Topical Fibronectin in the Treatment of Keratoconjunctivitis Sicca J. Daniel Nelson, M.D., Judy F. Gordon, D.V.M., and the Chiron Keratoconjunctivitis Sicca Study Group Topical fibronectin was evaluated for the treatment of keratoconjunctivitis sicca in a multicenter, double-masked, controlled study in which 272 patients were randomly assigned to treatment. Patients with documented clinical evidence of keratoconjunctivitis sicca received either fibronectin, a vehicle alone, or a commercially available artificial tear. Evaluation at baseline, 21, 42, and 63 days consisted of patient self-evaluation of symptoms, rose bengal and fluorescein staining, tear breakup time, Schirmer's testing, and conjunct!val impression cytology. Although all groups showed improvements in most study variables during the course of the study, there were no statistically significant differences found between any of the groups. Topical fibronectin does not appear to be more effective than artificial tears in the treatment of keratoconjunctivitis sicca. 1HERAPY for keratoconjunctivitis sicca remains symptomatic, and includes artificial tear solu tions, lubricating ointments, ocular inserts, and punctal occlusion. Topical application of artifi cial tear solutions remains the primary method of treatment. An ophthalmic solution contain ing a therapeutic agent effective in the treat ment of dry eye would be desirable. Fibronectin is an adhesive glycoprotein that is a component of the basement membrane. In addition to its activity as a binding agent that
Accepted for publication July 23, 1992. From the Department of Ophthalmology, Ramsey Clinic, St. Paul-Ramsey Medical Center, St. Paul, Minne sota; and Department of Ophthalmology, University of Minnesota, Minneapolis, Minnesota (Dr. Nelson); and Chiron Ophthalmics, Irvine, California (Dr. Gordon and Chiron Keratoconjunctivitis Sicca Study Group). Dr. Gordon is an employee of Chiron Ophthalmics. Reprint requests to J. Daniel Nelson, M.D., Depart ment of Ophthalmology, St. Paul-Ramsey Medical Cen ter, 640 Jackson St., St. Paul, MN 55101.
promotes cell-to-cell and cell-to-substrate ad hesion, fibronectin also has a role in cell migra tion, wound healing, and cytoskeletal organiza tion. Fibronectin has been shown to promote the attachment of rabbit corneal endothelial cells to plastic culture dishes. 1 The addition of fibronectin to cultures of transformed cells al lows the cells to adhere to and move across the surface of the plastic culture plates. 2,8 In vitro studies have shown that endogenously pro duced fibronectin forms a matrix over the un derlying stroma of corneas subjected to superfi cial keratectomy, thus allowing epithelial cell migration. 4 6 The in vivo induction of corneal lesions has also been shown to be accompanied by fibronectin deposition during the subse quent healing period. 710 The presence of endogenous fibronectin on the surface of the injured cornea led to clinical application of exogenous fibronectin in pa tients with keratoconjunctivitis sicca. Kono and associates 11 used autologous fibronectin in treating a group of 12 patients with keratocon junctivitis sicca associated with Sjögren's syn drome. Treatment with fibronectin resulted in a reduction in rose bengal and fluorescein stain ing as well as improvement in the subjective symptoms of dry eye. On the basis of data that suggested a role of fibronectin in the healing of ocular surface disorders, including keratoconjunctivitis sicca, a multicenter double-masked, vehicle-con trolled and placebo-controlled trial was con ducted to study the safety and efficacy of fibro nectin in the treatment of patients with keratoconjunctivitis sicca.
Material and Methods Patients—Over a 16-month period (from April 1988 to August 1989), 272 patients with keratoconjunctivitis sicca were enrolled in this clinical trial. Patients eligible for enrollment
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were required to meet at least one of the follow ing entry criteria: (1) wetting of less than 5 mm in ßve minutes when Schirmer's testing is per formed without topical anesthesia and a super ficial punctate staining score of at least 2 (on a scale of 1 to 4); (2) wetting of less than 5 mm in five minutes when Schirmer's testing is per formed without topical anesthesia and a rose bengal staining score greater than 3 (on a scale of 1 to 9); (3) rose bengal staining score greater than 3 (on a scale of 1 to 9) and a patient self-evaluation score of at least 50 on a visual analogue scale of 0 to 100 on three of nine dry-eye symptoms (pain, burning, itching, dryness, foreign-body sensation, tearing, blurring, photophobia, and other); and (4) superficial punctate staining score of at least 2 and a patient self-evaluation score of at least 50 on three dry-eye symptoms. In addition to meeting these diagnostic criteria, all patients were re quired to be of legal age and to have read and signed a written informed consent form. Patients with dry eye associated with congen ital alacrima, vitamin A deficiency, StevensJohnson syndrome, ocular pemphigoid, druginduced pseudopemphigoid, ocular chemical burns, neurogenic hyposecretion, lagophthalmos, inadequate eyelid closure, trachoma, ocu lar surface irregularities, trichiasis, or ocular radiation injury were excluded from the study. Other exclusionary criteria were uncontrolled blepharitis; rosacea keratoconjunctivitis; corneal perforations; preexisting blindness, glau coma, or uveitis; administration of medications known to cause ocular dryness; uncontrolled ophthalmic or systemic infections; serious im mune deficiency or immunosuppressive thera py; pregnancy; and lactation. Patients with a history of viral hepatitis or infection with hu man immunodeficiency virus were also ex cluded. Study design—The study was a doublemasked, controlled parallel group trial in which patients were randomly assigned to treatment and was conducted at 33 clinical sites. The experimental protocol was reviewed and ap proved by the Institutional Review Board at each study center. After determination of eligibility, all patients were required to complete 14 days of washout, during which they administered only an unpreserved lubricating agent (Refresh, Allergan Pharmaceuticals, Irvine, California). At the end of this 14-day period, patients were examined again to determine eligibility. The unpreserved
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lubricating agent was used to eliminate the effect of preservatives on the patient's clinical signs and symptoms. Patients who had an im provement in clinical signs and symptoms dur ing the washout period, and who no longer satisfied the entry criteria, were not enrolled in the study. Eligible patients were randomly assigned to one of three treatment groups: fibronectin oph thalmic solution (3.5 mg/ml); vehicle solution, consisting of all components of the fibronectin solution, without fibronectin; or a commercial ly available artificial tear product (Liquifilm Tears, Allergan Pharmaceuticals). The identity of the treatment was masked to investigators and patients. On day 1 of the study, each patient adminis tered one drop of the study medication (fibro nectin, vehicle, or artificial tear) into each eye six to eight times daily. Treatment was contin ued for 42 days, after which patients returned to administration of only an unpreserved lubri cating agent (Refresh) for three weeks (that is, through day 63). Separate computer-generated randomization codes were prepared for each clinical site, with treatment groups in blocks such that an equal number of patients at each center were treated with fibronectin, vehicle, and artificial tear. No other topical ophthalmic medication was allowed at any time during the study, and Refresh was provided to all study patients to ensure compliance. Systemic medi cations that were considered necessary for the patients' welfare, and that did not interfere with the study, were allowed. Patients who were self-administering oral beta blockers, diu retics, antidepressants, or estrogens were in structed to make no changes in their treatment regimen to avoid any possible effects on the level of ocular dryness. Study medications—The fibronectin ophthal mic solution (3.5 mg/ml) consisted of lyophilized plasma fibronectin reconstituted in sterile saline solution that contained 0.5% chlorobutanol as a preservative. The lyophilized plasma fibronectin contained albumin and sucrose as stabilizers, and sodium chloride, sodium dihydrogen phosphate, and sodium hydrogen phos phate as buffering agents. The vehicle solution consisted of the stabiliz ers and buffering agents found in fibronectin ophthalmic solution (that is, albumin, sucrose, sodium chloride, sodium dihydrogen phos phate, and sodium hydrogen phosphate). These materials were reconstituted in sterile saline
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solution that contained 0.5% chlorobutanol as a preservative. The artificial tear solution consisted of Liquifilm Tears, a commercially available artificial tear solution that contained polyvinyl alcohol 1.4%, sodium chloride, and chlorobutanol 0.5% in purified water. Schedule of observations—Patients were ex amined 14 days before enrollment in the study, and then on day 1 to determine eligibility for the study. During the 42-day treatment period, patients were examined on day 21 (three weeks) and day 42 (six weeks). All patients were re quired to return for a follow-up visit on day 63 (nine weeks). Each patient visit included a com plete ocular examination, which consisted of slit-lamp biomicroscopy, dilated fundus exami nation (day 1 and day 42), Schirmer's testing without anesthesia, and measurement of visual acuity, intraocular pressure, tear meniscus height, and tear breakup time. The primary objective signs that were evalu ated at each examination were fluorescein staining and rose bengal staining. Fluorescein staining was scored by the investigator on a scale of 0 to 4 (absent to severe staining) on the basis of comparison of the investigator's findings with a panel of standardized diagrams of staining provided on each data form. To evaluate fluorescein staining, five areas of the cornea (central cornea and then four equal peripheral areas) were individually scored, with a total maximum score of 20 per eye (40 for both eyes). Rose bengal staining was scored similarly, on a scale of 0 to 3 (absent to severe staining), with the degree of staining recorded separately for the temporal conjunctiva, cor nea, and nasal conjunctiva. The total rose ben gal staining score for each eye was 9, or 18 for both eyes. For both rose bengal and fluorescein staining in which only one eye was treated, the score was doubled to produce a comparable value for analysis. A subset of the full study population also underwent serial conjunctival impression cy tology, with samples obtained from the superi or bulbar, temporal bulbar, nasal bulbar, and inferior palpebrai conjunctiva to evaluate the degree of squamous metaplasia. Each region yielded a score ranging from 0.0 to 3.0 (normal cells to severe squamous metaplasia). Temporal and nasal specimens were considered a single specimen. The maximum impression cytology score was 18.0 for both eyes, with the score doubled for analysis when only one eye was
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treated. All impression cytology samples were evaluated in a masked fashion by a single read ing center, according to previously described cytologie techniques for grading conjunctival squamous metaplasia. 12 In addition to these clinical signs, a question naire was completed by the patients at each examination. Subjective symptoms were evalu ated by each patient on a visual analogue scale of 0 to 100 (0 represents absent, 100 represents severe). Scores for nine symptoms (pain, burn ing/stinging, itching, dryness, foreign-body sensation, tearing, blurring of vision, light sen sitivity, and other) were averaged to produce an overall symptom score. Additionally, patients rated their overall improvement on a visual analogue scale of 0 to 100 (no improvement to marked improvement). Compliance levels were gauged through questions directed to the patient (whether the patient adhered to the treatment regimen all the time, most of the time, or not at all), as well as by measurement of the residual volume of solution in each dropper bottle returned by the patient. Statistical analysis-Extensive statistical anal yses were performed on the data generated in this study. The initial analysis was a direct comparison of the treatment groups at each time point in the study, conducted on the full study population. A comparison of the change from baseline for each treatment group was also performed for the full study population. Additional analyses were performed on numer ous subgroups of the overall population (pa tients 60 years old and older, patients younger than 60 years, patients with Sjögren's syn drome-related keratoconjunctivitis sicca, pa tients with arthritis, patients with age-related keratoconjunctivitis sicca, a subgroup of pa tients who were severely affected, and a sub group of patients whose keratoconjunctivitis sicca was determined on the basis of impression cytology findings). Results of the subgroup analyses were not significantly different when compared to the results of the overall popula tion analysis. Mean values were determined with standard deviations. Continuous variables and ordered categorical variables were analyzed by using individual f-test comparisons of group means at each visit. Differences between groups in change from baseline were analyzed by using the chi-square test. For some analyses, continu ous variables were converted to categorical var-
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TABLE 1 PATIENT DEMOGRAPHICS AND DISPOSITION
MEAN AGE (YRS) (± STANDARD DEVIATION)
TREATMENT
Fibronectin Vehicle Control Total
60 61 69 63
± ± ± ±
14 14 14 15
NO. OF PATIENTS FEMALES
ENTERED
COMPLETED
DISCONTINUED
TERMINATED
94 59 82 235
103 69 95 267
80 55 86 221
11 6 7 24
12 8 2 22
iables and analyzed by using the chi-square test.
Results Patient population—We enrolled 267 patients in this study. Of these 267 patients, 221 pa tients completed the study. Demographic characteristics and disposition of patients were analyzed (Table 1). For the most part, all treat ment groups were comparable at baseline, al though there was a disproportionate number of females enrolled in the study, as compared to males (235 vs 32). Clinical signs—Both fluorescein and rose bengal staining improved in all groups during the course of the study (Tables 2 and 3). There were, however, no statistically significant dif ferences between any of the groups. Tear break up times and Schirmer's test scores did not change significantly during the study period. At baseline, mean impression cytology scores were similar for all treatment groups (Table 4). These scores remained virtually unchanged for the duration of the study, with no significant differences seen between treatment groups at any of the time points. Similarly, the change from baseline was not substantial for any group.
Total mean symptom scores were similar for the four treatment groups at baseline (Table 5). All groups had a decrease in total symptom scores during the study. There were, however, no statistically significant differences between any of the groups. Data on patient compliance suggested that most patients in the study complied with the treatment regimen all of the time. The mean volume of investigational material used per day between the three groups ranged from 0.82 to 0.93 g/day. Most of the reported adverse reactions could be categorized as ocular irritation that included pain, burning, stinging, itching, redness, irrita tion, and foreign-body sensation. More fibronectin-treated and vehicle-treated patients ter minated from the study because of adverse reactions than patients in the artificial tear group.
Discussion The results of this double-masked, placebocontrolled, multicenter study in which patients were randomly assigned to treatment demon strate that fibronectin had no greater therapeu tic efficacy in patients with keratoconjunctivitis sicca than did the vehicle solution or Liquifilm
TABLE 2 MEAN SUPERFICIAL PUNCTATE STAINING SCORES* FIBRONECTIN
VEHICLE NO. OF
PLACEBO
DAY
NO. OF PATIENTS
MEAN
STANDARD DEVIATION
PATIENTS
MEAN
STANDARD DEVIATION
NO. OF PATIENTS
MEAN
STANDARD DEVIATION
1 21 42 63
103 91 83 79
11.7 8.4 7.9 6.4
8.6 7.8 7.8 6.8
69 61 58 56
10.4 7.8 6.6 6.4
8.3 7.0 5.8 6.5
94 90 88 86
12.7 9.1 8.1 8.0
8.3 7.7 7.6 7.9
•Maximum superficial punctate staining score for each eye was 20 (40 for both eyes).
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TABLE 3 MEAN ROSE BENGAL STAINING SCORES* FIBRONECTIN NO. OF
VEHICLE NO. OF
PLACEBO
DAY
PATIENTS
MEAN
STANDARD DEVIATION
PATIENTS
MEAN
STANDARD DEVIATION
PATIENTS
NO. OF MEAN
STANDARD DEVIATION
1 21 42 63
103 91 83 79
8.5 6.8 6.2 5.5
4.0 4.9 4.6 4.1
69 61 58 56
8.8 6.6 6.0 5.9
4.2 4.7 4.5 4.2
95 90 88 86
9.1 6.5 5.6 6.3
4.2 4.1 4.5 4.4
'Maximum rose bengal staining score for each eye was 9 (18 for both eyes).
tears, a commercially available artificial tear solution. The total patient population (all treat ment groups) had marked improvements from baseline with regard to the major clinical varia bles associated with keratoconjunctivitis sicca, that is, superficial punctate staining, rose ben gal staining, and symptoms. However, these effects were no greater in the fibronectintreated patients than in the control patients. The extensive analyses performed on the data generated in this trial did result in scattered statistically significant effects, but there was no pattern of findings that could suggest superior efficacy of any of the treatments tested. Adverse reactions were more frequent in the fibronectin-treated and vehicle-treated groups. These consisted of foreign-body sensation and burning, which suggested that the fibronectin and vehicle preparations may have been more irritating than the control preparations. Fibronectin was proposed as a potential ther apeutic agent for keratoconjunctivitis sicca on the basis of experimental evidence that sup ports the involvement of this glycoprotein in corneal wound healing. The presence of a ma trix of endogenous fibronectin forming over corneas after superficial keratectomy 46 and the temporal relationship between fibronectin de position, cellular migration, and corneal heal ing8 suggest a role for fibronectin in ocular sur
face disease associated with a compromised corneal epithelium, such as severe keratocon junctivitis sicca. The data generated in this study suggest that the corneal substratum in patients with kerato conjunctivitis sicca, even those with a compro mised corneal epithelium as evidenced by staining with fluorescein and rose bengal, dif fers substantially from the corneal stroma after superficial keratectomy or other types of injury. Although patients with keratoconjunctivitis sicca may have intense staining of the cornea or conjunctiva, or both, at initial examination, these areas of missing cells apparently heal rapidly with appropriate lubrication. This is in contrast to other cases in which the corneal epithelium has been damaged, and is resistant to healing. In more serious types of corneal damage, the loss of epithelium is accompanied by increased proteolytic activity, resulting in degradation of fibronectin from the anterior stroma and destruction of basement mem brane. In such cases, treatment with exogenous fibronectin may be effective in promoting gene ration and adhesion of new epithelium. In the keratoconjunctivitis sicca syndrome, the loss of epithelial cells may not be followed by further degradation of the underlying tissue. If endoge nous production of fibronectin is normal in these patients, and loss of epithelium is related
TABLE 4 MEAN CONJUNCTIVAL IMPRESSION CYTOLOGY SCORES* VEHICLE
FIBRONECTIN NO. OF DAY
PATIENTS
MEAN
STANDARD DEVIATION
1 42 63
33 23 29
7.6 7.9 7.7
3.6 3.2 2.8
NO. OF PATIENTS
21 16 17
PLACEBO
MEAN
STANDARD DEVIATION
NO. OF PATIENTS
MEAN
STANDARD DEVIATION
7.9 8.0 7.4
2.6 3.6 3.5
29 24 24
7.5 7.0 7.4
3.6 3.4 3.3
»Maximum impression cytology score was 9 (18 for both eyes).
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TABLE 5 MEAN OVERALL SYMPTOM SCORES* FIBRONECTIN NO. OF
VEHICLE
PLACEBO
DAY
PATIENTS
MEAN
STANDARD DEVIATION
NO. OF PATIENTS
MEAN
STANDARD DEVIATION
PATIENTS
NO. OF MEAN
STANDARD DEVIATION
1 21 42 63
101 87 82 78
307.0 247.6 223.7 225.3
151.1 163.9 127.0 142.7
68 61 54 56
320.1 235.7 220.2 241.2
154.6 148.1 138.7 150.0
92 89 88 82
305.2 214.6 213.7 216.5
152.3 138.8 150.9 159.7
"Nine subjective symptoms were scored by each patient on a scale of 0 to 100 (0 represents absent and 100 represents severe), which were then averaged to produce an overall symptom score.
primarily to the combined physical factors of aqueous deficiency and eyelid movement, addi tion of exogenous fibronectin would not be expected to be more efficacious than appropri ate and frequent tear replacement with an arti ficial tear formulation. Even though patient symptoms and clinical findings improved over the course of the study, the ocular surface did not improve as assessed by conjunctival impression cytology. This has previously been observed.1'3 The reason for this is not known. There may be a primary ocular surface disorder in patients with keratoconjunctivitis sicca that does not respond to topical lubrication or may require certain, as yet un known, substances to reverse squamous meta plasia. Reversal of squamous metaplasia in keratoconjunctivitis sicca may take longer than six weeks.14 Although the full study population, includ ing the artificial tear control group, had signifi cant improvements from baseline in rose bengal and fluorescein staining, as well as in symptoms, the artificial tear solution used as the placebo control is not necessarily more efficacious in the treatment of keratoconjunctivitis sicca than the products used by the pa tients before enrollment. In fact, Liquifilm tears was one of the multitude of commercial dry-eye products used by patients before the study in attempting to alleviate their condition. These findings suggest that participation in a clinical trial, with the frequent examinations and in creased level of attention given to the patients, results in a high level of compliance with the treatment regimen, and a more positive re sponse to a previously used artificial tear.
THE CHIRON KERATOCONJUNCTIVITIS SICCA STUDY GROUP
The participants in the Chiron Keratoconjunctivitis Sicca Study Group are as follows: Principal Investigators: Penny A. Asbell, M.D., New York, New York; Alvan Baient, M.D., Fort Lauderdale, Florida; Richard Bowe, M.D., Tacoma, Washing ton; Terry Burris, M.D., Portland, Oregon; Delmar Caldwell, M.D., New Orleans, Louisiana; Dwight H. Cavanagh, M.D., Dallas, Texas; Donald J. Cinotti, M.D., Jersey City, New Jersey; Stuart Cole, M.D., Long Beach, California; William Constad, M.D., Jer sey City, New Jersey; Richard M. Davis, M.D., Co lumbia, South Carolina; Deborah Di Stefano, M.D., Chattanooga, Tennessee; Peter C. Donshik, M.D., West Hartford, Connecticut; Steven Dunn, M.D., Southfield, Michigan; Randy Epstein, M.D., Chicago, Illi nois; R. Linsey Farris, M.D., New York, New York; S. Lance Forstot, M.D., Littleton, Colorado; Gary Foulks, M.D., Durham, North Carolina; Bradley Fouraker, M.D., St. Louis, Missouri; M. H. Friedlaender, M.D., La Jolla, California; David Heidemann, M.D., Southfield, Michigan; Richard Keates, M.D., Irvine, California; Richard Lembach, M.D., Columbus, Ohio; Michael A. Lemp, M.D., Washing ton, D.C.; Lawrence Lohman, M.D., Kent, Ohio; Daniel Long, M.D., Gretna, Louisiana; Osvaldo Lo pez, M.D., Chicago, Illinois; William Mathers, M.D., Washington, D.C.; James P. McCulley, M.D., Dallas, Texas; Robert McCuIloch, M.D., Phoenix, Arizona; Alan Mindlin, M.D., Pontiac, Michigan; Richard Norden, M.D., Ridgewood, New Jersey; Stephen Pascucci, M.D., Scranton, Pennsylvania; Henry Per ry, M.D., Rockville Centre, New York; Francis Price, M.D., Indianapolis, Indiana; Peter A. Rapoza, M.D., Madison, Wisconsin; J. James Rowsey, M.D., Oklaho ma City, Oklahoma; H. Kaz Soong, M.D., Ann Arbor, Michigan; Michael Tapert, M.D, Mt. Pleasant, South Carolina; Michael Wagoner, M.D., Boston, Massa-
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chusetts; and William Whitson, M.D., Indianapolis, Indiana. Chiron Staff: Efraim Duzman, M.D., Medical Moni tor; Judy Gordon, D.V.M., Director, Clinical Re search; and Patti Lee, Senior Clinical Research Asso ciate.
References 1. Yamada, K. M., Yamada, S. S., and Pastan, I.: Cell surface protein partially restores morphology, adhesiveness and contact inhibition of movement to transformed fibroblasts. Proc. Nati. Acad. Sci. U.S.A. 73:1217, 1976. 2. AH, I. U., and Hynes, R. O.: Effects of LETS glycoprotein on cell motility. Cell 14:439, 1978. 3. Courtois, Y., Arruti, C , Barritault, D., Tassin, J., Olivie, M., and Hughes, R. C : Modulation of the shape of epithelial lens cells in vitro directed by a retinal extract factor. A model of interconversions and the role of active filaments and fibronectin. Differentiation 18:11, 1981. 4. Phan, T. M., Gipson, I. K., Foster, C. S., Zagachin, L., and Colvin, R. B.: Endogenous production of fibronectin in corneal stromal organ culture crossspecies transplant study. ARVO abstracts. Supple ment to Invest. Ophthalmol. Vis. Sci. Philadelphia, J. B. Lippincott, 1986, p. 52. 5. Nishida, T., Nakagawa, S., Ohaski, Y., Awata, T., and Manabe, R.: Fibronectin (FN) has been detect ed at the site of corneal wounds. Jpn. J. Ophthalmol. 26:410, 1982. 6. Phan, T., Colvin, R. B., and Foster, C. S.: Role of fibronectin in vitro healing of superficial keratectomies. ARVO abstracts. Supplement to Invest. Oph
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thalmol. Vis. Sci. Philadelphia, ]. B. Lippincott, 1985, p. 92. 7. Suda, T., Nishida, T., Ohashi, Y., Nakagawa, S., and Manabe, R.: Fibronectin appears at the site of the corneal stromal wound in rabbits. Curr. Eye Res. 1:553, 1982. 8. Fujikawa, L. S., Foster, C. S., Harris, T. J., Lanigan, J. M., and Colvin, R. B.: Fibronectin in healing rabbit corneal wounds. Lab. Invest. 45:120, 1981. 9. Ohashi, Y., Nakagawa, S., Nishida, T., Suda, T., Watanabe, K., and Manabe, R.: Appearance of fibro nectin in rabbit cornea after thermal burn. Jpn. J. Ophthalmol. 27:547, 1983. 10. Nishida, T., Ohashi, Y., Inoue, Y., Nakagawa, S., Awata, T., Suda, T., and Manabe, R.: Dynamics of fibronectin in corneal wound healing. Immunohistochemical study of experimental bullous keratopathy in rabbits. Cornea 1:311, 1982. 11. Kono, I., Mutsumoto, Y., Kono, K., Ishibashi, Y., Narushima, K., Kabashima, T., Yamane, K., Sakurai, T., and Kashiwagi, J.: Beneficial effect of topical fibronectin in patients with keratoconjuncti vitis sicca or Sjôgren's syndrome. J. Rheumatol. 12:487, 1985. 12. Nelson, J. D., Havener, V. R., and Cameron, J. D.: Cellulose acetate impressions of the ocular surface. Dry eye states. Arch. Ophthalmol. 101:1869, 1983. 13. Nelson, J. D., and Farris, R. L.: An evaluation of sodium hyaluronate and poly vinyl alcohol in kera toconjunctivitis sicca. Arch. Ophthalmol. 106:484, 1988. 14. Gilbard, J. P., Rossi, S. R., Gray, K. L., Hanninen, L. A., and Kenyon, K. R.: Tear film osmolarity and ocular surface disease in two rabbit models for keratoconjunctivitis sicca. Invest. Ophthalmol. Vis. Sci. 29:374, 1988.
Accepted for publication July 23, 1992. From the Department of Ophthalmology, Ramsey Clinic, St. Paul-Ramsey Medical Center, St. Paul, Minne sota; and Department of Ophthalmology, University of Minnesota, Minneapolis, Minnesota (Dr. Nelson); and Chiron Ophthalmics, Irvine, California (Dr. Gordon and Chiron Keratoconjunctivitis Sicca Study Group). Dr. Gordon is an employee of Chiron Ophthalmics. Reprint requests to J. Daniel Nelson, M.D., Depart ment of Ophthalmology, St. Paul-Ramsey Medical Cen ter, 640 Jackson St., St. Paul, MN 55101.
promotes cell-to-cell and cell-to-substrate ad hesion, fibronectin also has a role in cell migra tion, wound healing, and cytoskeletal organiza tion. Fibronectin has been shown to promote the attachment of rabbit corneal endothelial cells to plastic culture dishes. 1 The addition of fibronectin to cultures of transformed cells al lows the cells to adhere to and move across the surface of the plastic culture plates. 2,8 In vitro studies have shown that endogenously pro duced fibronectin forms a matrix over the un derlying stroma of corneas subjected to superfi cial keratectomy, thus allowing epithelial cell migration. 4 6 The in vivo induction of corneal lesions has also been shown to be accompanied by fibronectin deposition during the subse quent healing period. 710 The presence of endogenous fibronectin on the surface of the injured cornea led to clinical application of exogenous fibronectin in pa tients with keratoconjunctivitis sicca. Kono and associates 11 used autologous fibronectin in treating a group of 12 patients with keratocon junctivitis sicca associated with Sjögren's syn drome. Treatment with fibronectin resulted in a reduction in rose bengal and fluorescein stain ing as well as improvement in the subjective symptoms of dry eye. On the basis of data that suggested a role of fibronectin in the healing of ocular surface disorders, including keratoconjunctivitis sicca, a multicenter double-masked, vehicle-con trolled and placebo-controlled trial was con ducted to study the safety and efficacy of fibro nectin in the treatment of patients with keratoconjunctivitis sicca.
Material and Methods Patients—Over a 16-month period (from April 1988 to August 1989), 272 patients with keratoconjunctivitis sicca were enrolled in this clinical trial. Patients eligible for enrollment
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were required to meet at least one of the follow ing entry criteria: (1) wetting of less than 5 mm in ßve minutes when Schirmer's testing is per formed without topical anesthesia and a super ficial punctate staining score of at least 2 (on a scale of 1 to 4); (2) wetting of less than 5 mm in five minutes when Schirmer's testing is per formed without topical anesthesia and a rose bengal staining score greater than 3 (on a scale of 1 to 9); (3) rose bengal staining score greater than 3 (on a scale of 1 to 9) and a patient self-evaluation score of at least 50 on a visual analogue scale of 0 to 100 on three of nine dry-eye symptoms (pain, burning, itching, dryness, foreign-body sensation, tearing, blurring, photophobia, and other); and (4) superficial punctate staining score of at least 2 and a patient self-evaluation score of at least 50 on three dry-eye symptoms. In addition to meeting these diagnostic criteria, all patients were re quired to be of legal age and to have read and signed a written informed consent form. Patients with dry eye associated with congen ital alacrima, vitamin A deficiency, StevensJohnson syndrome, ocular pemphigoid, druginduced pseudopemphigoid, ocular chemical burns, neurogenic hyposecretion, lagophthalmos, inadequate eyelid closure, trachoma, ocu lar surface irregularities, trichiasis, or ocular radiation injury were excluded from the study. Other exclusionary criteria were uncontrolled blepharitis; rosacea keratoconjunctivitis; corneal perforations; preexisting blindness, glau coma, or uveitis; administration of medications known to cause ocular dryness; uncontrolled ophthalmic or systemic infections; serious im mune deficiency or immunosuppressive thera py; pregnancy; and lactation. Patients with a history of viral hepatitis or infection with hu man immunodeficiency virus were also ex cluded. Study design—The study was a doublemasked, controlled parallel group trial in which patients were randomly assigned to treatment and was conducted at 33 clinical sites. The experimental protocol was reviewed and ap proved by the Institutional Review Board at each study center. After determination of eligibility, all patients were required to complete 14 days of washout, during which they administered only an unpreserved lubricating agent (Refresh, Allergan Pharmaceuticals, Irvine, California). At the end of this 14-day period, patients were examined again to determine eligibility. The unpreserved
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lubricating agent was used to eliminate the effect of preservatives on the patient's clinical signs and symptoms. Patients who had an im provement in clinical signs and symptoms dur ing the washout period, and who no longer satisfied the entry criteria, were not enrolled in the study. Eligible patients were randomly assigned to one of three treatment groups: fibronectin oph thalmic solution (3.5 mg/ml); vehicle solution, consisting of all components of the fibronectin solution, without fibronectin; or a commercial ly available artificial tear product (Liquifilm Tears, Allergan Pharmaceuticals). The identity of the treatment was masked to investigators and patients. On day 1 of the study, each patient adminis tered one drop of the study medication (fibro nectin, vehicle, or artificial tear) into each eye six to eight times daily. Treatment was contin ued for 42 days, after which patients returned to administration of only an unpreserved lubri cating agent (Refresh) for three weeks (that is, through day 63). Separate computer-generated randomization codes were prepared for each clinical site, with treatment groups in blocks such that an equal number of patients at each center were treated with fibronectin, vehicle, and artificial tear. No other topical ophthalmic medication was allowed at any time during the study, and Refresh was provided to all study patients to ensure compliance. Systemic medi cations that were considered necessary for the patients' welfare, and that did not interfere with the study, were allowed. Patients who were self-administering oral beta blockers, diu retics, antidepressants, or estrogens were in structed to make no changes in their treatment regimen to avoid any possible effects on the level of ocular dryness. Study medications—The fibronectin ophthal mic solution (3.5 mg/ml) consisted of lyophilized plasma fibronectin reconstituted in sterile saline solution that contained 0.5% chlorobutanol as a preservative. The lyophilized plasma fibronectin contained albumin and sucrose as stabilizers, and sodium chloride, sodium dihydrogen phosphate, and sodium hydrogen phos phate as buffering agents. The vehicle solution consisted of the stabiliz ers and buffering agents found in fibronectin ophthalmic solution (that is, albumin, sucrose, sodium chloride, sodium dihydrogen phos phate, and sodium hydrogen phosphate). These materials were reconstituted in sterile saline
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solution that contained 0.5% chlorobutanol as a preservative. The artificial tear solution consisted of Liquifilm Tears, a commercially available artificial tear solution that contained polyvinyl alcohol 1.4%, sodium chloride, and chlorobutanol 0.5% in purified water. Schedule of observations—Patients were ex amined 14 days before enrollment in the study, and then on day 1 to determine eligibility for the study. During the 42-day treatment period, patients were examined on day 21 (three weeks) and day 42 (six weeks). All patients were re quired to return for a follow-up visit on day 63 (nine weeks). Each patient visit included a com plete ocular examination, which consisted of slit-lamp biomicroscopy, dilated fundus exami nation (day 1 and day 42), Schirmer's testing without anesthesia, and measurement of visual acuity, intraocular pressure, tear meniscus height, and tear breakup time. The primary objective signs that were evalu ated at each examination were fluorescein staining and rose bengal staining. Fluorescein staining was scored by the investigator on a scale of 0 to 4 (absent to severe staining) on the basis of comparison of the investigator's findings with a panel of standardized diagrams of staining provided on each data form. To evaluate fluorescein staining, five areas of the cornea (central cornea and then four equal peripheral areas) were individually scored, with a total maximum score of 20 per eye (40 for both eyes). Rose bengal staining was scored similarly, on a scale of 0 to 3 (absent to severe staining), with the degree of staining recorded separately for the temporal conjunctiva, cor nea, and nasal conjunctiva. The total rose ben gal staining score for each eye was 9, or 18 for both eyes. For both rose bengal and fluorescein staining in which only one eye was treated, the score was doubled to produce a comparable value for analysis. A subset of the full study population also underwent serial conjunctival impression cy tology, with samples obtained from the superi or bulbar, temporal bulbar, nasal bulbar, and inferior palpebrai conjunctiva to evaluate the degree of squamous metaplasia. Each region yielded a score ranging from 0.0 to 3.0 (normal cells to severe squamous metaplasia). Temporal and nasal specimens were considered a single specimen. The maximum impression cytology score was 18.0 for both eyes, with the score doubled for analysis when only one eye was
443
treated. All impression cytology samples were evaluated in a masked fashion by a single read ing center, according to previously described cytologie techniques for grading conjunctival squamous metaplasia. 12 In addition to these clinical signs, a question naire was completed by the patients at each examination. Subjective symptoms were evalu ated by each patient on a visual analogue scale of 0 to 100 (0 represents absent, 100 represents severe). Scores for nine symptoms (pain, burn ing/stinging, itching, dryness, foreign-body sensation, tearing, blurring of vision, light sen sitivity, and other) were averaged to produce an overall symptom score. Additionally, patients rated their overall improvement on a visual analogue scale of 0 to 100 (no improvement to marked improvement). Compliance levels were gauged through questions directed to the patient (whether the patient adhered to the treatment regimen all the time, most of the time, or not at all), as well as by measurement of the residual volume of solution in each dropper bottle returned by the patient. Statistical analysis-Extensive statistical anal yses were performed on the data generated in this study. The initial analysis was a direct comparison of the treatment groups at each time point in the study, conducted on the full study population. A comparison of the change from baseline for each treatment group was also performed for the full study population. Additional analyses were performed on numer ous subgroups of the overall population (pa tients 60 years old and older, patients younger than 60 years, patients with Sjögren's syn drome-related keratoconjunctivitis sicca, pa tients with arthritis, patients with age-related keratoconjunctivitis sicca, a subgroup of pa tients who were severely affected, and a sub group of patients whose keratoconjunctivitis sicca was determined on the basis of impression cytology findings). Results of the subgroup analyses were not significantly different when compared to the results of the overall popula tion analysis. Mean values were determined with standard deviations. Continuous variables and ordered categorical variables were analyzed by using individual f-test comparisons of group means at each visit. Differences between groups in change from baseline were analyzed by using the chi-square test. For some analyses, continu ous variables were converted to categorical var-
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444
TABLE 1 PATIENT DEMOGRAPHICS AND DISPOSITION
MEAN AGE (YRS) (± STANDARD DEVIATION)
TREATMENT
Fibronectin Vehicle Control Total
60 61 69 63
± ± ± ±
14 14 14 15
NO. OF PATIENTS FEMALES
ENTERED
COMPLETED
DISCONTINUED
TERMINATED
94 59 82 235
103 69 95 267
80 55 86 221
11 6 7 24
12 8 2 22
iables and analyzed by using the chi-square test.
Results Patient population—We enrolled 267 patients in this study. Of these 267 patients, 221 pa tients completed the study. Demographic characteristics and disposition of patients were analyzed (Table 1). For the most part, all treat ment groups were comparable at baseline, al though there was a disproportionate number of females enrolled in the study, as compared to males (235 vs 32). Clinical signs—Both fluorescein and rose bengal staining improved in all groups during the course of the study (Tables 2 and 3). There were, however, no statistically significant dif ferences between any of the groups. Tear break up times and Schirmer's test scores did not change significantly during the study period. At baseline, mean impression cytology scores were similar for all treatment groups (Table 4). These scores remained virtually unchanged for the duration of the study, with no significant differences seen between treatment groups at any of the time points. Similarly, the change from baseline was not substantial for any group.
Total mean symptom scores were similar for the four treatment groups at baseline (Table 5). All groups had a decrease in total symptom scores during the study. There were, however, no statistically significant differences between any of the groups. Data on patient compliance suggested that most patients in the study complied with the treatment regimen all of the time. The mean volume of investigational material used per day between the three groups ranged from 0.82 to 0.93 g/day. Most of the reported adverse reactions could be categorized as ocular irritation that included pain, burning, stinging, itching, redness, irrita tion, and foreign-body sensation. More fibronectin-treated and vehicle-treated patients ter minated from the study because of adverse reactions than patients in the artificial tear group.
Discussion The results of this double-masked, placebocontrolled, multicenter study in which patients were randomly assigned to treatment demon strate that fibronectin had no greater therapeu tic efficacy in patients with keratoconjunctivitis sicca than did the vehicle solution or Liquifilm
TABLE 2 MEAN SUPERFICIAL PUNCTATE STAINING SCORES* FIBRONECTIN
VEHICLE NO. OF
PLACEBO
DAY
NO. OF PATIENTS
MEAN
STANDARD DEVIATION
PATIENTS
MEAN
STANDARD DEVIATION
NO. OF PATIENTS
MEAN
STANDARD DEVIATION
1 21 42 63
103 91 83 79
11.7 8.4 7.9 6.4
8.6 7.8 7.8 6.8
69 61 58 56
10.4 7.8 6.6 6.4
8.3 7.0 5.8 6.5
94 90 88 86
12.7 9.1 8.1 8.0
8.3 7.7 7.6 7.9
•Maximum superficial punctate staining score for each eye was 20 (40 for both eyes).
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445
TABLE 3 MEAN ROSE BENGAL STAINING SCORES* FIBRONECTIN NO. OF
VEHICLE NO. OF
PLACEBO
DAY
PATIENTS
MEAN
STANDARD DEVIATION
PATIENTS
MEAN
STANDARD DEVIATION
PATIENTS
NO. OF MEAN
STANDARD DEVIATION
1 21 42 63
103 91 83 79
8.5 6.8 6.2 5.5
4.0 4.9 4.6 4.1
69 61 58 56
8.8 6.6 6.0 5.9
4.2 4.7 4.5 4.2
95 90 88 86
9.1 6.5 5.6 6.3
4.2 4.1 4.5 4.4
'Maximum rose bengal staining score for each eye was 9 (18 for both eyes).
tears, a commercially available artificial tear solution. The total patient population (all treat ment groups) had marked improvements from baseline with regard to the major clinical varia bles associated with keratoconjunctivitis sicca, that is, superficial punctate staining, rose ben gal staining, and symptoms. However, these effects were no greater in the fibronectintreated patients than in the control patients. The extensive analyses performed on the data generated in this trial did result in scattered statistically significant effects, but there was no pattern of findings that could suggest superior efficacy of any of the treatments tested. Adverse reactions were more frequent in the fibronectin-treated and vehicle-treated groups. These consisted of foreign-body sensation and burning, which suggested that the fibronectin and vehicle preparations may have been more irritating than the control preparations. Fibronectin was proposed as a potential ther apeutic agent for keratoconjunctivitis sicca on the basis of experimental evidence that sup ports the involvement of this glycoprotein in corneal wound healing. The presence of a ma trix of endogenous fibronectin forming over corneas after superficial keratectomy 46 and the temporal relationship between fibronectin de position, cellular migration, and corneal heal ing8 suggest a role for fibronectin in ocular sur
face disease associated with a compromised corneal epithelium, such as severe keratocon junctivitis sicca. The data generated in this study suggest that the corneal substratum in patients with kerato conjunctivitis sicca, even those with a compro mised corneal epithelium as evidenced by staining with fluorescein and rose bengal, dif fers substantially from the corneal stroma after superficial keratectomy or other types of injury. Although patients with keratoconjunctivitis sicca may have intense staining of the cornea or conjunctiva, or both, at initial examination, these areas of missing cells apparently heal rapidly with appropriate lubrication. This is in contrast to other cases in which the corneal epithelium has been damaged, and is resistant to healing. In more serious types of corneal damage, the loss of epithelium is accompanied by increased proteolytic activity, resulting in degradation of fibronectin from the anterior stroma and destruction of basement mem brane. In such cases, treatment with exogenous fibronectin may be effective in promoting gene ration and adhesion of new epithelium. In the keratoconjunctivitis sicca syndrome, the loss of epithelial cells may not be followed by further degradation of the underlying tissue. If endoge nous production of fibronectin is normal in these patients, and loss of epithelium is related
TABLE 4 MEAN CONJUNCTIVAL IMPRESSION CYTOLOGY SCORES* VEHICLE
FIBRONECTIN NO. OF DAY
PATIENTS
MEAN
STANDARD DEVIATION
1 42 63
33 23 29
7.6 7.9 7.7
3.6 3.2 2.8
NO. OF PATIENTS
21 16 17
PLACEBO
MEAN
STANDARD DEVIATION
NO. OF PATIENTS
MEAN
STANDARD DEVIATION
7.9 8.0 7.4
2.6 3.6 3.5
29 24 24
7.5 7.0 7.4
3.6 3.4 3.3
»Maximum impression cytology score was 9 (18 for both eyes).
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TABLE 5 MEAN OVERALL SYMPTOM SCORES* FIBRONECTIN NO. OF
VEHICLE
PLACEBO
DAY
PATIENTS
MEAN
STANDARD DEVIATION
NO. OF PATIENTS
MEAN
STANDARD DEVIATION
PATIENTS
NO. OF MEAN
STANDARD DEVIATION
1 21 42 63
101 87 82 78
307.0 247.6 223.7 225.3
151.1 163.9 127.0 142.7
68 61 54 56
320.1 235.7 220.2 241.2
154.6 148.1 138.7 150.0
92 89 88 82
305.2 214.6 213.7 216.5
152.3 138.8 150.9 159.7
"Nine subjective symptoms were scored by each patient on a scale of 0 to 100 (0 represents absent and 100 represents severe), which were then averaged to produce an overall symptom score.
primarily to the combined physical factors of aqueous deficiency and eyelid movement, addi tion of exogenous fibronectin would not be expected to be more efficacious than appropri ate and frequent tear replacement with an arti ficial tear formulation. Even though patient symptoms and clinical findings improved over the course of the study, the ocular surface did not improve as assessed by conjunctival impression cytology. This has previously been observed.1'3 The reason for this is not known. There may be a primary ocular surface disorder in patients with keratoconjunctivitis sicca that does not respond to topical lubrication or may require certain, as yet un known, substances to reverse squamous meta plasia. Reversal of squamous metaplasia in keratoconjunctivitis sicca may take longer than six weeks.14 Although the full study population, includ ing the artificial tear control group, had signifi cant improvements from baseline in rose bengal and fluorescein staining, as well as in symptoms, the artificial tear solution used as the placebo control is not necessarily more efficacious in the treatment of keratoconjunctivitis sicca than the products used by the pa tients before enrollment. In fact, Liquifilm tears was one of the multitude of commercial dry-eye products used by patients before the study in attempting to alleviate their condition. These findings suggest that participation in a clinical trial, with the frequent examinations and in creased level of attention given to the patients, results in a high level of compliance with the treatment regimen, and a more positive re sponse to a previously used artificial tear.
THE CHIRON KERATOCONJUNCTIVITIS SICCA STUDY GROUP
The participants in the Chiron Keratoconjunctivitis Sicca Study Group are as follows: Principal Investigators: Penny A. Asbell, M.D., New York, New York; Alvan Baient, M.D., Fort Lauderdale, Florida; Richard Bowe, M.D., Tacoma, Washing ton; Terry Burris, M.D., Portland, Oregon; Delmar Caldwell, M.D., New Orleans, Louisiana; Dwight H. Cavanagh, M.D., Dallas, Texas; Donald J. Cinotti, M.D., Jersey City, New Jersey; Stuart Cole, M.D., Long Beach, California; William Constad, M.D., Jer sey City, New Jersey; Richard M. Davis, M.D., Co lumbia, South Carolina; Deborah Di Stefano, M.D., Chattanooga, Tennessee; Peter C. Donshik, M.D., West Hartford, Connecticut; Steven Dunn, M.D., Southfield, Michigan; Randy Epstein, M.D., Chicago, Illi nois; R. Linsey Farris, M.D., New York, New York; S. Lance Forstot, M.D., Littleton, Colorado; Gary Foulks, M.D., Durham, North Carolina; Bradley Fouraker, M.D., St. Louis, Missouri; M. H. Friedlaender, M.D., La Jolla, California; David Heidemann, M.D., Southfield, Michigan; Richard Keates, M.D., Irvine, California; Richard Lembach, M.D., Columbus, Ohio; Michael A. Lemp, M.D., Washing ton, D.C.; Lawrence Lohman, M.D., Kent, Ohio; Daniel Long, M.D., Gretna, Louisiana; Osvaldo Lo pez, M.D., Chicago, Illinois; William Mathers, M.D., Washington, D.C.; James P. McCulley, M.D., Dallas, Texas; Robert McCuIloch, M.D., Phoenix, Arizona; Alan Mindlin, M.D., Pontiac, Michigan; Richard Norden, M.D., Ridgewood, New Jersey; Stephen Pascucci, M.D., Scranton, Pennsylvania; Henry Per ry, M.D., Rockville Centre, New York; Francis Price, M.D., Indianapolis, Indiana; Peter A. Rapoza, M.D., Madison, Wisconsin; J. James Rowsey, M.D., Oklaho ma City, Oklahoma; H. Kaz Soong, M.D., Ann Arbor, Michigan; Michael Tapert, M.D, Mt. Pleasant, South Carolina; Michael Wagoner, M.D., Boston, Massa-
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Fibronectin in Keratoconjunctivitis Sicca
chusetts; and William Whitson, M.D., Indianapolis, Indiana. Chiron Staff: Efraim Duzman, M.D., Medical Moni tor; Judy Gordon, D.V.M., Director, Clinical Re search; and Patti Lee, Senior Clinical Research Asso ciate.
References 1. Yamada, K. M., Yamada, S. S., and Pastan, I.: Cell surface protein partially restores morphology, adhesiveness and contact inhibition of movement to transformed fibroblasts. Proc. Nati. Acad. Sci. U.S.A. 73:1217, 1976. 2. AH, I. U., and Hynes, R. O.: Effects of LETS glycoprotein on cell motility. Cell 14:439, 1978. 3. Courtois, Y., Arruti, C , Barritault, D., Tassin, J., Olivie, M., and Hughes, R. C : Modulation of the shape of epithelial lens cells in vitro directed by a retinal extract factor. A model of interconversions and the role of active filaments and fibronectin. Differentiation 18:11, 1981. 4. Phan, T. M., Gipson, I. K., Foster, C. S., Zagachin, L., and Colvin, R. B.: Endogenous production of fibronectin in corneal stromal organ culture crossspecies transplant study. ARVO abstracts. Supple ment to Invest. Ophthalmol. Vis. Sci. Philadelphia, J. B. Lippincott, 1986, p. 52. 5. Nishida, T., Nakagawa, S., Ohaski, Y., Awata, T., and Manabe, R.: Fibronectin (FN) has been detect ed at the site of corneal wounds. Jpn. J. Ophthalmol. 26:410, 1982. 6. Phan, T., Colvin, R. B., and Foster, C. S.: Role of fibronectin in vitro healing of superficial keratectomies. ARVO abstracts. Supplement to Invest. Oph
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thalmol. Vis. Sci. Philadelphia, ]. B. Lippincott, 1985, p. 92. 7. Suda, T., Nishida, T., Ohashi, Y., Nakagawa, S., and Manabe, R.: Fibronectin appears at the site of the corneal stromal wound in rabbits. Curr. Eye Res. 1:553, 1982. 8. Fujikawa, L. S., Foster, C. S., Harris, T. J., Lanigan, J. M., and Colvin, R. B.: Fibronectin in healing rabbit corneal wounds. Lab. Invest. 45:120, 1981. 9. Ohashi, Y., Nakagawa, S., Nishida, T., Suda, T., Watanabe, K., and Manabe, R.: Appearance of fibro nectin in rabbit cornea after thermal burn. Jpn. J. Ophthalmol. 27:547, 1983. 10. Nishida, T., Ohashi, Y., Inoue, Y., Nakagawa, S., Awata, T., Suda, T., and Manabe, R.: Dynamics of fibronectin in corneal wound healing. Immunohistochemical study of experimental bullous keratopathy in rabbits. Cornea 1:311, 1982. 11. Kono, I., Mutsumoto, Y., Kono, K., Ishibashi, Y., Narushima, K., Kabashima, T., Yamane, K., Sakurai, T., and Kashiwagi, J.: Beneficial effect of topical fibronectin in patients with keratoconjuncti vitis sicca or Sjôgren's syndrome. J. Rheumatol. 12:487, 1985. 12. Nelson, J. D., Havener, V. R., and Cameron, J. D.: Cellulose acetate impressions of the ocular surface. Dry eye states. Arch. Ophthalmol. 101:1869, 1983. 13. Nelson, J. D., and Farris, R. L.: An evaluation of sodium hyaluronate and poly vinyl alcohol in kera toconjunctivitis sicca. Arch. Ophthalmol. 106:484, 1988. 14. Gilbard, J. P., Rossi, S. R., Gray, K. L., Hanninen, L. A., and Kenyon, K. R.: Tear film osmolarity and ocular surface disease in two rabbit models for keratoconjunctivitis sicca. Invest. Ophthalmol. Vis. Sci. 29:374, 1988.
