Cornea 19(5): 644–649, 2000.
`
`© 2000 Lippincott Williams & Wilkins, Inc., Philadelphia
`
`The Diagnosis and Management of Dry Eye
`A Twenty-five–Year Review
`
`Stephen C. Pflugfelder, M.D., Abraham Solomon, M.D., and
`Michael E. Stern, Ph.D.
`
`Purpose. To review the advances in the diagnosis, pathogenesis,
`and management of dry eye disease in the past 25 years. Methods.
`Literature review. Results. The preocular tear film is a hydrated
`mucus gel that contains soluble antimicrobial proteins and growth
`factors that protect and support the ocular surface. The final com-
`mon pathway in dry eye is a perturbation of the integrated ocular
`surface/lacrimal gland reflex unit. Diagnostic tests evaluating tear
`composition and clearance appear to show stronger correlation
`with the severity of ocular irritation symptoms and keratoconjunc-
`tivitis sicca (KCS) than the conventional Schirmer tests. KCS is a
`condition of abnormal differentiation and mucus production by the
`ocular surface epithelium that results in a poorly lubricated, ab-
`normally permeable ocular surface that has increased susceptibility
`to environmental insults. Chronic subclinical ocular surface in-
`flammation appears to play a key role in the pathogenesis of KCS.
`New therapeutic strategies are aimed at reducing the ocular surface
`inflammation of dry eye disease. Conclusions. There has been a
`tremendous increase in knowledge regarding dry eye disease in the
`past 25 years that has resulted in improved diagnostic classifica-
`tion and new targeted therapies.
`Key Words: Dry eye—Tear film—Keratoconjunctivitis sicca—
`Meibomian gland disease.
`
`Perhaps there is no other area in the field of Cornea and External
`Disease where more advances have been made in the past 25 years
`than in the diagnosis, pathogenesis, and treatment of dry eye and
`tear film disorders. This article will review the major advances in
`this area.
`
`TEAR FILM STRUCTURE AND COMPOSITION
`
`The precorneal tear film traditionally was reported to consist of
`three separate layers: mucus, water, and oil. Imaging studies per-
`
`Submitted February 8, 2000. Revision received May 22, 2000. Accepted
`May 30, 2000.
`From the Ocular Surface and Tear Center (S.C.P., A.S.), Bascom Palmer
`Eye Institute, Department of Ophthalmology, University of Miami School
`of Medicine, Miami, Florida; and Allergan, Inc. (M.E.S.), Irvine, Califor-
`nia, U.S.A.
`Supported in part by Public Health Service Research Grant EY11915,
`Department of Health and Human Services, National Eye Institute,
`Bethesda Maryland, an unrestricted grant from Research to Prevent Blind-
`ness and the Drs. David and Maureen Smith Ocular Surface and Tear
`Research Fund.
`Address correspondence and reprint requests to Dr. Stephen C. Pflug-
`felder, Bascom Palmer Eye Institute, 900 N.W. 17th Street, Miami, FL
`33136, U.S.A. E-mail: spflugfelder@bpei.med.miami.edu
`
`formed in rodents and humans indicate that the mucus, protein, and
`aqueous components in the precorneal tear film combine to form a
`hydrated gel (Fig. 1).1–3 The composition of this mucus gel has not
`been firmly established but appears to consist of a mixture of
`soluble and gel-forming mucins that are produced by the lacrimal
`glands, stratified ocular surface epithelium, and conjunctival gob-
`let cells (Table 1). These are anchored by chemical bonds to the
`underlying membrane-bound mucins (glycocalyx) on the superfi-
`cial cornea epithelium.4 The integrity of the precorneal mucus gel
`is essential for maintaining high quality vision and ocular comfort.
`Experimental removal of the mucus gel results in a transient al-
`teration of corneal barrier function to fluorescein dye, a decrease in
`contrast sensitivity and an increase in corneal surface irregularity.
`These findings are similar to those observed in eyes with kerato-
`conjunctivitis, the ocular surface pathology of dry eye that is as-
`sociated with decreased and abnormal production of mucins by the
`ocular surface epithelium.
`We have learned that the tear fluid consists of much more than
`just water and electrolytes. The tear fluid is a complex biochemical
`mixture of mucins, immunoglobulins, antimicrobial proteins (ly-
`sozyme and lactoferrin), and growth factors such as transforming
`growth factor-alpha, epidermal growth factor, and hepatocyte
`growth factor.5–8 These growth factors regulate cellular processes
`through their interaction with specific cell surface receptors. The
`exact biological function of these peptide factors has not been
`firmly established, but it is likely that some function to maintain
`ocular surface homeostasis, whereas others regulate wound healing
`after ocular surface trauma.
`There has also been a tremendous increase in knowledge re-
`garding the composition and structure of the Meibomian gland
`lipid composition. Hydrophilic polar lipids, including phospholip-
`ids (phosphatidylcholine, phosphatidylethanolamine), sphingomy-
`elin, ceramides, and cerebrosides, form the lipid layer adjacent to
`the aqueous–mucin gel.9,10 The bulk of the tear lipid layer consists
`of the overlying nonpolar hydrophobic lipids (wax esters, choles-
`terol esters, triglycerides, free fatty acids, and hydrocarbons) that
`are associated with the polar phase by means of hydrophobic
`bonds.11
`
`LACRIMAL GLAND-OCULAR
`SURFACE INTERACTION
`
`It is now recognized that the ocular surface (cornea, conjunctiva,
`accessory lacrimal glands, and Meibomian glands) and the inter-
`connecting neural reflex arcs function as an integrated or “func-
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`FIG. 1. Proposed structure of precorneal tear film that
`consists of a superficial lipid layer, a middle aqueous/
`mucin phase that contains soluble mucins, aqueous
`fluid, electrolytes, and proteins that are secreted by the
`lacrimal glands, and ocular surface epithelium. This
`layer is anchored to the underlying superficial corneal
`epithelium by chemical attractions to the epithelial
`membrane mucins (glycocalyx).
`
`tional” unit.12 Communication between these tissues occurs
`through their sensory/autonomic neural reflex loop (Fig. 2).
`The majority of tear secretion by the lacrimal glands is reflexive.
`Tear flow is engendered through stimulation (mostly subcon-
`scious) of the ocular surface and nasal mucosa.13,14 This initiates
`sensory afferent nerve traffic along the ophthalmic branch of the
`trigeminal nerve (CN-V) to the central nervous system in the area
`of the pons. It is here where the nerve signals are integrated with
`input from emotional (cortical) and various other appropriate cen-
`tral nervous system centers. Efferent nerves consist of parasym-
`pathetic fibers that travel in the facial (CN-VII) nerve and sym-
`pathetic fibers that emanate from the paraspinal sympathetic chain.
`The parasympathetic nerves synapse in the pterygopalatine gan-
`glion en route to the main and accessory lacrimal glands. The
`sympathetic fibers course along with the parasympathetic nerves,
`without synapsing at the pterygopalatine ganglion. Both types of
`nerve fibers cause water movement and tear secretion.
`
`DEFINITION OF DRY EYE
`
`For years, there has been considerable confusion regarding the
`definition of dry eye as well as the diagnostic classification of dry
`
`eye conditions. To address these issues, specialists in the field of
`dry eye participated in two workshops held at the National Eye
`Institute (December 13–14, 1993 and December 5–6, 1994) at
`which time a definition and classification scheme for dry eye dis-
`ease was proposed.15 Dry eye was defined as “a disorder of the tear
`film due to tear deficiency or excessive evaporation that causes
`damage to the interpalpebral ocular surface and is associated with
`symptoms of discomfort.”15 Consistent with this definition is a
`proposed classification scheme (Fig. 3) that stratifies patients with
`dry eye from those with decreased aqueous tear production by the
`lacrimal glands to those with increased evaporative loss.
`
`DIAGNOSIS OF DRY EYE DISEASE
`
`Most clinicians still rely on traditional diagnostic tests, such as
`fluorescein tear breakup time, the Schirmer test, and ocular surface
`dye staining, for diagnosis of dry eye disease. It is now recognized
`that a diagnostic algorithm using many diagnostic tests is better for
`identifying a patient’s dry eye condition than a single test alone.16
`One such approach that we have found useful for diagnostic clas-
`sification is presented in Figure 4.16 Many new diagnostic tests
`with improved sensitivity and specificity over these conventional
`
`TABLE 1. Mucins detected in the ocular surface epithelium and tear fluid
`
`Mucins
`
`MUC-1
`
`SMC/MUC-4 (protein,
`mRNA)
`
`MUC-4
`
`MUC-5AC
`
`H185
`
`MEM
`
`Source sample (detection
`method)
`
`Corneal and conjunctival
`specimens (PCR, in situ
`hybridization, IHC)
`Conjunctival specimens (IHC)
`
`Conjunctival and corneal
`epithelium, tear fluid (IHC,
`immunoprecipitation, western
`blotting, northern blotting)
`Conjunctiva (northern blotting and
`in situ hybridization)
`Conjunctival goblet cells (in situ
`hybridization)
`Conjunctiva and tear fluid
`(RT-PCR, IHC, western
`blotting)
`Conjunctival impression cytologies
`(IHC)
`Conjunctival epithelium (IHC)
`
`Normal
`
`KCS
`
`Reference
`
`Found in conjunctival and corneal
`epithelium
`
`—
`
`Inatomi et al., 199556
`
`—
`
`Found in the tear fluid and in the
`corneal and conjunctival
`epithelium
`
`Found in the stratified conjunctival
`epithelium
`Found in conjunctival goblet cells
`
`Found in conjunctival goblet cells,
`superficial conjunctival
`epithelium and tear fluid
`Mosaic staining pattern
`
`Decreased expression
`of glycosylated form
`NA
`
`Jones et al., 199820
`
`Pflugfelder et al., 20004
`
`NA
`
`NA
`
`—
`
`Inatomi et al., 199657
`
`Inatomi et al., 199657
`
`Jumblatt et al., 199958
`
`Starry sky pattern
`
`Danjo et al., 199821
`
`—
`
`Sjo¨gren’s syndrome
`
`Pflugfelder et al., 199722
`
`NA, not evaluated; MEM, mucosal epithelial membrane mucin; SMC, sialomucin complex; RT-PCR, reverse transcription polymerase chain
`reaction; IHC, immunohistochemistry.
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`duced expression of secretory and membrane-bound mucins by the
`superficial ocular surface epithelial cells.22,23
`An exact mechanism for the development of these pathologic
`changes has not been elucidated. The severity of KCS worsens as
`aqueous tear secretion decreases and as the ability to reflex tear in
`response to sensory stimulation is lost.16 Ocular surface epithelial
`function and differentiation may be adversely affected by the in-
`crease in tear film osmolarity that occurs in dry eye.24 Trauma to
`a poorly lubricated and unprotected ocular surface from environ-
`mental factors and blinking may be another contributing factor
`because KCS is most severe in the exposure zone that is subjected
`to these stresses.15,22 If KCS represents a chronic wound-healing
`response, then it may be perpetuated, in part, by reduced levels of
`lacrimal gland-secreted growth factors in the tear fluid.25,26
`Inflammation may represent another important causative factor
`for KCS. Decreased aqueous tear production and tear clearance
`leads to chronic inflammation of the ocular surface.27,28 Evidence
`exists that the etiology of this disease is facilitated through a loss
`of the antiinflammatory environment within the lacrimal glands
`provided through the presence of circulating androgens.29,30 At the
`time of menopause or due to various pathologic conditions, the
`available level of circulating androgens may drop below a thresh-
`old, leaving the tissues vulnerable for the initiation of immune-
`based inflammation. This is supported by a significantly increased
`incidence of dry eye in women.31 Any condition that results in
`rapid firing of the “functional unit” can now cause the initiation of
`neurogenic inflammation within acini of the lacrimal glands re-
`sulting in antigen presentation and cytokine secretion by these
`epithelial cells.32,33 This will draw the attention of vigilantly traf-
`ficking T cells within the lacrimal glands. In the normal individual,
`trafficking lymphocytes—finding no inflammation present—
`would undergo apoptosis (programmed cell death) as they leave
`the tissue en route to local lymph nodes. In the presence of in-
`flammatory signals, the apoptotic process is aborted (allowing the
`accumulation of lymphocytes within the glands) and the lympho-
`cytes become activated and secrete proinflammatory cytokines that
`will result in the “homing” of additional T cells to the tissue and
`an increase in the level of inflammation.34
`It has also been demonstrated that the normally stable, termi-
`
`FIG. 3. Diagnostic classification scheme for dry eye
`disease proposed by the National Eye Institute/
`Industry Workshop. Adapted from CLAO J 1995;21:
`222–32.15
`
`FIG. 2. Integrated ocular surface/lacrimal gland functional unit. Af-
`ferent sensory nerve impulses from the ocular surface and nasal
`mucosa synapse in the brainstem with efferent autonomic secreto-
`motor nerve impulses that innervate the lacrimal gland. Stimulation
`of this reflex loop results in delivery of tear fluid and proteins on
`demand to the ocular surface.
`
`tests have been reported over the past 25 years. These techniques
`include tear film osmolarity, tear fluid protein immunoassays, and
`fluorescein tear clearance.17–20 Although none of these tests has
`yet to gain widespread clinical acceptance, the results of studies
`evaluating these tests indicate that tear composition and clearance
`appear to show stronger correlation with the severity of ocular
`surface irritation symptoms and ocular surface disease than the
`Schirmer test that evaluates tear production alone.
`
`PATHOGENESIS OF DRY EYE DISEASE
`
`There has been a considerable increase in knowledge regarding
`the pathogenesis of the keratoconjunctivitis sicca (KCS) that oc-
`curs in dry eye disease. The pathologic features of this condition
`include increased epithelial stratification and proliferative index
`and abnormal differentiation with maintenance of a basal pheno-
`type.21 Cells throughout the abnormally thickened conjunctival
`epithelium express cytokeratin 14, a cytoskeletal protein normally
`produced by the basal epithelial cells. Furthermore, there is re-
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`FIG. 4. Algorithm for diagnosis of dry eye disease.
`
`nally differentiated, acinar epithelial cells of the lacrimal gland
`undergo abnormal (pathologic) apoptosis.34 Tears now secreted to
`the ocular surface will contain proinflammatory cytokines and will
`inflame the tissues of the ocular surface. Abnormal apoptosis has
`also been detected within the epithelial cells and lymphocytes of
`the ocular surface.34 This ocular surface inflammatory response
`consists of inflammatory cell infiltration, activation of the ocular
`surface epithelium with increased expression of adhesion mol-
`ecules, inflammatory cytokines and pro-apoptotic factors, in-
`creased concentrations of inflammatory cytokines in the tear fluid,
`and increased activity of matrix-degrading enzymes in the tear
`
`fluid (summarized in Table 2).26–28,35–39 Significant positive cor-
`relation has been observed between the levels of inflammatory
`cytokines in the conjunctival epithelium and the severity of ocular
`irritation symptoms, corneal fluorescein staining, and the severity
`of conjunctival squamous metaplasia in patients with Sjo¨gren’s
`syndrome KCS.26
`
`THERAPY OF DRY EYE DISEASE
`
`There have been many recent advances in the treatment of dry
`eye disease. First, there is increasing recognition that the frequent
`
`TABLE 2. Cytokines, growth factors and matrix degrading enzymes detected in the ocular surface tissues and in the tear fluid of patients
`tear film disorders
`
`Dry eye diseases
`
`Reference
`
`Cytokine/growth factor
`
`Source sample
`(detection method)
`
`EGF (protein)
`
`Tear fluid (ELISA)
`
`IL-1 ␣ (mRNA)
`
`IL-6 (mRNA)
`IL-8 (mRNA)
`TNF-␣ (mRNA)
`TGF-␤1 (mRNA)
`HGF (protein)a
`IL-1 ␣ (protein)
`Pro-MMP-9
`IL-1 ␣ (protein)
`IL-6 (protein)
`Plasmin
`MMP-3 (protein)
`MMP-9 (protein)
`TIMP-1 (protein)
`
`Conjunctival epithelium (RT-PCR on
`conjunctival cytology specimens)
`
`Decreased in ocular surface diseases and
`Sjo¨gren’s syndrome
`Sjo¨gren’s syndrome increased
`
`Tear fluid (ELISA)
`Tear fluid (ELISA)
`Tear fluid (ELISA)
`Tear fluid (ELISA)
`Tear fluid (ELISA)
`Fluorometric assay
`Tear fluid (ELISA)
`
`(Hypothesized to be deceased)
`Increased in ocular rosacea
`
`Increased in ocular rosacea
`Increased in Sjo¨gren’s syndrome
`Increased in Sjo¨gren’s syndrome
`Increased in ocular rosacea
`
`Van Setten 199125
`Pflugfelder et al., 199926
`Pflugfelder et al., 199926
`
`Jones et al., 199435
`
`Li et al., 19968
`Afonso et al., 199927
`
`Barton et al., 199728
`Tishler et al., 199838
`Virtanen et al., 199759
`Sobrin et al., 200039
`
`a Normal (after surgery).
`EGF, epidermal growth factor; MMP, matrix metalloproteinase; TNF, tumor necrosis factor; TGF, transforming growth factor; HGF, hepa-
`tocyte growth factor; TIMP, tissue inhibitor of metalloproteinase; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription
`polymerase chain reaction.
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`
`use of artificial tear solutions that are preserved with benzalko-
`nium chloride may be toxic to the ocular surface epithelium.40,41
`To avoid the risk of preservative toxicity to the abnormal ocular
`surface epithelium of eyes with KCS, there has been an increase in
`trend over the past decade to use unit-dose nonpreserved artificial
`tears in patients who must frequently instill artificial tears to con-
`trol their irritation symptoms. Laboratory studies have shown that
`certain electrolyte compositions and the use of a bicarbonate buff-
`ering system in artificial tear solutions promotes normal ocular
`surface differentiation and homeostasis.24,42 Currently, there are
`many commercially available artificial tear preparations with these
`features. Several different multidose artificial tear preparations
`with disappearing preservatives have also been released.
`Although artificial tears have been reported to improve irritation
`symptoms and to decrease ocular surface dye staining in eyes with
`KCS, their use has not been found to improve conjunctival squa-
`mous metaplasia.43 For this reason, there is increasing interest in
`developing therapeutic agents for KCS. Extracellular uridine tri-
`phosphate and other nucleotides that stimulate P2Y2 purinergic
`receptors, which have been reported to increase chloride, fluid, and
`mucin secretion by the conjunctiva.44,45 These agents may prove
`to have therapeutic benefit for treatment of dry eye disorders.
`Consistent with an immunologic mechanism for development of
`KCS, antiinflammatory therapy has been reported to improve both
`irritation symptoms and ocular surface disease. Cyclosporin is a
`potent immunomogulatory agent that inhibits T cell activation and
`downregulates the production of many inflammatory cytokines.
`Topically applied cyclosporin has been reported to significantly
`improve signs of KCS in dogs that develop this condition and it
`has been approved for treatment of veterinary dry eye disease.46 A
`prospective double-masked randomized placebo-controlled clini-
`cal trial evaluating a topical cyclosporin emulsion showed signifi-
`cant improvement in the severity of ocular irritation symptoms and
`ocular surface disease in the cyclosporin treated group compared
`to those treated with vehicle.47 Furthermore, when compared to the
`vehicle, cyclosporin was found to decrease many inflammatory
`markers and the level of the inflammatory cytokine interleukin-6
`(IL-6) in the conjunctiva.48
`Our center has reported that topically applied corticosteroids
`remarkably improve the irritation symptoms and ocular surface
`signs of KCS.49,50 Furthermore, we have found that steroids de-
`crease levels of the chemotactic cytokine IL-8 in the conjunctival
`epithelium.26 We currently reserve the use of topical corticoste-
`roids for treatment of those patients who experience intolerable
`irritation symptoms despite maximal aqueous enhancement
`therapy (artificial tears and punctual occlusion). The fact that cor-
`ticosteroids have been found to have excellent efficacy for treat-
`ment of KCS provides proof of the principle that inflammation
`plays a key role in the pathogenesis of dry eye disease.
`Female gender has been reported to be a risk factor for devel-
`oping KCS, particularly that associated with Sjo¨gren’s syn-
`drome.51 Experimental evidence indicates that androgen hormones
`improve lacrimal gland secretory function.52 Furthermore, sys-
`temically administered androgenic hormones have been reported
`to significantly reduce lacrimal gland lymphocytic infiltration and
`improve lacrimal gland function in murine models of Sjo¨gren’s
`syndrome.53,54 At the present time, there have been no controlled
`clinical trials of androgen hormones for treatment of dry eye dis-
`ease in humans.
`The normal human tear fluid contains many biologically active
`
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`
`growth factors, such as epidermal growth factor, that are secreted
`by the lacrimal glands. The concentration of these factors have
`been reported to decrease in patients with lacrimal gland dysfunc-
`tion. Autologous human serum, which contains many of the
`growth factors that are present in tear fluid, has been reported to
`improve KCS and stimulate conjunctival mucus production.55 Re-
`cent advances in the field of biotechnology provide the opportunity
`to formulate tear replacements containing recombinant forms of
`these cytokine growth factors. Future research will hopefully de-
`fine whether topical application of one or more of these factors
`alone will duplicate the therapeutic benefits of serum.
`
`REFERENCES
`
`1. Nichols BA. Demonstration of the mucous layer of the tear film by
`electron microscopy. Invest Ophthalmol Vis Sci 1985;26:464–73.
`2. Chen HB. Structure and composition of rat precorneal tear film. A
`study by an in vivo cryofixation. Invest Ophthalmol Vis Sci 1997;38:
`381–7.
`3. Prydal JI, Artal P, Woon H, et al. Study of human precorneal tear film
`thickness and structure using laser interferometry. Invest Ophthalmol
`Vis Sci 1992;33:2006–11.
`4. Pflugfelder SC, Liu Z, Monroy D, et al. Detection of sialomucin com-
`plex (MUC4) in human ocular surface epithelium and tear fluid. Invest
`Ophthalmol Vis Sci 2000;41:1316–26.
`5. Van Setten GB, Schultz GS. Transforming growth factor alpha is a
`constant component of tear fluid. Graefes Arch Clin Exp Ophthalmol
`1994;232:523–6.
`6. Ohashi Y, Motokura M, Kinoshita Y, et al. Presence of epidermal
`growth factor in human tears. Invest Ophthalmol Vis Sci 1989;30:
`1879–82.
`7. Van Setten GB, Viinikka L, Tervo T. Epidermal growth factor is a
`constant component of normal human tear fluid. Graefes Arch Clin
`Exp Ophthalmol 1989;22:184–7.
`8. Li Q, Weng J, Mohan RR, et al. Hepatocyte growth factor and hepa-
`tocyte growth factor receptor in the lacrimal gland, tears and cornea.
`Invest Ophthalmol Vis Sci 1996;37:727–39.
`9. Greiner JV, Glonek T, Korb DR, et al. Phospholipids in meibomian
`gland secretion. Ophthalmic Res 1996;28:44–9.
`10. McCulley J, Shine WE. Meibomian gland secretions in chronic
`blepharitis. Adv Exp Med Biol 1998;430:319–26.
`11. Shine WE, McCulley J. The role of wax and fatty alcohols in chronic
`blepharitis. Invest Ophthalmol Vis Sci 1993;34:3521–5.
`12. Stern ME, Beuerman R, Fox RI, et al. The pathology of dry eye: the
`interaction between the ocular surface and lacrimal glands. Cornea
`1998;17:584–9.
`13. Heigle T, Pflugfelder SC. Aqueous tear production in patients with
`neurotrophic keratitis. Cornea 1996;15:135–8.
`14. Jordan A, Baum J. Basic tear flow? Does it exist? Ophthalmology
`1980;87:920–30.
`15. Lemp MA. Report of the National Eye Institute/Industry Workshop on
`Clinical Trials in Dry Eyes. CLAO J 1995;21:222–32.
`16. Pflugfelder SC, Tseng SCG, Sanabria O, et al. Evaluation of subjective
`assessments and objective diagnostic tests for diagnosing tear-film
`disorders known to cause ocular irritation. Cornea 1998;17:38–56.
`17. Gilbard JP, Farris RL, Santamaria IJ. Osmolarity of tear microvolumes
`in keratoconjunctivitis sicca. Arch Ophthalmol 1978;96:677–81.
`18. McCollum CJ, Foulks GN, Bodner B, et al. Rapid assay of lactoferrin
`in keratoconjunctivitis sicca. Cornea 1994;13:505–8.
`19. Danjo Y. Ocular surface damage and tear lactoferrin in dry eye syn-
`drome. Acta Ophthalmol (Copenh) 1994;72:433–7.
`20. Afonso A, Monroy D, Stern ME, et al. Correlation of tear clearance
`and Schirmer test scores with ocular irritation symptoms Ophthalmol-
`ogy 1999;106:803–10.
`21. Jones DT, Ji A, Monroy D, et al. Evaluation of ocular surface cytokine,
`mucin, and cytokeratin expression in Sjo¨gren’s Syndrome. Adv Exp
`Med Biol 1998;438:533–6.
`22. Danjo Y, Watanabe H, Tisdale AS, et al. Alteration of mucin in human
`
`FAMY CARE - EXHIBIT 1030-0005
`
`

`
`DRY EYE REVIEW
`
`649
`
`conjunctival epithelia in dry eye. Invest Ophthalmol Vis Sci 1998;39:
`2602–9.
`23. Pflugfelder SC, Tseng SCG, Yoshino K, et al. Correlation of goblet
`cell density and mucosal epithelial membrane mucin expression with
`rose bengal staining in patients with ocular irritation. Ophthalmology
`1997;104:223–35.
`24. Gilbard JP, Rossi SR, Heyda KG. Ophthalmic solutions, the ocular
`surface and a unique artificial tear formulation. Am J Ophthalmol
`1989;107:348–55.
`25. Van Setten GB. Ocular disease leads to decreased concentrations of
`epidermal growth factor in the tear fluid. Curr Eye Res 1991;10:523–7.
`26. Pflugfelder SC, Jones DT, Ji Z, et al. Altered cytokine balance in the
`tear fluid and conjunctiva of patients with Sjogren’s syndrome kera-
`toconjunctivitis sicca. Curr Eye Res 1999;19:201–11.
`27. Afonso A, Sobrin L, Monroy D, et al. Tear fluid pro-gelatinase B
`activity correlates with tear clearance and IL-1 concentration in pa-
`tients with ocular rosacea. Invest Ophthalmol Vis Sci 1999;40:2506–
`12.
`28. Barton K, Monroy D, Nava A, et al. Inflammatory cytokines in tears
`of patients with ocular rosacea. Ophthalmology 1997;104:1868–74.
`29. Azzarolo AM, Wood RL, Mircheff A, et al. Androgen influence on
`lacrimal gland apoptosis, necrosis, and lymphocytic infiltration. Invest
`Ophthalmol Vis Sci 1999;40:592–602.
`30. Sullivan DA, Krenzer KL, Sullivan BD, et al. Does androgen insuffi-
`ciency cause lacrimal gland inflammation and aqueous tear defi-
`ciency? Invest Ophthalmol Vis Sci 1999;40:1261–5.
`31. McCarty CA, Bansal AK, Livingston PM, et al. The epidemiology of
`dry eye in Melbourne, Australia. Ophthalmology 1998;105:1114–9.
`32. Mircheff A, Gierow JP, Yang T, et al. Sjogren’s autoimmunity: how
`perturbation of recognition in endomembrane traffic may provoke
`pathological recognition at the cell surface. J Mol Recognit 1998;11:
`40–8.
`33. Meggs WJ. Neurogenic inflammation and sensitivity to environmental
`chemicals. Environ Health Perspect 1993;101:234–8.
`34. Gao J, Schwalb TA, Addeo JV, et al. The role of apoptosis in the
`pathogenesis of canine keratoconjunctivitis sicca: the effect of topical
`Cyclosporin A therapy. Cornea 1998;17:654–63.
`35. Jones DT, Monroy D, Ji Z, et al. Sjogren’s syndrome: cytokine and
`Epstein-Barr virus gene expression within the conjunctival epithelium.
`Invest Ophthalmol Vis Sci 1994;35:3493–504.
`36. Baudouin C, Brignole F, Becquet F, et al. Flow cytometry in impres-
`sion cytology specimens. Invest Ophthalmol Vis Sci 1997;38:1458–64.
`37. Bourcier T, Saint-Jean M, Brignole F, et al. Expression of CD40 and
`CD40 ligand in the human conjunctival epithelium. Invest Ophthalmol
`Vis Sci 2000;41:120–6.
`38. Tishler M, Yaron I, Geyer D, et al. Elevated tear interleukin-6 levels
`in patients with Sjogren’s syndrome. Ophthalmology 1998;105:
`2327–9.
`39. Sobrin L, Liu Z, Monroy D, et al. Activators and inhibitors of matrix
`metalloproteinase-9 in human tear fluid and corneal epithelial culture
`supernatant. Invest Ophthalmol Vis Sci 2000;41:1703–9.
`40. Gobbels M, Spitznas M. Corneal epithelial permeability of dry eyes
`before and after treatment with artificial tears. Ophthalmology 1992;
`99:873–8.
`41. Ichijima H, Petrol WM, Jester JV, et al. Confocal microscopic studies
`of living rabbit cornea treated with benzalkonium chloride. Cornea
`1992;11:221–5.
`
`42. Ubels JL, McCartney MD, Lantz WK, et al. Effects of preservative-
`free artificial tear solutions on corneal epithelial structure and function.
`Arch Ophthalmol 1995;113:371–8.
`43. Nelson JD, Farris RL. Sodium hyaluronate and polyvinyl alcohol ar-
`tificial tear preparations: a comparison in patients with keratoconjunc-
`tivitis sicca. Arch Ophthalmol 1988;106:484–7.
`44. Jumblatt JE, Jumblatt MM. Regulation of ocular mucin secretion by
`P2Y2 nucleotide receptors in rabbit and human conjunctiva. Exp Eye
`Res 1998;67:341–6.
`45. Hoyasa K, Ueda H, Kim KJ, et al. Nucleotide stimulation of Cl(−)
`secretion in the pigmented rabbit conjunctiva. J Pharmacol Exp Ther
`1999;291:53–9.
`46. Kazwan RL, Salisbury MA, Ward DA. Spontaneous canine kerato-
`conjunctivitis sicca:a useful model for human keratoconjunctivitis
`sicca. Treatment with cyclosporin eye drops. Arch Ophthalmol 1988;
`106:484–7.
`47. Sall K, Stevenson OD, Mundorf TK, et al. Two multicenter, random-
`ized studies of the efficacy and safety of cyclosporine ophthalmic
`emulsion in moderate to severe dry eye disease. Ophthalmology 2000;
`107:631–9.
`48. Turner K, Pflugfelder SC, Ji Z, et al. Interleukin-6 levels in the con-
`junctival epithelium of patients with dry eye disease treated with cy-
`closporine ophthalmic emulsion. Cornea 2000, in press.
`49. Prabhasawat P, Tseng SCG. The frequent association of tear clearance
`in ocular irritation. Br J Ophthalmol 1998;82:666–75.
`50. Marsh P, Pflugfelder SC. Topical non-preserved methylprednisolone
`therapy of keratoconjunctivitis sicca in Sjogren’s syndrome. Ophthal-
`mology 1999;106:811–6.
`51. Pflugfelder SC, Whitcher J, Daniels T. Sjogren’s syndrome. In: Pepose
`JS, Holland GN, Wilhelmus KR, eds. Ocular infection and immunity.
`St. Louis: Mosby, 1996:1043–7.
`52. Sullivan DA, Block L, Pena JD. Influence of androgens and pituitary
`hormones on the structural profile and secretory activity of the lacrimal
`gland. Acta Ophthalmol Scand 1996;74:421–35.
`53. Sullivan DA, Edwards JA. Androgen stimulation of lacrimal gland
`function in mouse models of Sjogren’s syndrome. J Steroid Biochem
`Mol Biol 1997;60:237–45.
`54. Sullivan DA, Wickham LA, Rocha EM, et al. Androgens and dry eye
`in Sjogren’s syndrome. Ann N Y Acad Sci1999;876:312–24.
`55. Tsubota K, Goto E, Fujita H, et al. Treatment of dry eye by autologous
`serum application in Sjogren’s syndrome. Br J Ophthalmol 1999;83:
`390–5.
`56. Inatomi T, Spurr-Michaud S, Tisdale AS, et al. Human corneal and
`conjunctival epithelia express MUC1 mucin. Invest Ophthalmol Vis
`Sci 1995;36:1818–27.
`57. Inatomi T, Spurr-Michaud S, Tisdale AS, et al. Expression of secretory
`mucin genes by human conjunctival epithelia. Invest Ophthalmol Vis
`Sci 1996;37:1684–92.
`58. Jumblatt MM, McKenzie RW, Jumblatt JE. MUC5AC mucin is a
`component of the human precorneal tear film. Invest Ophthalmol Vis
`Sci 1999;40:43–9.
`59. Virtanen T, Konttinen YT, Honkanen N, et al. Tear fluid plasmin
`activity of dry eye patients with Sjogren’s syndrome. Acta Ophthalmol
`Scand 1997;75:137–41.
`
`Cornea, Vol. 19, No. 5, 2000
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`FAMY CARE - EXHIBIT 1030-0006