The Role of Elastase in Corneal Epithelial Barrier Dysfunction Caused by Pseudomonas aeruginosa Exoproteins.

Purpose: To investigate the role of elastase in corneal epithelial barrier dysfunction caused by the exoproteins secreted by Pseudomonas aeruginosa. Methods: Exoproteins obtained from Pseudomonas aeruginosa culture supernatant were analyzed by shotgun proteomics approach. In vitro multilayered rabbit corneal epithelial barrier model prepared by air-liquid interface technique (CECs-ALI) were treated with 2 µg/ml exoproteins and/or 8 mM elastase inhibitor. Then the epithelial barrier function was evaluated by transepithelial electrical resistance (TEER) assay and tight junction proteins immunofluorescence. Cell viability and the apoptosis rate were examined by CCK8 assay and flow cytometry. TNF-α, IL-6, IL-8, and IL-1ß levels were measured by ELISA. Mice cornea treated with exoproteins and/or elastase inhibitor were evaluated in vivo and in vitro. Results: Elastase (24.2%) is one of the major components of exoproteins. After 2 µg/ml exoproteins were applied to CECs-ALI for two hours, TEER decreased from 323.2 ± 2.7 to 104 ± 6.8 Ω/cm2 (P < 0.001). The immunofluorescence results showed a distinct separation in tight junction and significant degradation of ZO-1 and occludin (P < 0.05). Elastase inhibitor (8 mM) alleviated the decrease in TEER value (234 ± 6.8 Ω cm2) induced by exoproteins. Inhibition of elastase decreased the apoptosis rate of CECs treated with exoproteins from 30.2 ± 3.8% to 7.26 ± 1.3% and the levels of inflammatory factors (P < 0.05). Mice corneal epithelium defect could be induced by exoproteins and protected by elastase inhibitor. Conclusions: Elastase plays a critical role in corneal epithelial barrier dysfunction caused by Pseudomonas aeruginosa exoproteins via damaging tight junctions. The inhibition of elastase could protect the corneal epithelial barrier via reducing virulence and inflammation.

Mechanism of Pseudomonas aeruginosa Small Protease (PASP), a Corneal Virulence Factor.

Purpose: Pseudomonas aeruginosa is the leading cause of contact lens-associated bacterial keratitis. Secreted bacterial proteases have a key role in keratitis, including the P. aeruginosa small protease (PASP), a proven corneal virulence factor. We investigated the mechanism of PASP and its importance to corneal toxicity. Methods: PASP, a serine protease, was tested for activity on various substrates. The catalytic triad of PASP was sought by bioinformatic analysis and site-directed mutagenesis. All mutant constructs were expressed in a P. aeruginosa PASP-deficient strain; the resulting proteins were purified using ion-exchange, gel filtration, or affinity chromatography; and the proteolytic activity was assessed by gelatin zymography and a fluorometric assay. The purified PASP proteins with single amino acid changes were injected into rabbit corneas to determine their pathological effects. Results: PASP substrates were cleaved at arginine or lysine residues. Alanine substitution of PASP residues Asp-29, His-34, or Ser-47 eliminated protease activity, whereas PASP with substitution for Ser-59 (control) retained activity. Computer modeling and Western blot analysis indicated that formation of a catalytic triad required dimer formation, and zymography demonstrated the protease activity of the homodimer, but not the monomer. PASP with the Ser-47 mutation, but not with the control mutation, lacked corneal toxicity, indicating the importance of protease activity. Conclusions: PASP is a secreted serine protease that can cleave proteins at arginine or lysine residues and PASP activity requires dimer or larger aggregates to create a functional active site. Most importantly, proteolytic PASP molecules demonstrated highly significant toxicity for the rabbit cornea.

Cutting edge: IL-1ß processing during Pseudomonas aeruginosa infection is mediated by neutrophil serine proteases and is independent of NLRC4 and caspase-1.

To examine the role of caspase-1 and the NLRC4 inflammasome during bacterial infection, C57BL/6, IL-1ß(-/-), caspase-1(-/-), and NLRC4(-/-) mouse corneas were infected with ExoS/T- or ExoU-expressing Pseudomonas aeruginosa. We found that IL-1ß was essential for neutrophil recruitment and bacterial clearance and was produced by myeloid cells rather than resident cells. In addition, neutrophils were found to be the primary source of mature IL-1ß during infection, and there was no significant difference in IL-1ß processing between C57BL/6 and caspase-1(-/-) or NLRC4(-/-) infected corneas. IL-1ß cleavage by human and mouse neutrophils was blocked by serine protease inhibitors and was impaired in infected neutrophil elastase (NE)(-/-) corneas. NE(-/-) mice also had an impaired ability to clear the infection. Together, these results demonstrate that during P. aeruginosa infection, neutrophils are the primary source of mature IL-1ß and that IL-1ß processing is dependent on serine proteases and not NLRC4 or caspase-1.

Testican-1 promotes resistance against Pseudomonas aeruginosa-induced keratitis through regulation of MMP-2 expression and activation.

PURPOSE: Testican-1 (or SPOCK) is a highly conserved chimeric proteoglycan encoded by the SPOCK1 gene. Protease regulatory activity has recently been demonstrated by this molecule and its family members testican-2 and -3. The present study tested the hypothesis that testican-1 regulates corneal matrix metalloproteinase (MMP)-2 expression, thus improving disease outcome after Pseudomonas aeruginosa-induced keratitis. METHODS: C57BL/6 (B6) and BALB/c mice were routinely infected with P. aeruginosa and were evaluated at various postinfection (pi) times for corneal expression of testican-1 and MMP-2, by PCR array, real-time RT-PCR, ELISA, activity assays, zymography, and immunohistochemistry. Next, B6 mice were treated with recombinant human (rh) testican-1, and expression was knocked down in BALB/c ice by siTestican-1 treatment, to determine the relationship between the two molecules. RESULTS: BALB/c versus B6 mice expressed significantly higher mRNA and protein levels of testican-1 after P. aeruginosa-induced ocular infection. MMP-2 expression and activation was also disparate between the two mouse strains. After rhTestican-1 treatment in B6 mice, overall disease response was significantly improved, whereas siRNA treatment of BALB/c mice converted the normally resistant response to susceptible. Testican-1 was shown to influence MMP-2 expression, activation, and regulation, as well. CONCLUSIONS: This study demonstrates corneal expression of testican-1 and its temporal regulation of MMP-2 expression and activation after induction of bacterial keratitis. Furthermore, the data collectively indicate that testican-1 is a novel target for disease treatment to promote better disease outcome regarding chronic inflammation and infection and diseases involving pathologic tissue destruction.

Properties of PASP: a Pseudomonas protease capable of mediating corneal erosions.

PURPOSE: To analyze PASP in terms of its gene distribution and expression, its corneal pathologic effects, its enzymatic properties, and the protectiveness of the immune response to this protease. METHODS: Twenty-five strains of P. aeruginosa were analyzed for the PASP gene and secreted protein by PCR and Western blot analysis, respectively. Active recombinant (r)PASP (10 microg/20 microL) or heat-inactivated rPASP was intrastromally injected into rabbit corneas. Pathologic changes were monitored by slit lamp examination (SLE) and histopathology. Purified rPASP was assayed for cleavage of collagens and susceptibility to TLCK. Rabbit antibody to rPASP was produced and tested for enzyme inactivation, and actively immunized rabbits were challenged by intrastromal injection of active rPASP (5 microg). RESULTS: All 25 strains of P. aeruginosa analyzed were positive for the PASP gene and protein. SLE scores of eyes injected with active rPASP were significantly higher than control eyes at all postinjection times (PI; P or= 10,000) was produced, but this antibody did not protect against active rPASP challenge. CONCLUSIONS: PASP is a commonly produced Pseudomonas protease that can cleave collagens and cause corneal erosions.

Matrix metalloproteinase-9 amplifies the immune response to Pseudomonas aeruginosa corneal infection.

PURPOSE: The purpose of this study was to determine the role of matrix metalloproteinases (MMP) in Pseudomonas aeruginosa keratitis. METHODS: Gene array and selective real-time PCR examined MMP expression in the cornea of susceptible (C57BL/6, B6) versus resistant (BALB/c) mice before and after infection; zymography tested enzyme activity for MMP-2 and -9. Clinical score, Langerhans cell (LC), and Neutrophil (PMN) quantitation were done in recombinant (r) MMP-9, antibody neutralized, and MMP-9(-/-) mice. The chemotactic potential of MMP-9 was tested in a Boyden chamber assay; light and transmission microscopy and immunostaining for collagen IV and MMP-9 were used to examine the effects and the source of MMP-9 after infection. ELISA was used to assess IL-1beta and MIP-2 levels. RESULTS: Gene array (confirmed by PCR) revealed sixfold more MMP-9, and zymography showed greater enzyme activity in the infected cornea of B6 over BALB/c mice. rMMP-9 injection of BALB/c mice enhanced, whereas MMP-9 antibody neutralization in B6 mice and its absence in MMP-9(-/-) mice decreased corneal disease. MMP-9(-/-) and antibody neutralized mice had fewer LCs in cornea; rMMP-9-treated mice had more. A myeloperoxidase (MPO) assay showed a similar pattern for PMN. MMP-9 was not chemotactic for LC or PMN. The basement membrane was more intact in MMP-9(-/-) over wild-type infected mice and correlated with staining for collagen IV; PMN was a source of MMP-9. IL-1beta and MIP-2 were increased in rMMP-9 but decreased in MMP-9 antibody neutralized and MMP-9(-/-) over control groups. CONCLUSIONS: MMP-9 regulates immune function in cornea by proteolysis, potentiating P. aeruginosa keratitis by degrading collagen IV and upregulating chemotactic cytokines/chemokines IL-1beta and MIP-2.

Pathogenesis of Staphylococcus in the rabbit anterior chamber.

PURPOSE: To investigate the host defense against Staphylococcus in the rabbit anterior chamber. METHODS: The bactericidal activity of rabbit aqueous humor was investigated in vitro. Rabbit anterior chambers were injected with viable Staphylococcus aureus or Staphylococcus epidermidis (1,000 or 500,000 colony-forming units [CFU]), killed bacteria, culture supernatants of either organism, or purified S. aureus alpha-toxin. CFU as well as phospholipase (PLA(2)) and myeloperoxidase (MPO) activities of aqueous humor were determined up to 25 hours postinfection (PI). RESULTS: The number of viable S. aureus or S. epidermidis was significantly reduced when incubated with aqueous humor for 30 minutes (P

Regulation of Pseudomonas aeruginosa corneal infection in IL-1 beta converting enzyme (ICE, caspase-1) deficient mice.

PURPOSE: Antibody neutralization studies have shown that in Pseudomonas aeruginosa corneal infection, IL-1 beta is critical to regulation of the host inflammatory response, but mechanisms remain undetermined. To elucidate these mechanisms, caspase-1 knockout (ICE(-/-)) mice, that do not release mature IL-1 beta after endotoxin challenge, were tested. METHODS: Clinical scores, MPO activity (for PMN quantitation), bacterial plate count, semiquantitative RT-PCR, ELISA and TUNEL staining were used to characterize the inflammatory response after infection in knockout and C57BL/6 (B6) wild type mice. RESULTS: Clinical scores were significantly reduced in ICE(-/-) vs. B6 mice at 3, 5 and 7 days postinfection (p.i.). The decreased inflammatory response of ICE(-/-) mice was striking at 1 day p.i., and bacterial load also was significantly reduced in the cornea of the knockout mice at 3-7 days p.i. Knockout mice exhibited significantly increased mRNA and protein levels for IL-1Ra, the physiological regulator of IL-1 activity, and in addition, a significant increase in the number of apoptotic cells were quantitated in the corneal epithelium of ICE(-/-) vs. B6 mice at 1 day p.i. CONCLUSIONS: These data provide evidence that bacterial infection in the cornea of ICE(-/-) mice induces a reduced inflammatory response by: reduction in PMN and cytokines and chemokines that attract these cells to the cornea; enhanced apoptotic cell death in the infected epithelium; and increased IL-1Ra levels. The data also confirm the importance of IL-1 regulation in this model and suggest that ICE inhibition may be an attractive ancillary therapeutic strategy to control the host response to this pathogen.

The galU Gene of Pseudomonas aeruginosa is required for corneal infection and efficient systemic spread following pneumonia but not for infection confined to the lung.

Acute pneumonias and corneal infections due to Pseudomonas aeruginosa are typically caused by lipopolysaccharide (LPS)-smooth strains. In cystic fibrosis patients, however, LPS-rough strains of P. aeruginosa, which lack O antigen, can survive in the lung and cause chronic infection. It is not clear whether an LPS-rough phenotype affects cytotoxicity related to the type III secretion system (TTSS). We previously reported that interruption of the galU gene in P. aeruginosa results in production of a rough LPS and truncated LPS core. Here we evaluated the role of the galU gene in the pathogenesis of murine lung and eye infections and in cytotoxicity due to the TTSS effector ExoU. We studied galU mutants of strain PAO1, of its cytotoxic variant expressing ExoU from a plasmid, and of the inherently cytotoxic strain PA103. The galU mutants were more serum sensitive than the parental strains but remained cytotoxic in vitro. In a corneal infection model, the galU mutants were significantly attenuated. In an acute pneumonia model, the 50% lethal doses of the galU mutants were higher than those of the corresponding wild-type strains, yet these mutants could cause mortality and severe pneumonia, as judged by histology, even with minimal systemic spread. These findings suggest that the galU gene is required for corneal infection and for efficient systemic spread following lung infection but is not required for infection confined to the lung. Host defenses in the lung appear to be insufficient to control infection with LPS-rough P. aeruginosa when local bacterial levels are high.

Murine ocular heparanase expression before and during infection with Pseudomonas aeruginosa.

PURPOSE: To demonstrate the constitutive expression and regulation of heparanase (heparan sulfate endoglycosidase) in the normal mouse eye and in mice intracorneally infected with Pseudomonas aeruginosa. METHODS: Naïve (unimmunized) and immunized C57BL/6J mice were infected with P. aeruginosa, and corneal heparanase gene and protein expression were detected by semiquantitative RT-PCR and immunoblot analysis. Immunohistochemistry was also applied to characterize corneal heparanase in naïve mice. RESULTS: Heparanase mRNA and protein expression were detected in uninfected corneas of C57BL/6J mice. Immunohistochemical studies indicated heparanase protein expression was primarily in the corneal epithelium before corneal infection and was also in the corneal stroma after infection. Immunohistochemical studies of uninfected and infected whole eyes of naïve mice indicated heparanase protein expression in most layers of the retina, but the expression did not appear to be upregulated during corneal infection. Staining was most intense in the inner photoreceptor layer of the retina. CONCLUSIONS: Heparanase was constitutively expressed in both the corneal epithelium and several retinal layers before intracorneal infection with P. aeruginosa. Temporal upregulation of corneal heparanase protein expression was detected in naïve mice during infection, most likely due to heparanase positive infiltrating cells, but the protein was not upregulated in corneas from immunized mice because they had a lower inflammatory response, associated with the restoration of corneal clarity. There did not appear to be temporal upregulation of heparanase expression in the retina of infected mice, as determined by immunohistochemistry.

Expression of protein gene product 9.5 in lepromatous eyes showing ciliary body nerve damage and a "dying back" phenomenon in the posterior ciliary nerves.

BACKGROUND/AIM: Peripheral nerve destruction is the hallmark of leprosy. Ocular complications form a substantial part of the clinical manifestations but histopathology of nerve destruction within ocular structures has not been shown satisfactorily. The role of protein gene product (PGP) 9.5 in identifying nerve destruction in the ciliary body and posterior ciliary nerves of lepromatous eyes is shown. METHODS: Serial sections from two lepromatous eyes and two non-lepromatous eyes were stained with PGP 9.5. Histopathological comparison was done on the expression of the PGP 9.5 stain in nerves within the ciliary body, posterior ciliary nerves adjacent to the optic nerve, and nerves tracking through the sclera. RESULTS: In non-lepromatous eyes, PGP 9.5 was expressed in nerves within the ciliary body, the nerves within the sclera, and posterior ciliary nerves adjacent to the optic nerve. In lepromatous eyes no PGP 9.5 was expressed, signifying nerve destruction. CONCLUSIONS: Nerve destruction in lepromatous eyes has been confirmed histopathologically by the absence of or patchy staining with PGP 9.5. Nerve destruction in the ciliary body can extend to the posterior ciliary nerves by an ascending axonopathy. This "dying back" phenomenon is akin to the "glove and stocking" anaesthesia found in lepromatous leprosy.

Regulation of MMPs and TIMPs by IL-1beta during corneal ulceration and infection.

PURPOSE: This study was conducted to investigate the role of IL-1beta in the regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in a mouse model of experimental keratitis and corneal injury. METHODS: Mice were injected subconjunctivally with 10 micro g of anti-mouse IL-1beta antibody 2 hours before challenge with Pseudomonas aeruginosa (strain 6294). Control animals received an equal volume and concentration of isotype control antibody at the same time. Eyes were enucleated at 0, 8, 24, and 72 hours, after bacterial challenge and processed for histologic examination. Some eyes were homogenized and used to evaluate production of MMP-2, MMP-9, TIMP-1, and TIMP-2 protein, by zymography and reverse zymography. RESULTS: Injury without bacterial infection resulted in increases in both MMP-2 and -9 and a slight but significant downregulation of TIMP-1. Administration of anti-IL-1beta just before injury and without bacterial infection resulted in a significant reduction in expression of MMP-2 (at 8 hours), MMP-9 (at 8 hours), TIMP-1 (at 8 and 72 hours), and TIMP-2 (at 8 hours). Mice treated with anti-IL-1beta antibody, before bacterial challenge, demonstrated markedly reduced corneal damage compared with the severe corneal injury and massive neutrophil infiltration observed in infected mice treated with control antibody. Administration of the neutralizing anti-IL-1beta antibody resulted in a significant reduction of MMP-9 and a change in the time course of TIMP-1 and -2 expression. The reduction in MMP-9 by anti-IL-1beta during infection was much greater than the reduction without infection. CONCLUSIONS: The results imply that IL-1beta has a central role in corneal destruction during bacterial keratitis and suggests that targeting IL-1beta may be a novel therapeutic strategy for microbial keratitis.

Phospholipase A2 activity in normal and Staphylococcus aureus-infected rabbit eyes.

PURPOSE: To quantify phospholipase A(2) (PLA(2)) activity in normal rabbit eyes and in eyes with Staphylococcus aureus keratitis. METHODS: PLA(2) was assayed by the killing of S. aureus at 33 degrees C or by the release of arachidonic acid from S. aureus labeled with radioactive oleic acid. Rabbit corneas were intrastromally injected with 100 log phase colony-forming units (CFU) of S. aureus 8325-4. The activity of myeloperoxidase (MPO) and PLA(2) were quantified in ocular tissues. RESULTS: The PLA(2)-mediated killing of S. aureus by normal rabbit tears decreased by more than 70% as the rabbits aged from 10 to 28 weeks and by nearly 50% from early morning to afternoon. In rabbits with S. aureus keratitis, the activity of PLA(2) and MPO increased proportionally with time from 5 to 25 hours postinfection (PI), as measured in ocular tissues. PLA(2) activity increased fivefold in tears from infected eyes collected at 25 hours PI compared with normal tears (P < or = 0.0001), whereas a ninefold increase was found in aqueous humor of infected eyes at 25 hours PI (P < or = 0.0001). Infected eyes demonstrated a significant increase in MPO activity compared with uninfected eyes beginning at 10 hours PI for the aqueous humor (P = 0.03), at 16 hours PI for the tear film (P = 0.0024) and at 22 hours PI for the corneal homogenate (P = 0.0007). CONCLUSIONS: The decrease in PLA(2) activity in the rabbit eye with age or after sleep and its increase during sleep or with the progression of infection are consistent with its role as an innate host defense factor.

Membrane-type matrix metalloproteinases in mice intracorneally infected with Pseudomonas aeruginosa.

PURPOSE: To establish the presence of membrane-type matrix metalloproteinases (MT-MMPs) in the cornea and their expression in naive and immunized mice intracorneally infected with Pseudomonas aeruginosa. METHODS: Naive (unimmunized) and immunized C57BL/6J mice were infected with P. aeruginosa, and gene expression of MT-MMPs were detected by RT-PCR. Immunoblot analysis and immunostaining were also used to characterize the MT-MMP response in both sets of animals. RESULTS: Expression of MT1-MMP, MT2-MMP, and MT3-MMP (MMP 14, 15, and 16) was detected by RT-PCR and immunoblot analysis. Of the three MT-MMPs detected, MT1-MMP exhibited the greatest expression at protein levels. In general, a bell-shaped curve was obtained for each of the MT-MMPs in naive mice, but all of them showed much less expression in the immunized mice. MT1-MMP was localized in the epithelial tissue of the cornea, whereas MT2-MMP and MT3-MMP were mainly found in the interface between the epithelium and substantia propria. CONCLUSIONS: MT1-MMP was detected and expressed to a greater extent in naive mice than MT2-MMP and MT3-MMP. Peak expression of all three MT-MMPs showed a good correlation with the overall inflammatory response.

Acute inflammation of the eyelid and cornea in Staphylococcus keratitis in the rabbit.

PURPOSE: The inflammatory response during Staphylococcus keratitis was analyzed biochemically and histologically to determine the source of the neutrophils infiltrating the tear film and cornea. METHODS: Rabbit eyes were swabbed and then examined by slit-lamp microscopy at 0, 5, 10, 15, 20, and 25 hours after intracorneal inoculation with Staphylococcus aureus. Bacterial colony-forming units were quantified in the cornea, eyelid, and acute inflammatory exudate. Myeloperoxidase activity of ocular swabs of acute inflammatory exudate, corneal homogenates, and eyelid homogenates was determined. Gross and microscopic examinations of corneas and eyelids were performed. RESULTS: The colony-forming units per cornea exceeded 10(7) after 10 hours, whereas no bacteria were cultured from the eyelid until 15 hours postinfection. Slit-lamp examination revealed progressive pathology, and the myeloperoxidase activities of ocular swabs, corneas, and eyelids increased markedly by 15 hours postinfection. Corneas showed a wave of neutrophils moving from the tear film toward bacteria in the central corneal stroma and early neutrophil migration from the limbus into the stroma. In the eyelid, neutrophils migrated from the stromal vessels to the tear film. CONCLUSIONS: Staphylococcus keratitis in the rabbit causes acute inflammation in the overlying eyelid. Neutrophils of the acute inflammatory exudate interact with the infected cornea, whereas neutrophils migrating through the cornea from the limbus remained distant from the site of infection.

Effect of nitric oxide synthase inhibition in experimental Pseudomonas keratitis in rabbits.

PURPOSE: The aim of this study was to assess the efficacy of the nitric oxide synthase inhibitor L-NG-nitro-arginine-methyleser (L-NAME) in the treatment of experimental Pseudomonas keratitis. METHODS: Twelve young New Zealand white rabbits were given intrastromal injections of Pseudomonas aeruginosa strains. Twenty-four hours later, the rabbits were randomly divided into four groups: group 1 was treated with topical 0.3% ciprofloxacin drops and a single subconjunctival injection of L-NAME (150 mg/kg); group 2 received topical 0.3% ciprofloxacin drops alone; group 3 received a single subconjunctival injection of L-NAME alone; group 4, the control group, was treated with topical balanced salt solution (BSS) drops. One drop of either ciprofloxacin of BSS was applied at each treatment interval. Twenty-six hours after the start of treatment, the eyes were examined by slit lamp to assess inflammation. Aqueous humor specimens were collected from each eye to measure the nitric oxide concentration. Corneas were removed to count bacteria results. RESULTS: Slit lamp examination cell scores were significantly lower for groups 1 and 3 than groups 2 and 4 eyes (p = 0.002 to p = 0.004). No viable bacteria were detected in the corneas of groups 1 and 2. Group 3 corneas had significantly fewer bacteria (6.33 +/- 0.42 x 10(3)) than group 4 (5.94 +/- 0.16 x 10(4)) (p < 0.05). Nitric oxide levels in the aqueous humor were significantly lower for group 1 eyes than for groups 2, 3, or 4 (p = 0.02, p = 0.01, and p = 0.003, respectively). CONCLUSIONS: We conclude that nitric oxide synthase inhibitors may be a useful adjunct but are not a replacement for traditional antibiotic drop therapy.

Effect of vitamin A deficiency on the early response to experimental Pseudomonas keratitis.

PURPOSE: Vitamin A-deficient humans and animals are more susceptible to infections than are healthy humans and animals. This study compares the early corneal response (within 24 hours) to an experimental Pseudomonas aeruginosa infection between vitamin A deficient and control rats. METHODS: Male WAG/Rij/MCW rats were fed either a vitamin A- deficient diet (A-) or the same diet with retinyl palmitate added back in a nonrestricted manner (N) or under pair-fed conditions (A+) to yield weight-matched rats. Some A-rats were repleted wih retinyl palmitate 16 days before being killed and then given free access to the retinyl palmitate-supplemented diet (R). Twenty-four hours before being killed, the corneas of anesthetized rats were scratched and P. aeruginosa organisms were applied to the corneal surface. The rats were killed using an overdose of sodium pentobarbital. Corneas were either processed for light and electron microscopic examination or extracted for proteinase and myeloperoxidase determination. Corneal myeloperoxidase concentrations relative to neutrophil myeloperoxidase concentrations were used to determine the number of neutrophils in the cornea. Zymography was used to study caseinases, gelatinases, and plasminogen activators. Reverse zymography was used to detect proteinase inhibitors. Similar results were noted at early, mid, and late weight plateau stages of vitamin A deficiency. RESULTS: Ulceration occurred within 24 hours when low numbers of P. aeruginosa (10(4) cpu) were applied topically onto scratched A- corneas, whereas no ulceration was observed in the A+, R, and N corneas. When higher numbers of P. aeruginosa (10(7)-10(8)) were applied to the scratched corneas, all corneas became ulcerated within 24 hours. The extent of ulceration in the control corneas was greater than that in A- corneas by a factor of two. Only the A- corneas contained inflammatory cells with unusual striated deposits in phagolysosomes. The total number of neutrophils in the cornea and the concentrations of caseinases, plasminogen activators, and gelatinases in the infected corneal extracts were similar; however, the concentrations of cysteine proteinase inhibitors were elevated under A- conditions. CONCLUSIONS: Vitamin A deficiency alters the response of the cornea to a P. aeruginosa infection during the first 24 hours. The alterations observed are probably due to multiple factors: an insufficient tear film for bacterial clearance and migration of neutrophils, epithelial keratinization, alterations in corneal wound healing, and changes in polymorphonuclear function.

In vivo bacterial protease production during Pseudomonas aeruginosa corneal infection.

PURPOSE: To establish if active pseudomonal proteases are present in vivo during corneal infection with Pseudomonas aeruginosa and to determine if the mouse strains used in these and previous studies have the ability to mount a nonocular antibody response to the purified proteases because antibodies to the bacterial proteases were not detected previously during in vivo ocular infection. METHODS: At certain times after corneal infection with P. aeruginosa, corneas were harvested and supernatants from the corneal homogenates were analyzed for proteolytic activity by zymography and immunoreactivity by immunoblotting. The efficiency of the extraction procedures used in these studies was determined by incubating uninfected corneal homogenates with the purified proteases. The resultant supernatants were analyzed for alkaline protease and elastase activity. Additionally, mice were immunized intraperitoneally with the purified proteases with and without adjuvant to determine if the animals could mount a nonocular antibody response. RESULTS: Corneas infected with P. aeruginosa demonstrated the presence of alkaline protease, but not elastase, by the two methods examined. The kinetics of the in vivo alkaline protease response closely parallels previously reported bacterial clearance studies in that peak alkaline protease activity was detected in corneal tissue when peak bacterial numbers also were observed in the eye, and it was absent when the eyes were sterile or nearly sterile. In addition, C57BL/6J mice were capable of mounting a nonocular antibody response to microgram quantities of both proteases only in the presence of adjuvant. CONCLUSIONS: In the model described, enzymatically active alkaline protease, but not elastase, was demonstrated in corneal tissues during in vivo infection. Concentrations of these proteases were much lower than those required to stimulate an antibody response.

Tear lysozyme levels in bacterial, fungal and viral corneal ulcers.

Low levels of tear lysozyme were observed in patients with infective corneal ulcers, when compared with controls. Lowest levels were seen in patients with bacterial corneal ulcers. The levels of tear lysozyme showed a corresponding decrease with the increase in Schirmer test values; meaning thereby, that in ocular conditions associated with increased rate of tear flow, the lysozyme content in tears tends to be low.