Keratoconjunctivitis Sicca Patients Research Articles

JAK1, SKI, ZBTB16 as potential biomarkers mediate the inflammatory response in keratoconjunctivitis sicca

Keratoconjunctivitis sicca (KCS) is an ocular condition characterized by insufficient tear production and inflammatory irritation, with Sjögren’s syndrome (SS) being a major causative factor. This study aimed to extract patient transcriptomic data from the GEO database to identify signature genes associated with the diagnosis and treatment of KCS and the expression of three key genes were experimentally verified.We performed a difference analysis on the SS patient dataset and performed a Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on the resulting genes. Additionally, a Weighted Gene Co-expression Network Analysis (WGCNA) was constructed. Machine learning techniques were employed to analyze the most strongly correlated gene modules with SS traits. These findings were further validated using KCS immune-correlation microarrays as a validation set. The correlation of the three identified genes with 22 immune cells was assessed through immune infiltration analysis. Subsequently, a rat model of desiccated keratoconjunctivitis was established, and the modeling situation and expression of characteristic genes were analyzed at the morphological, tissue, and molecular levels.Bioinformatic prediction revealed that the expression of JAK1, SKI, ZBTB16 not only differed in the machine learning validation set, but also correlated with some immune cells in the immune infiltration analysis. The results of animal experiments showed that the transcription and expression levels of these three genes were significantly different in rat KCS tissues and normal tissues, and there were also differences in the expression of JAK1 and SKI in rat peripheral blood, as well as significant up-regulation of the expression of related inflammatory factors in KCS tissues. Through bioinformatics prediction and animal experimental validation, this study identified three differentially expressed genes in SS mediated KCS patients, which provide new potential biological targets for the diagnosis and treatment of KCS.

Association of serum 25-hydroxyvitamin D concentrations with Schirmer tear test 1 and tear film breakup time in dogs.

The association between vitamin D and canine keratoconjunctivitis sicca (KCS) has not been investigated in dogs. To investigate the association of serum 25-hydroxyvitamin D [25(OH)D] concentrations with Schirmer tear test 1 (STT-1) and tear film breakup time (TFBUT) in dogs. Sixty-one clinically healthy, client-owned dogs were enrolled. STT-1 and TFBUT were measured in 122 (61dogs) and 82 (41 dogs out of total 61 dogs) eyes, respectively. Serum 25(OH)D concentrations were evaluated by quantitative chemiluminescent immunoassay. The dogs were classified into 6 groups according to the evaluations (STT-1: group 1, normal [≥ 15 mm/min] in both eyes; group 2, normal in one eye and abnormal [< 15 mm/min] in the fellow eye; group 3, abnormal in both eyes; TFBUT: group 4, normal [≥ 20 sec] in both eyes; group 5, normal in one eye and abnormal [< 20 sec] in the fellow eye; group 6, abnormal in both eyes). STT-1 was positively correlated with TFBUT (p < 0.001). Among the STT-1 groups, the mean serum 25(OH)D concentration in group 1 was significantly higher than in groups 2 and 3 with positive correlation (p < 0.001). However, there were no significant differences among the TFBUT groups 4, 5, and 6. In dogs, it was found that serum 25(OH)D concentrations had a greater effect on quantitative KCS than qualitative KCS. Therefore, it is considered that measurement of serum 25(OH)D concentration could be included in the diagnostic tests in canine quantitative KCS patients.

Topical applications of allogeneic adipose-derived mesenchymal stem cells ameliorate the canine keratoconjunctivitis sicca

BackgroundCanine keratoconjunctivitis sicca (KCS) is predominantly an immune-mediated disease. Current therapy of canine KCS is mainly by immunosuppressant, but the effectiveness was limited in some patients. In the past few years, some studies showed the results of the use of mesenchymal stem cells in treating canine KCS via periocular injections. However, the periocular injection procedure requires sedation or general anesthesia, and may lead to iatrogenic or incidental injury during the injection process. The aim of this study was to investigate the efficacy of topical allogenic canine adipose-derived mesenchymal stem cells (cAD-MSCs) in clinical patients of canine KCS.ResultsThe cAD-MSCs used in this study were characterized for their capability of tri-lineage differentiation and immunomodulatory properties. In addition, preparation methods for eye drops of cAD-MSCs was developed and its optimal preservation was tested. The canine KCS patients were recruited for clinical trial and divided into two groups based on their history of previous treatment. All patients received topical cAD-MSCs treatment once per week for 6 consecutive weeks and complete ophthalmic examinations were performed 1 week before treatment (week 0) and at 3rd, 6th, 9th weeks, respectively. The results showed that the quantity and quality of tears have improved significantly following topical cAD-MSCs treatment based on Schirmers tear test-1 and tear break-up time. More than half of all patients were found improved in the tear quantity. In particular, 56.5% of the patients that were unresponsive to prior immunosuppressant therapy had an effective increase in tear volume. The severity of clinical signs was also ameliorated according to the numeric rating scale score from both patient owners and the clinician.ConclusionTo sum up, topical cAD-MSCs may be beneficial especially in KCS patients with poor owner compliance for frequent daily use of eye drops or those who are unresponsive to immunosuppressant therapy.

Nuclear parameters and chromatin remodeling in epithelial cells and lymphocytes from the palpebral conjunctiva of dogs with keratoconjunctivitis sicca.

To study parameters related to nuclear morphology and chromatin remodeling in epithelial cells and lymphocytes from the inferior palpebral conjunctiva of dogs with and without keratoconjunctivitis sicca (KCS). Thirty-two dogs (64 eyes) were included in the study. Based on the tear production measured by Schirmer tear test 1, the dogs were distributed into control and KCS groups. Epithelial cells and lymphocytes were collected by conjunctival brush cytology, fixed on glass slides, and subjected to the Feulgen reaction, a topochemical method specific for DNA/chromatin. Feulgen-stained cells were studied by microscopy and video image analysis to establish nuclear size (area and perimeter) and shape (relative nuclear roundness factor=RNRF), DNA content (ploidy), and compaction and texture of chromatin. Conjunctival samples in the KCS group showed infiltration of inflammatory and immune cells. Micronuclei, snake-like chromatin, aberrant chromosomes, and goblet cells were not detected. Compared with the controls, cells on the conjunctival surface of dogs with KCS showed altered nuclei. Conjunctival epithelial cells were more affected by KCS (changes in nuclear size, shape, DNA content, and chromatin compaction) than lymphocytes (changes in chromatin compaction, only). Significant chromatin decompaction was observed in both conjunctival epithelial cells and lymphocytes. Our results show that KCS promotes chromatin remodeling in epithelial cells and lymphocytes on the conjunctival surface of dogs. The changes described in this study are different from those reported for conjunctival cell nuclei of human KCS patients.

Significance of laser confocal tomography in diagnosis and monitoring of keratoconjunctivitis sicca

Laser confocal tomography of the cornea enables studying ultrathin sections of corneal layers, which provides additional reliable information on tissue changes in keratoconjunctivitis sicca (KCS). To assess the significance of laser confocal tomography of the cornea in the diagnosis and monitoring of KCS. We investigated 38 eyes of 30 patients with severe KCS. The patients were divided into two groups. Group 1 (15 patients, 19 eyes) was prescribed cyclosporine А 0.05% instillations 2 times daily, artificial tears, and soft contact lenses. Group 2 (15 patients, 19 eyes) received only instillations of cyclosporine А 0.05% 2 times daily and artificial tears. Besides standard ophthalmic examination, additional tests were performed, namely Schirmer's test, tear break-up time test, fluorescein eye stain test, tear osmolarity test (TearLab System, USA), and Heidelberg retinal tomography of the cornea (HRT, Heidelberg Engineering GmbH, Germany). HRT findings revealed a 3 times shorter epithelization period and faster recovery of corneal transparency in group 1 as compared to group 2 (1.5 and 4.5 months, respectively). There was also an evident reduction in the number of immune cells in the cornea, most pronounced in group 1 at 3 months, which is indicative of inflammation termination. The use of HRT of the cornea in KCS patients allows real-time cellular level observation of corneal changes, which together with clinical findings and diagnostic tests not only confirms the diagnosis but also determines treatment effectiveness. It has been also found that soft contact lenses accelerate epithelization of the cornea and relieve inflammation of the ocular surface in KCS patients under cyclosporine A 0.05% instillation therapy. Transparency of financial activity: the authors have no financial interest in the submitted materials and methods.

Evaluation of conjunctival epithelial damage in dry eye

Using sulphorhodamine B, which is highly sensitive in detecting damaged ocular surface epithelium, we evaluated conjunctival epithelial damage in dry eye patients. The subjects were 99 eyes of 50 dry eye patients (41 eyes of 21 patients with Sjögren's syndrome and 58 eyes of 29 keratoconjunctivitis sicca patients without Sjögren's syndrome). We also investigated the relation between sulphorhodamine B and fluorescein staining in ocular surface epithelium. The conjunctival epithelial damage stained with sulphorhodamine B showed a high correlation with corneal epithelial damage stained with fluorescein. In addition, conjunctival damage in Sjögren's syndrome tended to be more severe than in patients without Sjögren's syndrome.

Study of autoantibodies in patients with keratoconjunctivitis sicca infected by the human T cell lymphotropic virus type 1

Human T cell lymphotropic virus type 1 (HTLV-1) is endemic in many regions of the world, including Brazil, and has been associated to several immunological manifestations such as arthritis, uveitis, dermatitis and Sjögren's syndrome. This study was intended to evaluate the frequency of autoantibodies in patients infected with HTLV-1 and manifesting keratoconjunctivitis sicca (KCS). HTLV-1 patients with KCS, enrolled in a reference ambulatory of the city of Salvador, were tested for autoantibodies such as antinuclear antibodies, rheumatoid factor, anti-SSA/Ro and anti-SSB/La. Two comparison groups were also included: (a) HTLV-1 patients without KCS and (b) seronegative patients with KCS. Correlation of proviral load (PVL) in HTLV-1 patients with presence or absence of KCS was also assessed. No autoantibodies were detected in HTLV-1 patients with KCS. The PVL of HTLV-1 patients was higher in patients with KCS without other clinical manifestations customarily associated to HTLV-1. In conclusion, in this study, no changes were observed in humoral immunity concerning production of certain autoantibodies in HTLV-1-infected patients with KCS, which suggests that other mechanisms may be involved in the pathogenesis of this manifestation. Additionally, PVL may be a marker of KCS development in these patients.

Presence of Snake-like Chromatin in Epithelial Cells of Keratoconjunctivitis Sicca Followed by a Large Number of Micronuclei

To evaluate the number of micronuclei in snake-like chromatin (SLC) cells in the conjunctival epithelium of keratoconjunctivitis sicca (KCS) patients. To elucidate possible correlations between SLC cell numbers and KCS intensity.Impression cytology specimens from the bulbar conjunctiva of healthy controls and KCS patients were harvested and divided into 3 groups: group 1, controls; group 2, KCS SLC-negative; and group 3, KCS SLC-positive. The number of micronuclei (MNi) in SLC-negative and SLC-positive epithelial cells of each group was counted.The number of MNi in SLC-negative cells of groups 1 and 2 did not exceed 1 MNi/1,000 cells. A significant increase in the frequency of micronuclei in the upper bulbar conjunctiva was noted in SLC-positive (14.75 +/- 8.09 MNi/1,000 cells) as well as SLC-negative cells (4.0 +/- 3.83 MNi/1,000 cells) of group 3.We demonstrate here that the presence of MNi in the conjunctival epithelium of KCS patients could be a characteristic feature accompanying SLC cells. The fact that increased numbers of SLC cells correlates with impaired values in clinical test as well as decreased goblet and epithelial cell densities confirms that the presence of SLC cells correlates with KCS intensity.

Recurrence after Topical Nonpreserved Methylprednisolone Therapy for Keratoconjunctivitis Sicca in Sjögren's Syndrome

The aim of this study was to evaluate the prevalence of long-term recurrence after topical nonpreserved methylprednisolone pulse therapy for the treatment of keratoconjunctivitis sicca (KCS) with Sjögren's syndrome. A total of 106 eyes of 53 Sjögren's syndrome patients were included in the study. All patients were treated with topical nonpreserved 1% methylprednisolone solution. Initial therapy consisted of eyedrops 4 times a day for 2 weeks, and then patients were reevaluated and tapered off the medication every 2 weeks until discontinuation. Tear film breakup time (TBUT), Schirmer test, corneal fluorescein staining, and subjective symptom scores were measured. Additionally, impression cytology of the bulbar conjunctiva was performed. The over-all success rates and success period were determined using the Kaplan-Meier survival curve. A reduction in subjective symptoms and fluorescein staining, and an improvement in TBUT and Schirmer test results, was observed after treatment (P<0.001). Impression cytology specimens revealed a significantly increased number of periodic acid-Schiff-positive cells after treatment. After the first pulse therapy, mean survival was 56.6 weeks and 11 (20.8%) patients recurred. After the second pulse therapy, mean survival was 72.4 weeks and only 1 patient recurred. No serious complications, including intraocular pressure elevation and cataract formation, were encountered during the entire follow-up period. Topical nonpreserved methylprednisolone pulse therapy proved to be a safe, effective long-term treatment of improving subjective and objective dry eye factors in KCS patients with Sjögren's syndrome.

Morphological and immunocytochemical characterization of snake-like chromatin cells.

Snake-like chromatin (SLC) is a nuclear alteration occurring under various pathological conditions and in different tissues. The aim of this study was the morphological and immunocytochemical characterization of SLC-positive conjunctival epithelial cells from keratoconjunctivitis sicca (KCS) patients. Impression cytology specimens from the upper bulbar conjunctiva of 10 controls and 10 KCS patients with a high incidence of SLC cells were assessed, the morphology of SLC nuclei evaluated by light microscopy, and proliferation markers, nucleolar proteins, lamins and cytokeratin filaments detected immunocytochemically. In KCS patients, SLC cells with a normal nuclear shape, with nuclear membrane notching (2.3% of cells) and with binuclear dumb-bell structures (4.4% of cells) were observed. The most striking features of SLC cells were the absence of an A/C lamin signal, the redistribution of fibrillarin into two spots adjacent to SLC structures and cytokeratin 14 positivity in the strangulation belt of the dumb-bell structures. The deficiency of lamin A/C is the probable reason for the disintegration of chromatin from the nuclear lamina in SLC cells. The occurrence of SLC-positive cells, SLC-positive dumb-bell shaped nuclei and SLC-positive binucleated cells, together with the absence of mitotic markers, leads to the conclusion that the SLC phenomenon might be a form of nuclear segregation.

Topical 0.1% Prednisolone Lowers Nerve Growth Factor Expression in Keratoconjunctivitis Sicca Patients

To compare nerve growth factor (NGF) levels in tears and on the ocular surfaces of normal control and non-Sjögren's type keratoconjunctivitis sicca subjects, and to investigate the effect of 0.1% prednisolone eyedrops on NGF levels in keratoconjunctivitis sicca patients. Prospective, double-masked, randomized, comparative clinical trial. Forty-one keratoconjunctivitis sicca patients and 23 age- and gender-matched healthy subjects. Baseline tear NGF levels were measured in keratoconjunctivitis sicca patients and healthy control subjects using enzyme-linked immunosorbent assays. Keratoconjunctivitis sicca patients received 0.1% prednisolone drops in one eye and 0.1% hyaluronic acid drops in the other, 3 times a day for 28 days. Also, impression cytology (IC) and immunostaining for NGF on conjunctival epithelium were performed on both groups. Tear NGF/total tear protein (TP) concentration ratio, IC and NGF immunocytologic staining, subjective symptom scale, tear breakup time, and Schirmer values. Keratoconjunctivitis sicca patients were found to have baseline tear NGF concentrations higher than those of age- and gender-matched healthy control subjects (65.9+/-14.5 vs. 122.1+/-45.3 pg/mug, P<0.0001). In keratoconjunctivitis sicca patients, prednisolone treatment for 28 days resulted in a decrease in tear NGF levels, symptom scores, and IC scores, whereas hyaluronic acid treatment had no such effect (68.2+/-25.0 pg/mug vs. 108.0+/-43.4 pg/mug, P<0.0001 for tear NGF/TP ratio; 2.16+/-1.01 vs. 3.39+/-1.50, P = 0.0014 for symptom scale; 1.05+/-0.67 vs. 1.61+/-0.86, P = 0.0317 for IC). Measurements taken at both 14 and 28 days indicated that neither prednisolone nor hyaluronic acid treatment affected breakup time or Schirmer values. Keratoconjunctivitis sicca patients showed elevated levels of tear NGF, which were decreased by treatment with 0.1% prednisolone. These data suggest that ocular surface NGF may play an important role in ocular surface inflammation processes associated with dry eyes.

Corneal surface changes in keratoconjunctivitis sicca. Part I: The surface proper. A non-contact photomicrographic in vivo study in the human cornea.

To describe corneal surface changes in keratoconjunctivitis sicca (KCS). In all 23 patients with KCS were examined with the slit lamp and photographed by non-contact photomicrography. Without staining, grey surface cells and small epithelial cysts were visible. Application of fluorescein sodium resulted in two types of micropunctuate (15-30 microm) staining, a nonfluorescent (yellow/brown) and a fluorescent (green) one, in brilliantly green staining of cysts, and in enlarging fluorescent (green) flecks (70-1600 microm) with propensity to confluence. The flecks often developed in inconspicuous areas and showed abnormal subsurface cells. Application of rose bengal resulted in more or less intensively red-stained cells measuring about 30 mm in diameter, often less (15-20 microm), with or without a centrally located nucleus, individual or gathered in small groups or larger patches, and in confluent staining. Smaller and larger cells, with or without nuclei, weakly or strongly stained, were often located close to each other. The smaller green flecks showed a few centrally located red-stained cells, and the larger ones showed many almost uniform small red-stained nucleated ones. Corneal epithelial surface disease in the present KCS patients consisted of unspecific features-such as increased light-reflecting property (grey cells), disruptions of intercellular junctions (micropunctuate green fluorescein staining), pathological dye uptake (micropunctuate yellow/brown fluorescein and red rose bengal stainings), and epithelial oedema (microcysts)--and also of features that seemed to be disease specific. Such features were, with fluorescein sodium, focal disruptions of the epithelial barrier function manifesting in enlarging green flecks, and, with rose bengal, abundant nucleated cells, particularly small ones organised in foci. The nature of the abnormal subsurface cells present in the areas of the green flecks, and the mechanisms behind the focal involvement are unclear.

Cytologic evaluation of conjunctival epithelium using Cytobrush-S: value of slide preparation by ThinPrep technique.

Recent clinical trials have indicated that an automated smear apparatus (ThinPrep process) of sample preparation has great diagnostic sensitivity. In this study, conjunctival brush cytology prepared using the ThinPrep method was applied in ocular surface disorders especially for dry eye status. To assess its diagnostic value in cellular samples, 17 patients with keratoconjunctivitis sicca (KCS) and 10 normal volunteer patients were examined using this technique. Conjunctival cells from normal controls revealed fine chromatin and polyhedral cytoplasm without having keratinized cytoplasm. On the other hand, the cellular samples from KCS revealed increased keratinized cells with pyknotic nuclei. They also contained extremely elongated cells. In KCS patients, the mean number of keratinized cells was significantly higher (34.1 cells/300 cells) than that of the normal control group (0.2 cells/300 cells). In patients with KCS, inflammatory cell counts were also higher than those of normal controls. Conjunctival cytology by means of the ThinPrep method obviously deserves additional trials as an adjunct in the cytology of dry eye states, especially in quantitative ocular evaluation for various ocular lesions.

A comparison of the precorneal residence of an artificial tear preparation in patients with keratoconjunctivitis sicca and normal volunteer subjects using gamma scintigraphy

The precorneal residence of an artificial tear preparation has been compared in twelve patients with keratoconjunctivitis sicca (KCS) and twelve normal healthy volunteers. The artificial tear solution was radiolabelled by the inclusion of 1 MBq technetium-99m diethylenetriaminepentaacetic acid (99Tcm-DTPA), and 25 microliters was instilled into one eye only. Deposition of the preparation was followed by gamma scintigraphy. Precorneal clearance of the solution was found to follow bi-exponential kinetics in all subjects with no significant differences in clearance rates between the two study populations. There was also no significant difference in the area under the time-activity profile for the two study groups. This suggests that the reduced reflex tearing and basal lacrimation in KCS patients, indicated by the Schirmer's test, are not important in the clearance of isotonic solutions from the eye surface.

Sjogren??s Syndrome and Keratoconjunctivitis Sicca

Keratoconjunctivitis sicca patients diagnosed on the basis of a history, dry-eye symptoms, and definite clinical signs of keratoconjunctivitis sicca, with the associated symptoms of dry mouth and/or arthritis, had measurements of tear osmolarity, Schirmer tear test without anesthetic, stimulated parotid salivary flow, and serum analysis for the presence of autoantibodies associated with Sjogren's Syndrome. In contrast to previous studies, a lower incidence of SS-A or SS-B (1-3%), ANA (41-47%), DNA (11-16%), and RF (9-12%) serum antibodies was detected. Salivary-stimulated parotid flow was abnormally decreased in 59% of the patients. Sjogren's syndrome, as indicated by the presence of serum antibodies, appears to have a lower incidence in keratoconjunctivitis sicca than considered previously.

The precorneal residence of an artificial tear preparation in keratoconjunctivitis sicca patients and normal volunteers

The precorneal residence of an artificial tear preparation in keratoconjunctivitis sicca patients and normal volunteers

Topical Retinoid Therapy for Squamous Metaplasia of Various Ocular Surface Disorders: A Multicenter, Placebo-controlled Double-masked Study

In a prospective, double-masked clinical study, the authors evaluated the efficacy and safety of topically applied tretinoin ophthalmic ointment (0.01%) versus placebo in the treatment of squamous metaplasia associated with various ocular surface disorders involving dry eyes. Study parameters consisted of graded symptoms and signs, and serial impression cytologies. A total of 161 patients were enrolled in the study. Of these patients, 116 who had a minimum of 4 to 8 months of follow-up qualified for final statistical analysis. These patients were classified into two major groups: (1) keratoconjunctivitis sicca (KCS) and (2) conjunctival cicatricial diseases (Stevens-Johnson syndrome, inactive pemphigoid, radiation-induced dry eye, drug-induced pseudopemphigoid, and toxic epidermal necrolysis). An analysis of adjusted mean changes for KCS patients showed no statistically significant differences between active drug and placebo. A similar analysis of patients with conjunctival cicatricial diseases indicated a statistically significant (P < 0.05) reversal of conjunctival keratinization in the temporal bulbar site after treatment with active drug, however, clinical symptoms and signs showed no significant improvement with active drug relative to placebo. Side effects were limited to blepharoconjunctivitis and were reversible upon tapering or stopping the drug.

Alterations in goblet cells and in epithelial microvilli of the conjunctiva in human keratoconjunctivitis sicca patients: A SEM study

Alterations in goblet cells and in epithelial microvilli of the conjunctiva in human keratoconjunctivitis sicca patients: A SEM study

Tear Diluents in the Treatment of Keratoconjunctivitis Sicca

To determine the optimum solution concentration for lowering elevated tear film osmolarity in keratoconjunctivitis sicca (KCS), tear osmolarity was measured in four KCS patients before and after instillation of either an isotonic saline solution or one of four hypotonic saline solutions (range, 75–225 mOsm/L). Average tear osmolarity one minute after instillation was significantly lower with the hypotonic solutions than with the isotonic saline (mean ± SEM, 290 ± 3 mOsm/L vs. 317 ± 1 mOsm/L, P < 0.0005). Solutions 150 mOsm/L or less were most effective in lowering osmolarity; the 75 mOsm/L solution was occasionally associated with irritation. In 16 KCS patients, we then compared the therapeutic efficacy of the 150 mOsm/L solution with that of an otherwise identical isotonic solution in a two-week, double-masked, crossover study. The 150 mOsm/L solution was superior for symptom relief by nearly 2:1 (P = 0.01).

The relations between tear fluid concentrations of lysozyme, tear-specific prealbumin and lactoferrin

A degeneration of the tear gland leads to reduced lysozyme concentrations in the tear fluid. Tear lysozyme concentrations are generally determined by enzymatic methods. Lysozyme determination by radial immunodiffusion has some advantages above the enzymatic assays, and laboratory facilities are not required. The enzymatic properties of lysozyme that favoured its choice as a parameter for tear gland function, influenced to some extent the results of the radial immunodiffusion assay. Therefore we looked among the proteins originating from the lacrimal gland for alternative parameters of tear gland function. In 94 tear samples obtained from a heterogeneous group, ranging from persons with normal lacrimal gland function to severe kerato-conjunctivitis sicca patients, a high degree of correlation was found between lysozyme concentration and that of lactoferrin as well as tear-specific prealbumin. This indicates that the latter two proteins can be used as alternatives for lysozyme in tear gland function tests.