the cornea. Although other collagenous structures in the body are opaque (such as cartilage or skin), the corneal stroma is transparent. It is constructed from lamellar collagen fibrils that are small, uniform diameter, and equidistant. Some proteoglycans (PGs) and collagens are unique to the cornea, as is the total lack of blood vessels.

Corneal endothelium—The main physiologic function of this thin inner layer is water transport. Corneal transparency hinges on the precisely controlled hydration required to maintain the stromal matrix structures in their correct spatial organization. The endothelium forms a barrier from the aqueous humor, based on the tight junctions and gap junctions between individual cells, and it maintains a fluid pumping mechanism controlled by a membrane-associated enzyme (Na⁺,K⁺-ATPase) that transports excess water out of the stroma. Without this pumping action, the stroma would develop edema, becoming swollen with water and cloudy. The human corneal endothelium has no regenerative and only limited repair capabilities. If endothelial cells are destroyed by disease or trauma, the remaining cells must enlarge and migrate to maintain function. When sufficient endothelial cell loss occurs, corneal edema and blindness ensue, with corneal transplantation as the only available therapy.

In this country, corneal diseases and injuries are the leading cause of visits to eyecare clinicians. These are also some of the most painful ocular disorders. These facts alone underscore the need for laboratory and clinical research aimed at improving treatment for or preventing these diseases and injuries. Corneal problems result largely from the cornea's location as the outermost structure of the eye, but genetic disorders also contribute. Laboratory and clinical research performed during the last 5 years—largely funded by the National Eye Institute (NEI)—has made great progress in understanding and treating corneal disorders. Researchers now know many of the molecules involved in transparency and how these function. They know the origin of the cells that continuously replace those of the corneal epithelium. They also know some of the factors that may be involved in the cells' regulation, which has allowed for the amplification of these cells in culture. This knowledge has recently been applied to restoring the human corneal surface with cells grown from the patient's own eye. This has resulted in treatment for certain conditions that previously resulted in blindness. Procedures such as grafting of placental membrane or corneal limbal epithelial stem cells have aided epithelial healing in other difficult situations.

Research on the cornea has also developed knowledge that can be applied to problems in other organ systems. For example, the cornea has long been known to be favorable for transplantation, and this procedure has become routine. Studies on the properties of the eye that make the cornea such a "privileged" immune site raise the possibility that this property can be conferred to other tissues, thus facilitating the transplantation of other organs. Studies on the molecular structure of collagen fibrils in the corneal stroma have not only provided basic information on the assembly of this tissue, they have also contributed insight into certain developmental defects of the skeletal system and blistering diseases of the skin. Moreover, since the cornea is constantly exposed to ultraviolet (UV) light and oxidative stress, it has provided information on ways that cells can protect themselves from this damage (such as the production of antioxidative enzymes and nuclear ferritin). These mechanisms could provide ways to protect other cells and organs from similar environmental insults.

In Fiscal Year 1997, the NEI funded 179 extramural research projects in the Corneal Diseases Program at a total cost of $38,610,000. These projects covered six broad areas:

Physiology.These grants are devoted to increasing understanding of fluid and ion transport processes in the cornea and conjunctiva that affect transparency and wound healing. Research was also conducted on the structure and physiology of the tear film and ocular surface to develop rational treatments for dry-eye conditions.

Cell Biology.This area of research supports investigation of corneal growth, development, and wound healing.

Genetics.These projects study corneal gene expression, inheritance of corneal dystrophies, and identification of genes encoding risk factors for the development of corneal anomalies.

Immunobiology. This section supports studies of the etiology of ocular immune privilege and the corneal immune response, corneal transplantation, the nature and regulation of corneal inflammation, and the role of immune cell subpopulations in ocular disease.

Infectious Diseases. Work in this area focuses on the pathogenesis, diagnosis, and treatment of disease caused by potentially blinding agents such as viruses (particularly herpes simplex virus), bacteria, and parasites, and on conditions such as adenoviral epidemic keratoconjunctivitis (commonly known as pink eye), which are associated with very high morbidity and economic costs.

Correction of Refractive Error.Approximately 60 percent of Americans have refractive errors or defects in the ability of their corneas to focus light, which could be corrected to give them sharper vision. This subprogram supports projects to understand the topographic and biomechanical properties of the cornea that result in a normal refraction, to understand the biological effects of contact lens wear on the cornea, to develop instrumentation to measure and correct refractive error, to understand the response of the cornea to refractive surgery, and to investigate the epidemiology of refractive error in the American population.

Thus, the overall goal of the Corneal Diseases Program is to:

Understand the normal function of the cornea and apply this knowledge to the prevention and treatment of traumatic injury and disease.

ASSESSMENT OF PROGRESS

Molecular mechanism of corneal fluid transport.The eye contains a large mass of water and is composed of several specialized cell tissue layers that have fluid movement at the core of their function. The cornea possesses two such layers: the endothelium, which covers the internal side of the cornea, and the epithelium, which covers its external face. Corneal transparency hinges on a precisely controlled degree of hydration that is required to maintain corneal matrix structures in their correct spatial orientation. Although it is known that transport mechanisms present in the endothelium are responsible for corneal dehydration and transparency, details on the coupling between electrolyte transport and water movement have been elusive. The driving force for fluid transport is the osmotic gradient created by the transport of solutes, and it has long been recognized that cell membranes contain specialized structures, or pores, which provide selective passage for water molecules, but the details of solute-solvent coupling have remained obscure.

In the great majority of epithelia, the energy for water transport is derived from the operation of an Na+:K+ pump that, either directly or through activation of secondary transporters and ion channels, produces the appropriate osmotic environment. Although similarities exist between fluid transport among epithelia of various organs, each one presents a particular challenge to the investigator. In epithelial layers the cells are polarized so that the properties and elements of the basolateral and apical membranes are different. Thus, electrolytes and water must cross two distinctive membranes during transepithelial movement. This, and the multiple layers of cells in the corneal epithelium and conjunctiva, represent additional degrees of complexity in the study of these ocular membranes. A recent major advance in the study of corneal fluid transport has come from the discovery of the aquaporin (AQP) family of water channel or pore proteins. There has been an explosion of work to identify, clone, and sequence the AQP genes; to localize individual transporters; and to determine their function. Other membrane-spanning proteins such as the glucose transporter and K⁺ channel proteins exist in sufficient abundance that they might also contribute to the transcellular water movement. The lipid bilayer of the cell membrane also allows a substantial flow of water, but unlike protein channels, the bilayer is less susceptible to solute interaction and regulation.

To date, seven AQPs have been identified and cloned using techniques of molecular biology. AQP-0, which exhibits a lesser degree of water permeability than other family members, is known to be the major intrinsic protein of lens fiber cells. AQP-1 (a small channel-forming integral membrane protein) has been found in various ocular epithelia including the iris, ciliary body, and lens, and is constitutively expressed in the corneal endothelium. AQP-3, which may also transport glycerol, is found in the conjunctiva. AQP-4 is abundant in retinal Müller cells and in the brain, where it appears to function in cerebrospinal fluid absorption. AQP-5 is observed in the secretory lobule of the lacrimal gland and in the corneal epithelium. AQP-5 exhibits a structural feature similar to that in AQP-2, which is now recognized as a critical water-transporting element in the collecting duct of the kidney. AQP-6 is found in the retinal pigment epithelium.

Corneal dystrophies. The corneal dystrophies are a heterogeneous group of conditions that involve abnormal corneal development and result in defects in structure or clarity. These diseases are usually inherited and do not affect other parts of the body. They may begin early in life, but can also manifest with age.

The most common corneal dystrophy in the United States is keratoconus, a progressive thinning process that may be accompanied by scarring. Keratoconus leads to progressive nearsightedness, astigmatism, and a cone-shaped cornea. It has been estimated to occur at a rate of 1 in 2,000 people in the general population. Clinical care for keratoconus is time consuming for patients and doctors because of its chronic progression and the difficulty of achieving a stable contact lens fit for visual rehabilitation. Keratoconus is the most frequent reason for penetrating keratoplasty in the Western world, and it accounts for $50 million to $100 million a year in medical care costs in the United States.

Keratoconus has become better understood during the last 5 years through investigations into the genetic predisposition for the disease, detection of early forms of the disorder through computerized topographic analysis, initiation of an NEI-funded prospective assessment of the progression of the disease (the Collaborative Longitudinal Evaluation of Keratoconus Study or CLEK), and advances in understanding the enzymology that underlies corneal thinning. Diagnosis of the disease may become more certain in the future through the application of noninvasive tests and refinements in corneal topographic analysis. Numerous corneal topography and contact lens innovations are also being introduced to assist in diagnosis and treatment.

Recent biochemical investigation into the pathogenesis of this disorder suggests that the loss of corneal stroma could result from either increased levels of proteases and other catabolic enzymes or decreased levels of proteinase inhibitors. Studies of corneal α-1 proteinase inhibitor and α-2 macro- globulin support the hypothesis that degradative processes may be aberrant in keratoconus. Both inhibitors are markedly diminished in the epithelium of keratoconus corneas. These biochemical observations may merely reflect a more generalized keratocyte abnormality in keratoconus, since proteinases are released upon cell death from apoptosis (a regulated program of cell death). Compared with normal corneal keratocytes, those from keratoconus corneas have a fourfold greater number of cell surface receptors to the cytokine interleukin-1 (IL-1), an inducer of apoptosis. This increased receptor expression may sensitize keratocytes to IL-1-induced apoptotic death. This hypothesis is consistent with the relationship between keratoconus and eye rubbing, contact lens wear, and allergic hypersensitivity, as trauma could lead to an increased release of IL-1 from the epithelium.

The literature strongly suggests genetic influences in the pathogenesis of keratoconus, including family clusters, twin studies, bilaterality, and the symmetry of topographic alterations between eyes of individual patients. Although recent family studies suggest an autosomal dominant mode of inheritance with variable expression, additional genetic analyses are required to accurately define the role of genetic influences. Studies of families that demonstrate clear inheritance patterns could provide new insights into the pathogenesis of this disorder.

Genetic study of afflicted families has recently yielded new insight into the pathogenesis of other, rarer inherited corneal dystrophies (see Table 1 below).

Table 1.  Gene mapping of corneal dystrophies and disorders
Corneal disease	Gene locus
Rieger's Anomaly (iridocorneal mesodermal dysgenesis)	4q
Avellino, lattice type I, Reis Bucklers', and granular dystrophy	5q31
Groenouw type I (keratoephithelin protein)
Lattice type II dystrophy	9q34
Thiel-Behnke dystrophy	10q23-q24
Peter's Anomaly (Pax-6)	11p
Cornea plana	12
Meesmann's corneal dystrophy (keratin K3, K12)	12q and 17q
X-linked megalocornea	Xq
Fish-eye disease (x-lecithin: cholesterol acyl transferase)	16
Posterior polymorphous dystrophy	20

Linkage analysis has shown that four clinical types of corneal dystrophy result from mutations in a single gene. Granular, Reis Bucklers', lattice type I, and Avellino corneal dystrophies all map to the βig-h3 gene, which encodes the keratoepithelin adhesion protein. It appears that in these four corneal dystrophies, the mutated keratoepithelin forms amyloidogenic intermediates that precipitate in the cornea, causing a progressive opacification. Three other corneal diseases also involve amyloid-like deposits: polymorphic amyloid degeneration, lattice corneal dystrophy type IIIA, and gelatinous drop-like dystrophy. Keratoepithelin is a good candidate gene for further investigation in these families. Because of the accessibility of the cornea, these disorders represent excellent model systems for study of the molecular details of amyloid deposition in devastating diseases such as Alzheimer's disease.

Several other corneal dystrophies are being explored through genetic studies. Meesmann's corneal dystrophy, in which the cornea accumulates numerous small, round, debris-laden intraepithelial cysts, has been shown to be associated with defects in the cornea-specific keratin K3 or K12 genes on chromosomes 12 and 17. Posterior polymorphous corneal dystrophy, characterized by corneal endothelial vesicles and associated with glaucoma in 15 percent of cases, maps to a site on chromosome 20. Fish-eye disease, associated with large cloudy corneas, is a defect in the α-lecithin:cholesterol acyl transferase gene on chromosome 16. Segregation analysis has shown that severe astigmatism, a refractive error characterized by blurring of vision, is associated with a major autosomal dominant locus.

The challenge now is to refine the genetic loci associated with corneal dystrophies and clone the responsible genes. Continued molecular genetic studies of the corneal dystrophies will improve scientists' understanding of the etiology, pathogenesis, and diagnosis of their dystrophies and hopefully will suggest new avenues for therapy.

Development, growth, and wound healing.For the cornea, as well as many other ocular structures, the extracellular matrices provide the structural and frequently the functional organization. Virtually every structure of the eye is composed of one or more extracellular matrices or has an extracellular matrix associated with it. In the cornea, the matrices include the stroma, Bowman's layer, Descemet's membrane, and the corneal epithelial basement membrane. Scientists have made strides toward understanding the molecular composition and the details of the assembly of these matrices. They have also increased their understanding of the degradative enzymes, largely matrix metalloproteinases (MMPs), involved in corneal remodeling during growth and wound healing.

Corneal matrices are chiefly composed of two classes of proteins: collagens and PGs. Many new species have been identified recently, and certain structural features have been deduced using molecular biology techniques. The known collagens now number 20, and at least 13 of these participate in the development and assembly of the different corneal matrices. The number of known PGs is also increasing rapidly, as evidenced by the discovery that the keratan sulfate PGs that characterize the cornea comprise a family of molecules.

Functional studies on corneal collagens and PGs have suggested mechanisms for creating the thin, uniform-diameter collagen fibrils that are characteristic of the corneal stroma and are thought to be a factor essential for transparency. One model, for which there is both structural and functional evidence, involves the formation of heterotypic fibrils, coassemblies of more than one type of collagen, with one type serving a regulatory function. Heterotypic fibrils were first described in the cornea, but are now known to occur in other connective tissues. Other types of collagen, such as the fibril-associated species that bind to the fibril surface, have been identified in the cornea. They are differentially expressed during development and wound healing, and studies suggest that they may alter the interactions between fibrils. Different types of PGs are also known to bind at specific sites along corneal collagen fibrils, and assembly studies suggest that these may also be involved in regulating fibril diameter. The assembly of corneal collagen fibrils in orthogonal arrays undoubtedly also contributes to transparency. Here again, PGs may play a role, but much remains to be learned about the mechanism by which more perfectly arranged fibrils can be induced to form during wound healing.

The MMPs are a family of 18 structurally related enzymes catalyzing cleavage of matrix components. They have recently been implicated in the remodeling that occurs during development, wound healing, or following laser surgery. They appear in a precisely controlled sequence that suggests they perform specific roles. Cellular sources of MMPs include resident corneal cells and invasive inflammatory cells. Regulation of MMP expression can occur at the level of synthesis, as certain transcription factors have been implicated in differentially regulating their expression. MMP levels can also be controlled by tissue inhibitors of metalloproteinases (TIMPs). Changes in the levels of MMPs and TIMPs have been correlated with pathologic conditions, such as stromal ulceration, keratoconus, and failure to reepithelialize. Animal studies suggest that recombinant TIMPs can ameliorate these conditions. MMP gene expression can be regulated by growth factors, inflammatory cytokines, and constituents of wounds such as fibronectin fragments.

Hemidesmosomes and transmembrane collagen. The hemidesmosome is a complex structure on the basal surface of corneal epithelium. Both the epithelium and the corneal fibroblasts participate in the hemidesmosome's development, the latter by producing matrix components that induce hemidesmosomes to differentiate. The hemidesmosome promotes epithelial attachment to the underlying extracellular matrix through transmembrane receptors that link hemidesmosomal components with keratin fibrils in the cell. New insights into the function of the hemidesmosome have come from the discovery of autoantibodies to a 180 KD protein (BP 180) in the serum of patients with the blistering disease called bullous pemphigoid. BP 180 is a transmembrane component that is associated with a subunit of the hemidesmosome α6β4 integrin. BP 180 is a novel transmembrane ligand of α6β4 that is necessary for normal hemidesmosome formation, and mutations in BP 180 and other hemidesmosomal components are likely to play a role in ocular blistering diseases. It has been suggested that abnormalities in these complexes are involved in persistent epithelial defects or recurrent epithelial erosions of the cornea. These conjectures need to be examined.

Stem cells. Biochemical and biological studies have suggested that the corneal epithelial stem cells necessary for normal regeneration reside in the limbus (see figure), the narrow peripheral zone of cornea bordering the conjunctiva. These stem cells are postulated to be the progenitor cells necessary for maintaining the normal corneal epithelium. Two recent observations are consistent with this hypothesis. First, removal of the limbal epithelium leads to a spectrum of corneal surface abnormalities such as conjunctival epithelial ingrowth, vascularization, and chronic inflammation. Second, limbal transplants have been shown to result in much better corneal epithelial repair than conjunctival transplants. In a recent report, limbal epithelial cells obtained from a patient's uninvolved eye were cultured, passaged, and grown to confluence to replace conjunctival epithelium removed from the diseased eye. The clinical results were promising, and this technique deserves continued development and evaluation.

An alternate approach that has been recently developed involves transplantation of amniotic membrane cells to cover the perilimbal environment before limbal allograft transplantation. This intervention results in decreased inflammation and vascularization and enhances graft survival.

Regulation of corneal cell division. The discovery of proteins called cyclins, which appear and disappear during the mitotic cycle, has provided a major insight into the mechanism of cell division. Cyclins act as regulatory subunits to activate enzymes known as cyclin-dependent kinases (Cdks). Progression from G1 to S phase of the mammalian cell cycle is regulated by Cdks and by cyclin E-dependent kinase (Cdk2). Subsequent transition through the cycle requires the action of at least three other cyclins.

Of considerable importance to corneal research is the recent finding that that fibrillar collagen inhibits cell proliferation by regulating Cdk2. When smooth-muscle cells are grown on polymerized collagen, phosphorylation of Cdk2 by its kinase is inhibited and levels of Cdk2 inhibitors increase. In contrast, cells grown on monomeric collagen divide normally. Experiments with blocking antibodies indicate that fibrillar collagen specifically regulates Cdk2 activity by stimulating the signaling by extracellular matrix receptors that leads to increased levels of Cdk2 inhibitors and inhibition of proliferation. In the developing cornea, a significant increase in the concentration of fibrillar collagen is associated with dramatic downregulation of keratocyte proliferation. This might be mediated by inhibition of Cdk2, and it is possible that changes in the fibrillar collagen of the stroma play a role in regulation of corneal fibroblast proliferation during corneal disease. If this is the case, it opens new avenues for therapy.

Corneal gene expression. A molecular analysis of developmental and cellular processes in the cornea has lagged behind that in other ocular tissues, largely from the lack of a tissue-specific promoter. However, progress has been made recently in studies of gene expression involved in creation of the corneal extracellular matrix (section C) and in the differentiation of corneal epithelium, as will be described here. First, at least two keratins (K3 and K12) appear to be produced specifically in the corneal epithelium, and studies are underway to identify the promoter elements that control the expression of their genes. Another opportunity comes from the high expression of certain enzymes in the corneal epithelial cells; the tissue-specific promoters for these genes are candidates for use in directing foreign gene products to the cornea.

The most highly studied abundant enzyme in the corneal epithelium of mammals is aldehyde dehydrogenase class 3 (ALDH3). It comprises up to 40 percent of the soluble corneal protein and can be induced by oxidative stress. Transketolase is another abundant corneal enzyme that is expressed in most other tissues at lower concentrations. Alpha-enolase is also found at unusually high concentrations in the cornea. This enzyme is of special interest because it is preferentially expressed in the circumferential limbal cells of the cornea, where the stem cells reside, with potential implications for delivering gene therapy to the cornea.

There has been less progress in the identification of specific gene products in endothelial cells. Several investigators are presently conducting experiments to identify highly expressed specific genes in the various layers of the cornea. These studies are beginning to be fruitful, and a number of novel genes have been found.

The status of gene expression in cornea differs from the high levels of expression of enzymes and crystallins in the lens, where regulation occurs through developmental processes rather than environmental induction. No specific transcription factors regulating corneal genes have been identified as yet, but Pax-6 is a major candidate for study. Pax-6 is highly expressed in the developing anterior segment; has been shown to have a direct role in the regulation of crystallin genes in the lens; and induces eye formation in all animals, including invertebrates. Moreover, Pax-6 mutations are associated with human anterior segment diseases such as aniridia (absence of the iris) and Peters' Anomaly (faulty developmental separation of the iris and cornea).

The high expression of enzymes in corneal epithelial cells is reminiscent of the recruitment of enzymes and stress proteins as refractive crystallins in the lens. In the lens, different enzymes are used as crystallins in different species, so the enzyme crystallins are taxon specific. Similarly, the abundant enzymes in the corneal epithelium may differ among species. For example, ALDH3 is found in mammalian corneas but not in chicken or fish corneas. The unexpectedly high concentrations of these corneal enzymes suggest that they may play both enzymatic and nonenzymatic roles in the cornea. This is analogous to the situation in the lens, where the abundant crystallins have refractive and nonrefractive functions—a strategy called gene sharing. ALDH3 appears to protect the corneal epithelium against oxidative damage resulting from the continual surface exposure to the environment, especially to UV light. It has also been suggested that ALDH3 protects the cornea, as well as the rest of the eye, from oxidative stress, by directly absorbing UV radiation. Indeed, since relatively few enzymes comprise the majority of the water-soluble proteins of the corneal epithelial cells, it has been proposed that they be collectively called "absorbins." Finally, it is even possible that, in addition to being UV filters, the abundant corneal enzymes contribute to transparency by minimizing concentration fluctuations and providing a continuous refractive index within the cytoplasm. The discovery of the possible multiple roles of the abundant corneal enzymes is an exciting challenge for future research.

Immunopathology of corneal infections. A growing body of evidence has demonstrated that the pathology of many corneal infections is mediated by the immune system. This is particularly clear in the case of the host immune response to corneal infection with herpes simplex virus-1 (HSV-1). HSV-1 infection of mouse strains deficient in T-cells (nude mice) fails to produce corneal inflammation or keratitis, and mice whose corneas are purged of antigen-presenting cells develop a milder and briefer keratitis than intact animals. Individuals infected with the human immunodeficiency virus (HIV) usually do not develop stromal keratitis, even after severe HSV-1 epithelial infection. Results from studies of repeated exposure to trachoma antigens implicate chronic delayed-type hypersensitivity responses in the production of the characteristic lesions in this blinding disease. Studies of corneal bacterial infections with Pseudomonas or Staphylococcus show that specific subsets of T-lymphocytes are crucial for the development of ocular pathology.

Herpetic disease. The NEI-sponsored Herpetic Eye Disease Study (HEDS) has recently provided valuable new information about the natural history of HSV-1 epithelial keratitis. Patients with a past history of HSV stromal keratitis or iritis were found to be significantly more likely to develop it again after an episode of dendritic keratitis, and thus should be followed closely. These patients should be taught to seek ophthalmic care promptly if they become symptomatic, since past HEDS results demonstrated the efficacy of treatment with topical corticosteroids and prophylactic antivirals. Ongoing HEDS protocols are examining the role of longer term, lower dose, systemic aciclovir in preventing recurrent manifestations of HSV keratitis and determining the risk factors for recurrences.

Corneal HSV-1 infection is potentially blinding, requires frequent office visits, and contributes to a substantial loss of work. Permanent structural damage to the cornea requires surgical intervention and is the cause of over 1,000 penetrating keratoplasties annually in the United States. Acute primary infection of the corneal surface produces virus-induced cell death. In contrast, stromal disease occurs as the result of recurrent infection and involves an immunopathologic process that often leads to scarring, ingrowth of blood vessels, endothelial dysfunction, and vision loss. Understanding the biology of the HSV-1 latency/reactivation/recurrence cycle, and then interfering with reactivation at the molecular level, is likely to be the most efficient means of ameliorating and preventing herpetic keratitis. Recent research has shown that latency-associated transcript (LAT), the only HSV-1 gene abundantly transcribed during latency, is essential for efficient spontaneous reactivation, and that this function maps to the first 20 percent of the LAT transcript.

Recent studies have indicated that the induction of programmed cell death (apoptosis) of lymphocytes by members of the tumor necrosis factor-fas (TNF-fas) receptor family may be a protective mechanism by which the eye limits the extent of inflammation caused by HSV-1 infection. The regulation of interactions between the apoptosis-inducing ligand and its receptor appear especially worthy of study to those interested in ocular immune privilege and the control of extensive ocular immunopathology. The eye remains an ideal organ to study these processes in general. It is readily accessible and mutant strains of small animals with specific genetic defects in members of the fas and TNF receptor families are now available.

Although neutrophils play a role in viral clearance, they also perpetuate T-cell-mediated inflammatory reactions. The cytokine interleukin-2 (IL-2) has recently been shown to mediate corneal inflammation by upregulating the local production of other cytokines that establish a neutrophil-chemotactic gradient and maintain neutrophil viability in the cornea. Molecular studies have recently described a host shutoff mutant of HSV-1 with a restricted ability to invade the corneal epithelium. Recent work in a mouse model suggests that vaccination with HSV glycoprotein gK exacerbates herpetic corneal scarring. This model may be useful in determining and characterizing the protective immune responses generated against HSV-1. Studies in rabbits indicate that local ocular vaccination is much more efficient than systemic vaccination at protecting against both primary and recurrent ocular HSV-1 shedding and corneal disease. This suggests that enhancing local ocular immunity should be targeted in developing a vaccine to combat HSV-1 ocular disease.

Bacterial infection.Corneal infection by Pseudomonas aeruginosa often results from contact lens wear and can lead to a highly destructive process resulting in loss of vision. The corneal destruction associated with this organism is thought to be due both to the response of the host and to bacterial proteases acting on corneal tissues. P. aeruginosa possesses a number of virulence factors, such as cell-associated pili and extracellular enzymes such as elastases, alkaline protease, and exotoxin A. Once infection has occurred, complex tissue reactions are initiated that include inflammation, formation of new blood vessels, and degradation of the stromal matrix. Various other host factors play a role in this tissue destruction, including enzymes from infiltrating inflammatory cells. Recent research in an aging mouse model has suggested that failure to upregulate the intercellular adhesion molecule-1 (ICAM-1) in corneal tissues may reflect a reduction of both IL-1 and γ-interferon levels in the infected cornea. This lack of ICAM-1 appears to result in delayed recruitment of neutrophils and other inflammatory cells into the cornea.

Staphylococcus aureus occupies a dominant position in bacterial diseases of the eye. In addition to causing direct infections of the external eye and intraocular tissues, it is also responsible for hypersensitivity diseases of the external eye. Animal models of these entities have been developed in rabbits, where ribitol teichoic acid was found to be the relevant antigen involved in an antibody-mediated immunopathogenesis.

Recent studies into the role of complement in bacterial endophthalmitis have shown that decomplemented guinea pigs demonstrated impaired host defense against Staphylococcus. Additionally, immunologic or chemical injury to human donor corneas showed that terminal components of the complement cascade can be generated in corneal tissue. The results of these studies suggest that complement is important to the host defense against ocular infection.

Immune compromise. Many immune responses are downregulated within the eye. (See Retinal Diseases Panel Report.) The prevailing teleological explanation is that the eye is designed to restrain inflammatory responses that could inflict collateral damage to innocent bystander cells in the eye, leading to a loss of transparency. This is a unique immunological adaptation because it disarms a major immunological effector mechanism that could be enlisted to protect the eye against myriad pathogens. Thus, limiting the extent of intraocular inflammatory responses represents a compromise in which vision-threatening immune responses are silenced at the risk of opportunistic infection.

Immunological privilege of the anterior chamber and the cornea is multifaceted and involves a large number of adaptations. These include anterior chamber-associated immune deviation; anti- inflammatory and inhibitory effects of aqueous humor constituents, such as transforming growth factor-β on the induction and expression of delayed-type hypersensitivity; low expression of major histocompatibility complex (MHC) class I antigens; wide expression of FasL (the fas ligand); presence of a potent inhibitor of natural killer cells in the aqueous humor; presence of complement-regulatory proteins in the aqueous humor and on the corneal endothelium; absence of lymphatics draining the anterior chamber; and absence of antigen-presenting cells, especially Langerhans' cells, in the central regions of the cornea.

New information about self-tolerance has emerged from genetic studies. The demonstration that strains of mice with variations in the immunoglobulin-heavy-chain locus are more resistant to HSV-1 keratitis than normal mice represents an important starting point for ocular studies. Analyzing differences in the immunoglobulin-heavy-chain between susceptible and resistant strains demonstrated the involvement of a self-antigen. This self-peptide could be used to confer resistance to HSV-1 keratitis. The importance of this observation relates to a general principle that sequences of peptides of some ocular tissues may be important for the preservation of self-tolerance and autoimmunity. Future studies should be directed at understanding the mechanism of tolerance induction and how to use this information to create new immunologically based pharmaceuticals to treat eye injuries. Recognition that the eye offers an extremely accessible model for study of the immune system is perhaps the most important development in the last several years and should continue as the understanding of immune privilege and tolerance advances.

The aqueous humor may limit pathogenic or autoimmune responses, since molecules found in the aqueous humor appear to dampen lymphocyte functions. How the levels of these molecules are regulated and whether they can be pharmacologically manipulated offer novel therapeutic approaches. Unique features of the accessible surfaces of the eye offer opportunities for development of small molecules that disrupt immunological or inflammatory processes. Wound healing of the cornea may be amenable to treatment with small, rationally designed peptidomimetics derived from receptors involved in inflammation. Study of the TNF family of receptors has recently led to the development of small molecules that interfere with cell death and may be topically active for treating corneal injuries. These studies illustrate the need for detailed structural understanding of the molecules involved in ocular injury and inflammation. Structural analysis of receptors required for microbial pathogenesis, immunity, and cell-cell contact are all likely to lead to new therapies.

Lacrimal gland physiology. The goals of research in this area have been to understand the causes of lacrimal insufficiency and to develop rational treatments for dry eye. The nature and regulation of tears is becoming better understood, and a great deal of work has been done on the structure and function of mucins, regulation of lacrimation by hormones, and the function of the lipid layer. However, the clinical diagnosis and treatment of dry eye, although often investigated, is still not well understood. Objective and more well-validated, clinically useful tests for dry eye are needed.

Several lines of investigation identified as priorities in the last national plan have progressed significantly. Identification of G proteins and protein kinase isoforms has increased understanding of the pathways that transduce stimulatory nerve signals into the intracellular chemical messages that control lacrimal glandular secretion. Changes in innervation of the lacrimal glands and in aspects of lacrimal gland electrophysiology have been shown to precede local autoimmune phenomena in mouse models for Sjögren's Syndrome. Recognizing the presence of growth factors with potential regulatory actions, both in the lacrimal gland and at the ocular surface, has expanded scientists' view of secretory control. Trophic actions of the innervation of the lacrimal glands have been revealed. A substantial body of evidence has been developed supporting the thesis that androgens and related steroid hormones influence immunosuppressive phenomena in the lacrimal glands.

The hypothesis that lacrimal gland secretory cells actively provoke Sjögren's Syndrome autoimmune responses has gained support from analyses of the intracellular traffic of histocompatibility molecules and autoantigens. This hypothesis appears to be gaining further support from new experiments based on autologous mixed cell reactions that may recreate autoimmune responses under defined cell culture conditions. A new autoantigen (the cytoskeletal component α-fodrin), implicated in Sjögren's Syndrome autoimmunity, has been identified. This autoantigen appears to have considerable specificity, since antibodies to it were found in the serum of 95 percent of patients with Sjögren's Syndrome. No antibodies were found in normal individuals or in patients with other autoimmune disorders such as systemic lupus erythematosus and rheumatoid arthritis. Thus, it may have considerable diagnostic potential. Moreover, neonatal vaccination with α-fodrin prevented development of the disease in mice, opening the possibility of new therapeutic approaches for Sjögren's Syndrome.

Unexpected discoveries have opened new lines of investigation that may hasten the discovery of more effective therapies for dry eye. Experimental studies with animal models and observational studies with humans have indicated that androgen sex hormones and prolactin modulate the functional status of the lacrimal gland. This work has inspired a general theory that explains why women should be vastly more likely than men to develop both primary lacrimal gland deficiency and Sjögren's autoimmunity, and it suggests hormone modulation therapies that may prevent and treat primary lacrimal deficiency. Moreover, there have been preliminary reports that the androgens modulate the function of the meibomian glands and the lacrimal glands, offering the possibility that hormone treatment may effectively treat dry eye related to lipid-layer deficiency. There have been preliminary reports that androgen withdrawal activates pathways leading to classical apoptotic death of interstitial cells and to nonapoptotic death of secretory epithelial cells in the lacrimal glands. These observations, if substantiated in further work, would help account for the lacrimal gland atrophy that has been reported to follow androgen loss. Moreover, since known autoantigens are present in apoptotic cell fragments, this phenomenon indicates another pathway that may lead to Sjögren's Syndrome autoimmunity.

Lipid mediators of inflammation and nonsteroidal anti-inflammatory drug therapy. Understanding how the cornea metabolizes lipids to form mediators of inflammation and wound healing has advanced markedly in recent years. A major avenue of investigation has focused on the metabolism of the fatty acid arachidonic acid. Arachidonic acid may be oxidized by the cornea using three distinct enzymatic pathways, all of which produce biologically active molecules.

The first pathway, cyclo-oxygenase pathway, transforms arachidonic acid into prostaglandins, thromboxanes, and prostacyclin. These compounds alter the permeability of blood vessels and the aggregation of platelets (blood cells involved in blood clotting). The enzyme that converts arachidonic acid to the precursor for the prostaglandins is inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin, ketorolac, and diclofenac. The anti-inflammatory properties of NSAIDs derives, in major part, from their ability to inhibit prostaglandin production. Several NSAIDs have been introduced into the clinical armamentarium during the past few years. These have proven beneficial in relieving symptoms of allergic conjunctivitis and in controlling pain and inflammation following refractive surgery or cataract extraction.

The second route, lipoxygenase pathways, produce arachidonic acid metabolites that appear to be involved in many aspects of inflammation and wound healing. In the cornea, products of these pathways have been implicated in recruiting inflammatory cells into the cornea and in regulating epithelial wound healing.

The third route for metabolism of arachidonic acid is by epoxygenase pathways. The cornea is a particularly fruitful system for studying these pathways, as evidenced by the seminal contributions to this field using the cornea. Epoxygenase pathway products may be involved in regulating the influx of white blood cells into the cornea following injury and may play a role in the infiltration of blood vessels into the cornea after severe or prolonged injury.

An alternative mechanism by which the cornea may regulate inflammation is by producing platelet-activating factor. Proinflammatory properties ascribed to platelet-activating factor include induction of platelet aggregation, constriction of blood vessels, enhanced release of other mediators that cause dilation and increased permeability of blood vessels, increased arachidonic acid metabolism by other cells, and increased movement of white blood cells.

Ongoing investigations funded through the NEI will further researchers' understanding of the role of these lipids in regulating how the cornea responds to injury. Moreover, pharmacological manipulation of arachidonic acid metabolism and platelet-activating factor production may prove beneficial in ameliorating corneal inflammation and limiting the ingrowth of blood vessels.

PROGRAM OBJECTIVES

The objectives for the Corneal Diseases Program include the following important areas of laboratory and clinical research:

• Explore the molecular basis of corneal transparency.
• Analyze the molecular nature of corneal inflammation and wound healing.
• Delineate the pathogenesis of corneal developmental anomalies and dystrophies.
• Improve the understanding of ocular surface physiology.

The needs and opportunities related to each of these objectives and the strategies for accomplishing them will now be considered.

Objective 1: Explore the molecular basis of corneal transparency.

Researchers must continue to discover the parameters required for transparency and then determine how to achieve these by manipulating the cellular, biochemical, and molecular mechanisms involved in their expression.

Research Needs and Opportunities

Researchers need to learn more about the mechanisms regulating corneal hydration. It is obvious that the various water-channel proteins are widely distributed in ocular tissues. It should now be possible to identify the cell-signaling mechanisms that regulate water permeability either directly through a given AQP channel or via a number of different channels. Their different protein structures suggest that each channel is uniquely regulated. Short-term goals should include measuring water permeability in intact ocular tissues, determining the effects of second messenger systems, and determining the regulation of AQP gene expression. It is important to develop new models to explain the nature of the endothelial fluid pump and to determine whether or not the expression of additional water channels in this cell layer could aid in stromal deturgescence. Longer term work should include obtaining high-resolution structural data so that the aqueous pathway traversing the water channel can be visualized. Such structural information is needed to develop inhibitors of specific water channels. Similarly, it is possible that increased water permeability in cell membranes of lacrimal, corneal epithelial, or conjunctival cells could aid in moving water into the tears in dry-eye conditions.

Better therapy is needed for corneal edema, which is among the leading indications for corneal transplant surgery in the United States. Corneal edema occurs in a variety of clinical settings, including Fuchs' endothelial dystrophy, trauma, inflammation of the iris, glaucoma, and postsurgical disorders. It is frequently associated with deposition of abnormal proteins, amyloid, and PGs, leading to loss of transparency and decreased visual acuity. Biochemical and molecular studies need to be undertaken to understand these clinically significant conditions. The appearance of growth factors and extracellular matrix abnormalities within these corneas needs to be further understood so that interventions can be developed.

Processes to improve the corneal graft should be developed. Underlying scarring, neovascularization, and tear deficiencies compromise transplant acceptance.

Cell populations for corneal grafting need to be produced, and the means to induce corneal cells to undergo changes facilitating tissue replacement and/or repair need to be discovered. It may even be possible to program other sources of cells to acquire the properties of those of the cornea, thus providing a limitless source of cells for corneal reconstruction and replacement. Unlike the donor corneas currently used for transplantation, such cell populations would always be available when needed, and they would be free of the potential problem of transmission of diseases harbored by a donor.

Strategic Research Questions

Can corneal endothelial cells be induced to divide and repair an injured endothelium? This question requires both cell culture and animal experiments, the use of growth factors, and the molecular analysis of the cyclins and their associated kinases. Transfections with recombinant DNAs and use of viral vectors need to be explored. Can researchers develop a visual quality-of-life instrument to measure functional corneal transparency? The measure for outcome of therapy for most corneal conditions is high-contrast visual acuity. Yet for many patients visual acuity may be normal even when visual function for real-life conditions (such as nighttime driving, hazy conditions, and strenuous physical activity) is compromised. Even though refractive error affects up to 60 percent of the American public, this condition has not been explicitly incorporated into past quality-of-life questionnaires. Improved versions should be developed.

Objective 2: Analyze the molecular nature of corneal inflammation and wound healing.

The cornea is uniquely organized to discourage the induction and expression of inflammatory responses, particularly immune effector mechanisms that inflict significant injury to adjacent cells. The creation of such an immunological blindspot should render the cornea vulnerable to opportunistic infections and neoplasms. However, the conspicuous absence of neoplasms and the relatively low incidence of opportunistic infections suggest the existence of an effective immunological surveillance at the corneal surface. Future work should focus on understanding the molecular mechanisms of corneal immune phenomena, wound healing, cell adhesion, and migration. These studies would prove beneficial for clinical outcomes of infection, refractive surgery, and management of diabetes.

Research Needs and Opportunities

Researchers need to determine which genes are responsible for imparting the characteristics unique to the corneal epithelium, stroma, and endothelium. Knowledge of the mechanisms responsible for their regulation will permit manipulation to correct genetic diseases and physical and chemical injuries. These genes can be identified by screening methods now available, including differential display, a variety of subtractive hybridization procedures, and direct sequencing of transcripts. Once identified, the regulation and roles of these genes can be studied by a variety of means, including cell transfections with gene constructs and retroviral vectors and transgenic mutations and gene knockouts. Using genetic engineering, this knowledge should be applied to correct mutations and alter the behavior of corneal cells during wound healing.

Further study of the mechanisms of mucosal immunity is called for. The IgA secreted on mucosal surfaces is the most abundant immunoglobulin. It has been assumed that tear IgA plays a significant role as a barrier to corneal infections, but direct evidence is lacking. The potential for mucosal vaccines to prevent respiratory and gastrointestinal infections is widely recognized and is a topic of intense research activity. By contrast, only a small number of investigators are actively involved in ocular mucosal vaccine research.

Improved management of herpes simplex keratitis is needed. Recurrent HSV-1 infection is a major cause of corneal blindness. Although antivirals are available to treat primary and recurrent disease, no therapy exists to eradicate recurrences or to protect at-risk populations. Current animal systems and molecular biology tools give researchers the ability to address these problems. The molecular mechanisms of the latency/reactivation/recurrence cycle need to be understood, and the efficacy of HSV-1 glycoproteins as vaccine candidates needs to be explored. Developing successful vaccines will require knowing the immunological parameters involved in resolving primary HSV-1 keratitis in naive animals, protecting previously vaccinated animals against ocular HSV-1 challenge, and protecting against recurrent infection. The focus should be on local ocular immunity and mucosal immunity as they appear more important than systemic immune responses. Developing therapeutic vaccines against recurrent ocular HSV-1 infection and herpetic keratitis should be explored.

Strategic Research Questions

What is the character and function of corneal stem cells? Investigation should be directed toward firmly establishing the location of conjunctival epithelial stem cells, determining the role of goblet cells in homeostasis, identifying molecules that regulate the growth and differentiation of epithelial cells, identifying molecules unique to stem cell populations, and determining the role of corneal nerves in the regenerative process.

What are the immunological sentries of the cornea? T-cells bearing the γδ cell receptor appear to act as immunological sentries at mucosal and epithelial surfaces, displaying cytotoxicity to a wide variety of tumor target cells and infectious agents. Unlike conventional effector T-cells, γδ T-cells kill a wide range of antigenically unrelated targets in an MHC-unrestricted manner. Thus, they appear to be ideally suited to serve as sentinels at the corneal surface. To date, it is not known if γδ T-cells are present in the cornea, but their presence could have profound importance in corneal immunobiology. It would also be worth investigating whether antigen-unspecific elements, such as defensins and natural killer cells, function in the cornea.

What are the interactions between stromal keratocytes and epithelial cells? Healing of the cornea following injury or refractive surgery is a complex and poorly understood process. Understanding the interactions between these cell types in the wound healing process is vital to improving the treatment of corneal injuries, including those exacerbated by other diseases such as diabetes, and the outcome of refractive surgery.

What specific host/pathogen interactions occur in ocular infectious disease? Further studies defining the components and mechanisms of host/pathogen interactions are warranted. If molecules used during interactions by the host and the infectious agent can be identified and characterized, molecular biologic methods could duplicate these molecules, leading to new therapeutic strategies.

What are the molecular mechanisms of ocular infectious diseases? Studies that define the molecular pathogenesis of infectious diseases are important. The ability to identify and regulate factors that control the production of inflammatory mediators and molecules may lead to fewer complications and the development of novel therapeutic strategies.

What processes generate immune privilege in the cornea? Regulating the immune response could have important uses in promoting corneal allograft survival. Further exploration and clarification are needed to determine which anti-inflammatory cytokines function in the cornea, which factors prevent the expression of delayed-type hypersensitivity, and how MHC class I antigen expression is downregulated. It would also be important to determine which antigen-presenting cells promote the development of anterior chamber associated immune deviation and how specific T-cell subtypes prevent the expression of delayed-type hypersensitivity. Investigation is also needed into the role of the spleen and of neuropeptides in sustaining immune privilege.

What cellular and molecular events occur during corneal wound healing? Areas of present interest include the mechanisms of migration, stratification, and differentiation of epithelial cells; apoptosis; migration and differentiation of keratocytes; stromal/epithelial interactions; and deposition and organization of the stroma. These studies should provide new insights into the maintenance of corneal clarity and control of topographic change after corneal surgery, infection, and trauma.

Objective 3: Delineate the pathogenesis of corneal developmental anomalies and dystrophies.

The normal processes of corneal development and differentiation need to be identified and understood at the cellular and molecular levels to be able to unravel their pathogenesis and develop appropriate therapies. A combination of methods using tissue culture, transfection, and transgenic mice will provide valuable new insights.

Research Needs and Opportunities

Resources to archive family pedigrees, tissue specimens, and cell lines from corneal dystrophies need to be established. Family pedigrees of the various corneal dystrophies have been collected by clinicians, many of whom have little access to molecular genetic technology. Conversely, there are many highly sophisticated molecular genetic laboratories that lack the appropriate clinical material to pursue disease studies. Archiving specimens of the various corneal dystrophies would provide a valuable resource to study these disorders in a cost-efficient manner.

Researchers need to determine the corneal mechanisms of inhibition of UV light damage and tumor induction. Primary carcinomas of skin epidermis are common, whereas they are rare in corneal epithelium, even though this tissue is exposed to similar amounts of UV light and other DNA-damaging agents. UV-induced oxidative damage to DNA is mutagenic through a process catalyzed by free iron. It has recently been shown that nuclear ferritin is an important antigen of corneal epithelial cells that protects against UV damage by its iron-sequestering action. Thus, it is important to investigate further the mechanism of action of endogenous ferritins of the cornea and the fluids that bathe it. It is also of interest to define further the putative role of corneal enzymes, such as ALDH3, in protection against UV light. The rarity of primary tumors arising in the cornea suggests that this tissue resists transformation by viral and chemical carcinogens. Does the cornea uniquely regulate the expression of tumor suppressor genes? Does the cornea resist transformation by oncogenes or oncogenic agents? Are apoptosis-inducing genes upregulated in the corneal endothelium? Researchers need to conduct further studies to answer these questions.

Corneal gene therapy should be developed. While the surface location of the cornea is an advantage for experimentation in this area of research, its cellular complexity and large acellular stroma add to the difficulties. Since the corneal epithelium possesses a mitotic potential and is continually renewed from the limbal stem cells, it should be a high-priority target for gene therapy. Because it can be maintained in standard culture conditions for considerable time prior to transplantation, the cornea affords the opportunity for genetic modification in the laboratory. Genetic manipulations could enhance the efficacy of limbal transplants in correcting corneal surface disorders. Appropriate corneal vectors need to be identified for the eventual implementation of gene therapy. Developing genetic engineering in the cornea will have ramifications beyond the actual treatment of disease. It will allow a molecular dissection of cellular processes involved in corneal development and wound healing and open possibilities for the development of disease model systems.

It will be important to increase scientists' knowledge of the structure and function of the corneal membranes. While much is known about the corneal stroma, little is known about Bowman's layer and Descemet's membrane. Emphasis should be placed on the structure and function of these matrices since they potentially influence epithelial and endothelial attachment. This will be especially significant for Bowman's layer, which may be partially or completely removed during refractive laser surgery.

Strategic Research Questions

What roles do the MMPs play in corneal development? The genes for most of the MMPs and TIMPs are cloned and mapped, and knockout mice are available for many. These resources should greatly facilitate functional studies. MMP inhibitors could potentially be a new way to treat a variety of disorders; however, few studies have examined these agents in detail using animal models of corneal injury.

What are the functions of the metabolic enzymes present at very high concentration in the cornea? The metabolic and detoxification functions of these enzymes should be explored, and the possibility that they might play structural roles should be investigated. Since many of the abundant corneal proteins are encoded by stress-responsive genes, it is important to establish whether inductive events regulate their expression.

What is the molecular genetic basis of keratoconus? Corneal topographic analyses have identified early forms of this disorder. These analyses need to be refined through longitudinal topographic analysis and used to construct pedigrees of the hereditary forms of the disorder. Gene loci should then be identified and cloned to provide clues to the pathogenesis and therapy.

Which gene products are responsible for the inheritance of the various corneal dystrophies and developmental anomalies? Understanding the pathogenesis of these disorders should lead to more effective means of treatment and diagnosis. Strategies to answer this question include differential display and subtractive hybridization. In addition to providing clues to disease processes, corneal-specific gene products will ultimately provide promoters for the use of gene therapy.

Which gene products are specific for corneal development, repair, and wound healing? To manipulate the behavior of corneal cells during development, wound healing, and regeneration, these cells' structure and function at the molecular level must be understood. Emphasis should be placed on regulating components involved in cellular adhesion, migration, and communication. Work should continue on the processes of molecular and supramolecular assembly, which result in the unique architecture of each corneal layer.

Objective 4: Improve the understanding of ocular surface physiology.

There is growing appreciation that the ocular surface and the lacrimal glands are intimately linked in a servomechanism that maintains the comfort and health of the ocular surface. The lacrimal gland interacts with the ocular surface via sensory and secretomotor pathways and lymphocytes trafficking throughout the mucosal immune system. The tear fluid and its many constituents influence the ocular surface and therefore modulate the information that returns to the lacrimal gland. A systemic understanding of these relationships should help unravel the mystifying relationship between the signs and symptoms of dry eye. The present lack of understanding frustrates the diagnosis of dry eye and poses a formidable impediment to epidemiological and interventional studies. Progress in this area should make it possible to more effectively characterize, diagnose, and treat dry-eye conditions.

Research Needs and Opportunities

Researchers need to develop cell lines that maintain lacrimal gland phenotypes. Primary cultures of lacrimal acinar cells that maintain much of the normal differentiated phenotype are being used in several laboratories. However, the use of primary cultures of lacrimal ductal epithelial cells is not yet widespread, and it is not clear whether they can be obtained in quantities large enough for biochemical studies. It will not be possible to exploit the full repertoire of modern molecular biology techniques until immortal cell lines that mimic the differentiated phenotypes are obtained.

The sources of individual variability in the function of the ocular surface-lacrimal gland system need to be determined. These are likely to include initiation of sensory signals, central integration and conscious perception of sensory input, generation of secretomotor output, signal transduction, intracellular signal integration, and functional status of the secretory epithelium. Examining individual variability from such a systemic perspective should make it possible to determine the relationship between signs and symptoms of dry eye.

More information is necessary on the function of tear film components. Relatively little is known about the function of some of the major protein components in the tear film. The interactions between the tear proteins, lipids, and mucins must be elucidated to understand how the tear film protects and lubricates the eye. Increased knowledge of the role of the normal tear components would provide insight into the molecular functional deficiencies in dry-eye disease and could lead to effective treatments.

Strategic Research Questions

Can researchers reconstitute interactions between nerve cells and the lymphoid and epithelial components of the lacrimal gland? It is hoped that progress in cellular and molecular neuroscience will be translated into progress on the physiology of the ocular surface-lacrimal gland system.

What are the details of fluid and electrolyte transport in the conjunctival epithelium? Preliminary reports indicate that the conjunctiva is a Cl^--secreting and Na⁺-absorbing epithelium, thereby implying that its cells have the potential to contribute toward or modify the composition and tonicity of the tear film. It is not known whether these conjunctival mechanisms coexist in the same cell or if there is a distinct dispersal of functional cell types within the bulbar or palpebral portions of this tissue. This issue needs to be addressed, as does the role of neural regulation.

What cell-cell interactions influence the function of the ocular surface? Coculture systems comprised of primary lacrimal secretory epithelial cells together with autologous lymphocytes offer new opportunities. Such systems could accelerate researchers' efforts to delineate the interactions that ultimately influence the function of the ocular surface-lacrimal gland system.

What is the relationship between early changes in innervation and cellular electrophysiology and the later onset of autoimmune phenomena of Sjögren's Syndrome in animal models? Understanding this relationship may provide important clues to the mechanisms underlying the disease in humans. Similarly, understanding immune regulation at the cellular and molecular levels could provide new medical interventions to control or reverse the autoimmune phenomena of Sjögren's Syndrome.

Can steroid hormones be used as therapy for dry-eye conditions? It is reasonable to explore therapeutic strategies based on steroid hormones. There should be continuing efforts to understand the molecular mechanisms underlying the influence of the hormonal environment on the ocular surface-lacrimal gland system. The lacrimal gland-ocular surface servomechanism has evolved to maintain the homeostasis of the ocular surface, and its function is influenced by systemic factors like hormones and medications, and by local factors like extracellular matrix constituents and cytokines released by infiltrating inflammatory cells. It is possible that the actions of sex hormones are mediated through networks of autocrine and paracrine interactions.

CORNEAL DISEASES PANEL

CHAIRPERSONS

Thomas Linsenmayer, Ph.D.
Tufts University
Boston, MA

Bartly Mondino, M.D.
Jules Stein Eye Institute
Los Angeles, CA

PANEL MEMEBER

Peter Agre, M.D.
Johns Hopkins School of Medicine
Baltimore, MD

Joseph T. Barr, O.D., M.S.
Ohio State University College of Optometry
Columbus, OH

Oscar Candia, M.D.
Mt. Sinai School of Medicine
New York, NY

Mark Greene, M.D., Ph.D
University of Pennsylvania School of Medicine
Philadelphia, PA

Elizabeth Hay, M.D.
Harvard Medical School
Boston, MA

Peter McDonnell, M.D.
University of Southern California School of Medicine
Los Angeles, CA

Austin Mircheff, Ph.D.
University of Southern California School of Medicine
Los Angeles, CA

Jerry Y. Niederkorn, Ph.D.
University of Texas Southwestern Medical Center
Dallas, TX

Joram Piatigorsky, Ph.D.
National Eye Institute, NIH
Bethesda, MD

Alan Proia, M.D., Ph.D.
Duke University Eye Center
Durham, NC

Yaron Rabinowitz, M.D.
Cedars-Sinai Medical Center
Los Angeles, CA

NEI STAFF

Loré Anne McNicol, Ph.D.
National Eye Institute, NIH
Bethesda, MD