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.

ACE2 Expression Is Upregulated in Inflammatory Corneal Epithelial Cells and Attenuated by Resveratrol.

Purpose: The ocular surface is considered an important route for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. The expression level of the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is vital for viral infection. However, the regulation of ACE2 expression on the ocular surface is still unknown. We aimed to determine the change in ACE2 expression in inflamed corneal epithelium and explore potential drugs to reduce the expression of ACE2 on the ocular surface. Methods: The expression of the SARS-CoV-2 receptors ACE2 and TMPRSS2 in human corneal epithelial cells (HCECs) was examined by qPCR and Western blotting. The altered expression of ACE2 in inflammatory corneal epithelium was evaluated in TNFα- and IL-1ß-stimulated HCECs and inflamed mouse corneal epithelium, and the effect of resveratrol on ACE2 expression in HCECs was detected by immunofluorescence and Western blot analysis. Results: ACE2 and TMPRSS2 are expressed on the human corneal epithelial cells. ACE2 expression is upregulated in HCECs by stimulation with TNFα and IL-1ß and inflamed mouse corneas, including dry eye and alkali-burned corneas. In addition, resveratrol attenuates the increased expression of ACE2 induced by TNFα in HCECs. Conclusions: This study demonstrates that ACE2 is highly expressed in HCECs and can be upregulated by stimulation with inflammatory cytokines and inflamed mouse corneal epithelium. Resveratrol may be able to reduce the increased expression of ACE2 on the inflammatory ocular surface. Our work suggests that patients with an inflammatory ocular surface may display higher ACE2 expression, which increases the risk of SARS-CoV-2 infection.

Antimicrobial Effects of Thymosin Beta-4 and Ciprofloxacin Adjunctive Therapy in Pseudomonas aeruginosa Induced Keratitis.

Prior work has indicated that thymosin beta 4 (Tß4) administered with ciprofloxacin markedly improves disease outcome for Pseudomonas aeruginosa (PA)-induced keratitis. As a result, the goal of the current study was to elucidate mechanisms by which Tß4 mitigates the corneal response; specifically, regarding its bactericidal influence and potential synergy with ciprofloxacin. An in vitro approach was carried out using minimum inhibitory concentration (MIC) assays to assess bactericidal activity against PA. In addition, antimicrobial peptide (AMP) production was evaluated at the mRNA levels using human corneal epithelial cells in response to lipopolysaccharide (LPS) challenge. The results of the MIC assays did not show direct bactericidal activity with Tß4 alone, although ciprofloxacin exhibited significant killing at concentrations far lower than clinically dosed. Tß4, however, displayed an indirect effect on bacterial killing, as shown by an upregulation of AMPs and related molecules. The cumulative data from this study indicate an indirect bactericidal role of Tß4, as well as a synergistic relationship with ciprofloxacin. Furthermore, ciprofloxacin alone was found to influence cellular functions that otherwise have yet to be reported. These results highlight a mechanism of intracellular communication for Tß4 and further strengthen its development as an adjunct therapy with antibiotics for corneal infections.

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.

Intravital imaging of the cellular dynamics of LysM-positive cells in a murine corneal suture model.

BACKGROUND: Corneal suturing is a surgical procedure used in patients with corneal trauma or transplants. It was reported that endogenous neutrophils are brightly labelled in gene-targeted mice expressing enhanced green fluorescent protein (eGFP) under the control of the endogenous lysozyme M promoter (LysM-eGFP mice). METHODS: We applied intravital imaging methods to analyse in vivo the dynamics of LysM-positive granulocytes (neutrophils) in LysM-eGFP mice with corneal sutures and examined their role in the elicitation of neutrophil infiltration. RESULTS: We found that in the presuturing state, neutrophils strongly positive for LysM were located in the periphery of the corneal stromal layer; none were present in the centre of the cornea. After introducing a corneal suture, neutrophils accumulated in limbal vessels and then migrated to the corneal side and the conjunctival side, suggesting that they derived from limbal vessels. Thereafter they accumulated towards the central corneal area, arriving at the suture about 7 h after its placement. Although corneal sutures may elicit the continuous infiltration of neutrophils, their number was markedly decreased by day 1 after suture removal and continued to decrease thereafter. CONCLUSIONS: Our results showed that corneal sutures may elicit the continuous infiltration of neutrophils.

Regulatory effect of caspase-11 on interleukin-1ß in the fungal keratitis.

Caused by fungus, fungal keratitis is a kind of infections corneal disease with high rate of blindness, which patients are mainly farmers in developing countries. Interleukin, as important proinflammatory cytokines, involve in immune defense process against fungal infection of cornea. The expression of interleukin in the pathogenesis of fungal keratitis, especially the main source of its cells, is not clear and the cell signaling pathways which regulate the synthesis and modification of interleukin is still unknown. Caspase-11 was obtained and cultured. And the ELISA and Western-blot methods were used to explore the regulatory effect of Caspse-11 on Interleukin-1ß in the fungal keratitis. neutrophils were the main cell lineage of IL-1ß to take part in the innate anti-fungi immunity in the cornea; IL-1ß generation induced by fungal infection might not be through the pre-excitation in the classical signal pathway; TLR4/TRIF pathway was not involved in pro-IL-1ß generation; while Dectin-1/syk pathway was involved in IL-1ß generation in the fungal keratitis; Caspase-l participated in the modification of IL-1ß to change from the precursor into the mature body; but NLRP3 inflammasome and ASC inflammasome were not involved in IL-1ß generation; Caspase-11 was involved in IL-1ß generation through regulating the modified process of Caspase-l to turning from precursor into mature body. TLR4/TRIF pathway and NLRP3 inflammasome and ASC inflammasome are not involved in the pro-IL-1ß generation, while Caspase-l, Caspase-11 and Dectin-1/syk pathway are involved in the IL-1ß generation.

Chitinase 3-Like 1 Promotes Candida albicans Killing and Preserves Corneal Structure and Function by Controlling Host Antifungal Responses.

Chitinase 3-like 1 (CHI3L1) has been shown to play a role in promoting antibacterial responses, decreasing tissue injury, and enhancing pulmonary repair. This study sought to elucidate the role of CHI3L1 in augmenting the corneal innate immune response to Candida albicans infection in an animal model of fungal keratitis. Flagellin applied topically 24 h prior to C. albicans inoculation significantly protected the corneal from C. albicans and induced CHI3L1 expression in C57BL/6 mouse corneas. CHI3L1, however, played a detectable but minor role in flagellin-induced protection. While C. albicans keratitis was more severe in the corneas treated with Chi3l1 small interfering RNA (siRNA), corneas treated with recombinant CHI3L1 before C. albicans inoculation had markedly ameliorated keratitis, reduced fungal load, and decreased polymorphonucleocyte (PMN) infiltration in an interleukin 13 receptor α2 (IL-13Rα2)-dependent manner. CHI3L1 treatment resulted in the induction of the antimicrobial peptides ß-defensin 3, CRAMP, and chemokine CXCL10 and its receptor CXCR3 in corneal epithelial cells. Importantly, CHI3L1 administered after C. albicans inoculation also had strong protection against fungal keratitis, suggesting a therapeutic window. This is the first report demonstrating that CHI3L1 is induced during fungal infection, where it acts as an immunomodulator to promote fungal clearance and to regulate antifungal innate immune responses in the cornea.

Assessment of Streptococcus pneumoniae capsule in conjunctivitis and keratitis in vivo neuraminidase activity increases in nonencapsulated pneumococci following conjunctival infection.

PURPOSE: The pneumococcal capsule is required for pathogenesis in systemic infections, yet reports show most conjunctivitis outbreaks are caused by nonencapsulated pneumococci, while keratitis infections are caused by encapsulated strains. This study aims to determine the effect of capsule in pneumococcal keratitis and conjunctivitis in rabbit models of infection. METHODS: A capsule-deficient isogenic mutant was created using homologous transformation. Parent and mutant strains were injected within the upper bulbar conjunctiva (conjunctivitis) or into the corneal stroma (keratitis) of New Zealand white rabbits. Clinical examinations were performed 24 and 48 hr post-infection at which time corneas or conjunctivae were removed, homogenized, and plated to determine the recovered bacterial load. Whole eyes were removed for histological examination. The neuraminidase activity was determined following in vitro and in vivo growth. RESULTS: There were no significant differences in clinical scores between the eyes infected with the parent or mutant for either infection, nor was there a difference in the amount of bacteria recovered from the cornea. In the conjunctivae, however, the mutant strain was cleared by the host faster than the parent strain. Histological examination showed slightly more infiltrating polymorphonuclear leukocytes (PMN) and macrophages in the conjunctivae infected with the parent strain. The neuraminidase activity of both strains was not significantly different when the strains were grown in vitro. However, the neuraminidase activity of the parent was significantly less than that of the mutant at 3 and 12 hr post conjunctival infection. CONCLUSIONS: Although more outbreaks of pneumococcal conjunctivitis are tied to nonencapsulated S. pneumoniae strains, this study showed that an encapsulated strain was capable of establishing conjunctivitis in a rabbit injection model and survive attack by the host immune system longer than its nonencapsulated isogenic mutant. Nonetheless, the nonencapsulated pneumococci had an increased neuraminidase activity level in vivo when compared to the parent strain.

Specific NFkappaB subunit activation and kinetics of cytokine induction in adenoviral keratitis.

PURPOSE: Corneal inflammation associated with ocular adenoviral infection is caused by leukocytic infiltration of the subepithelial stroma in response to expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by infected corneal cells. We have shown that these two chemokines are activated by the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38 for IL-8, and Jun-terminal kinase (JNK) for MCP-1. It is also well established that transcription of each of these chemokines is tightly controlled by the nuclear factor kappa B (NFkappaB) transcription factor family. Therefore, we sought to better understand the differential regulation of chemokine expression by NFkappaB in adenoviral infection of the cornea. METHODS: Primary keratocytes derived from human donor corneas were treated with signaling inhibitors and small interfering RNA specific to MAPKs, and infected with adenovirus for different time periods before analysis. Activation of specific NFkappaB subunits was analyzed by western blot, confocal microscopy, electromobility shift assay, and chromatin immunoprecipitation, and chemokine expression was quantified by enzyme-linked immunosorbent assay. RESULTS: Upon adenoviral infection, NFkappaB p65, p50, and cREL subunits translocate to the nucleus. This translocation is blocked by inhibitors of specific MAPK signaling pathways. Confocal microscopy showed that inhibitors of the p38, JNK, and ERK pathways differentially inhibited NFkappaB nuclear translocation, while PP2, an inhibitor of Src family kinases, completely inhibited NFkappaB nuclear translocation. Western blot analysis revealed that activation of specific NFkappaB subunits was time dependent following infection. Chromatin immunoprecipitation experiments indicated that binding of NFkappaB p65 and p50 subunits to the IL-8 promoter upon viral infection was differentially reduced by chemical inhibitors of MAPKs. Electromobility shift assay and luciferase assay analysis revealed that transactivation of IL-8 occurred with binding by the NFkappaB p65 homodimer or NFkappaB p65/p50 heterodimer as early as 1 h post infection, whereas MCP-1 expression was dependent upon the NFkappaB cREL but not the p65 subunit, and occurred 4 h after IL-8 induction. Finally, knockdown of NFkappaB p65 by short interfering RNA abrogated IL-8 but not MCP-1 expression after adenoviral infection. CONCLUSION: The kinetics of NFkappaB subunit activation are partly responsible for the observed pattern of acute inflammation in the adenoviral-infected cornea. MAPKs differentially regulate chemokine expression in adenoviral keratitis by differential and time-dependent activation of specific NFkappaB subunits.

Exacerbated corneal inflammation and neovascularization in the HO-2 null mice is ameliorated by biliverdin.

Heme oxygenase (HO-1 and HO-2) represents an intrinsic cytoprotective and anti-inflammatory system based on its ability to modulate leukocyte migration and to inhibit expression of inflammatory cytokines and proteins. HO-2 deletion leads to unresolved corneal inflammation and chronic inflammatory complications including ulceration, perforation and neovascularization. We examined the consequences of HO-2 deletion on hemangiogenesis and lymphangiogenesis in the model of suture-induced inflammatory neovascularization. An 8.0 silk suture was placed at the corneal apex of wild type and HO-2 null mice. Neovascularization was assessed by vital microscopy and quantified by image analysis. Hemangiogenesis and lymphangiogenesis were determined by immunofluorescence staining using anti-CD31 and anti-LYVE-1 antibodies, respectively. Inflammation was quantified by histology and myeloperoxidase activity. The levels of HO-1 expression and inflammatory cytokines were determined by real time PCR and ELISA, respectively. Corneal sutures produced a consistent inflammatory response and a time-dependent neovascularization. The response in HO-2 null mice was associated with a greater increase compared to the wild type in the number of leukocytes (827,600+/-129,000 vs. 294,500+/-57,510; p<0.05), neovessels measured by vital microscopy (21.91+/-1.05 vs. 12.77+/-1.55 mm; p<0.001) 4 days after suture placement. Hemangiogenesis but not lymphangiogenesis was more pronounced in HO-2 null mice compared to wild type mice. Induction of HO-1 in sutured corneas was greatly attenuated in HO-2 null corneas and treatment with biliverdin diminished the exaggerated inflammatory and neovascular response in HO-2 null mice. The demonstration that the inflammatory responses, including expression of proinflammatory proteins, inflammatory cell influx and hemangiogenesis are exaggerated in HO-2 knockout mice strongly supports the notion that the HO system is critical for controlling the inflammatory and neovascular response in the cornea. Hence, pharmacological amplification of this system may constitute a novel therapeutic strategy for the treatment of corneal disorders associated with excessive inflammation and neovascularization.

Neurotrophic keratitis in a patient with dihydroxypyrimidine dehydrogenase deficiency.

We describe a case of neurotrophic keratitis in association with dihydroxypyrimidine dehydrogenase (DHPD) deficiency. Ocular manifestations in patients with DHPD are rare and neurotrophic keratitis has never been reported before. A six-year-old boy who was a known case of DHPD deficiency and born of a consanguineous marriage presented to our clinic with non-healing corneal ulcers in both eyes. Reduced corneal sensations were detected and the patient was started on lubricating eye drops. The patient continues to be on lubricant eye drops and there has been no recurrence of the disease.

[Mental rationale for local use of angiotensin-converting enzyme in the treatment of inflammatory processes in the eye].

A rabbit model of deep alkaline-induced corneal burn was used to study the involvement of the local renin-angiotensin system of the eye in the development of an inflammatory process and wound healing. Corneal burn injury was shown to cause a significant increase in the activity of angiotensin-converting enzyme (ACE) in the tear and internal ocular tissue structures, promoting their microcirculatory disorders and inflammation development. The local use of ACE inhibitors as instillations substantially reduces an inflammatory reaction and the incidence of deep and extensive corneal ulcers. The study performed provides experimental rationale for the local use of ACE inhibitors for the treatment of inflammatory processes in the eye.

Heme oxygenase-1 induction attenuates corneal inflammation and accelerates wound healing after epithelial injury.

PURPOSE: Heme oxygenase (HO) is considered a fundamental endogenous immunomodulatory, cytoprotective, and anti-inflammatory system. This protective function is primarily ascribed to the inducible HO-1. The authors examined the effect of HO-1 induction on corneal inflammation and wound healing in mice undergoing epithelial injury. METHODS: C57BL6 mice were treated with SnCl(2) the day before epithelial injury and once daily thereafter. The corneal epithelium was removed with the use of a corneal rust ring remover in anesthetized mice. Reepithelialization was measured by fluorescein staining. The inflammatory response was examined by histology and was quantified by the myeloperoxidase assay. Inflammatory lipid mediators were detected and quantified by LC/MS/MS-based lipidomic analysis. HO-1 expression was assessed by real-time PCR, and HO activity was determined by measuring HO-dependent carbon monoxide production. RESULTS: Epithelial injury caused a time-dependent transient increase in HO-1 expression and HO activity that was significantly amplified by treatment with SnCl(2), resulting in a twofold to threefold increase in mRNA levels and a similar increase in corneal HO activity. Induction of HO-1 was associated with a significant acceleration of wound healing when compared with a vehicle-treated group and with attenuation of the inflammatory response, evidenced by a significant decrease in the number of infiltrating cells and by a significant reduction in the expression and production of proinflammatory lipid mediators and cytokines. CONCLUSIONS: Increased expression of HO-1 provides a mechanism that modulates inflammation and promotes wound closure; pharmacologic amplification of this system may constitute a novel strategy to treat corneal inflammation while accelerating wound repair after injury.

Modulation of matrix metalloproteinases by ultraviolet radiation in the canine cornea.

PURPOSE: To determine whether ultraviolet (UV) radiation can modulate expression and regulation of matrix metalloproteinases (MMP) in the canine cornea and to examine the expression of MMPs in canine chronic superficial keratitis (CSK). METHODS: Immunohistochemistry for MMP-2 and MMP-9 was performed on samples of CSK. In vitro, canine corneal epithelial cell (CEC) and stromal cell cultures were exposed to UV-irradiation. Following 2, 8 or 24 h, cells were harvested. MMP expression was examined by zymography, and RT-PCR was used to examine expression of Slug and Snail. CEC cultures treated with an EGFR inhibitor or a p38 inhibitor were UV-exposed and harvested 24 h later to examine expression of MMPs, Slug and Snail. RESULTS: Canine CSK had increased immunopositivity for both MMP-2 and MMP-9 compared to normal canine corneas. In vitro, CEC and stromal cell cultures exposed to UV showed generally increased expression of MMP-2, -9, Slug, and Snail; this response was dose and time dependent. Inhibition of the EGFR pathway did not prevent increased expression of MMP-2, -9, Slug or Snail in UV-exposed CEC; however, p38 inhibition did attenuate UV induction. CONCLUSIONS: We have found increased expression of MMPs in clinical samples of CSK compared to normal corneas. In addition, we have shown that there is a temporal association and dose dependency between UV exposure and production of MMPs, Slug, and Snail. These findings suggest that overexpression of MMPs due to UV-exposure may be linked to changes in the cornea that allow an influx of inflammatory cells and vascularization.

MyD88 functions as a negative regulator of TLR3/TRIF-induced corneal inflammation by inhibiting activation of c-Jun N-terminal kinase.

The adaptor molecule MyD88 is necessary for responses to all Toll-like receptors except TLR3 and a subset of TLR4 signaling events, which are mediated by the adaptor molecule TRIF. To determine the role of TRIF in host inflammatory responses, corneal epithelium of C57BL/6, TLR3(-/-), TRIF(-/-), and MyD88(-/-) mice was abraded and stimulated with the synthetic TLR3 ligand poly(I:C). We found that poly(I:C) induced a pronounced cellular infiltration into the corneal stroma, which was TLR3- and TRIF-dependent. Unexpectedly, the inflammatory response was exacerbated in MyD88(-/-) mice, with enhanced neutrophil and F4/80(+) cell infiltration into the corneal stroma and elevated corneal haze, which is an indicator of loss of corneal transparency. To determine whether MyD88-dependent inhibition of TLR3/TRIF responses is a general phenomenon, we examined cytokine production by MyD88(-/-) bone marrow-derived macrophages; however, no significant difference was observed between MyD88(+/+) or MyD88(-/-) macrophages. In contrast, human corneal epithelial cells (HCECs) transfected with MyD88 small interfering RNA had significantly increased (2.5-fold) CCL5/RANTES production compared with control HCECs, demonstrating a negative regulatory role for MyD88 in TLR3/TRIF responses in these cells. Finally, knockdown of MyD88 in HCECs resulted in increased phosphorylation of c-Jun N-terminal kinase (JNK), but not p38, IRF-3, or NF-kappaB. Consistent with this finding, the JNK inhibitor SP600125, but not p38 inhibitor SB203580, ablated this response. Taken together, these findings demonstrate a novel JNK-dependent inhibitory role for MyD88 in the TLR3/TRIF activation pathway.

[The roles of gelatinases in pathological changes of fungal keratitis in experimental rabbits].

OBJECTIVE: To study the roles of gelatinases, including matrix metalloproteinase (MMP)-2 and MMP-9, in pathological changes of fungal keratitis in rabbits. METHODS: Eighty New Zealand albino rabbits were randomly and equally divided into 4 groups, 3 of them were test groups, with Fusarium solani, Aspergillus fumigatus, and Candida albicans inoculated onto the right corneas, respectively. In the other group, sterile saline was injected onto the right corneas and was used as the control group. The source and activity of gelatinases were examined by immunohistochemistry and gelatin zymography, respectively. Hematoxylin-eosin stains was applied for observing infiltration of inflammatory cells and degradation of corneal extracellular matrixes (ECMs). Periodic acid-Schiff stain was used for studying hyphal growth patterns and the invasive depth in the cornea. RESULTS: MMP-2 was mainly produced by the keratocytes. Active MMP-2 was detected from day 5 after inoculation and increased greatly on day 8. MMP-9 was mostly produced by neutrophils, and active MMP-9 was detected from day 1 and increased to day 3, then decreased gradually. In A. fumigatus and C. albicans keratitis, the corneal ECMs were degraded obviously, the hyphae grew vertically, and the neutrophils were much more than those in F. solani keratitis whose hyphae grew horizontally and ECMs were degraded slightly. On day 8, the hyphae and neutrophils in F. solani and C. albicans keratitis decreased greatly compared with day 3, but did not change significantly in A. fumigatus keratitis. CONCLUSIONS: There is significant difference in gelatinase activities in the rabbits' corneas infected by F. solani, A. fumigatus, and C. albicans. Gelatinases play important roles in the degradation of corneal ECMs. Hyphal growth pattern and invasive depth are depended on the difference of degradations of ECMs and show difference in various fungal keratitis.

Matrix metalloproteinases (MMP-8, MMP-9) and the tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) in patients with fungal keratitis.

PURPOSE: To study the infiltrating cells and quantify the levels of matrix metalloproteinases (MMP-8, MMP-9) and tissue inhibitor of metalloproteinases (TIMP-1, TIMP-2) in the cornea, tear, and serum of patients with fungal keratitis. METHODS: Experimental study. Infected corneal tissue from 4 patients with fungal keratitis (group 1) scheduled to undergo therapeutic keratoplasty accounted for the histopathologic and cytospin smear analysis. Ten patients with fungal keratitis from group 2 served for the quantification of MMPs and TIMPs. Five patients with keratoconus undergoing penetrating keratoplasty and 5 cadaver corneas were chosen as controls for group 2. Corneal buttons obtained during keratoplasty, 15 to 20 microL of tears collected using the capillary flow method, and 3 mL of blood was obtained from patients with fungal keratitis and patients with keratoconus. Corneal button sections from group 1 were stained with hematoxylin and eosin and Grocott methenamine silver nitrate for the histopathologic studies and Giemsa staining for the cytospin smear analysis. Enzyme-linked immunosorbent assay was used for the quantification of total MMP-8, MMP-9, TIMP-1, and TIMP-2 in the corneal homogenates, tear, and serum samples of group 2. RESULTS: Corneal sections from group 1 revealed dense fungal filaments and a large proportion (91.4% +/- 38%) of polymorphonuclear leukocytes (PMNs). Significant elevation in the levels of MMP-8 and MMP-9 (P < 0.05) in the fungal keratitis corneas was observed in group 2 compared with the cadaver and keratoconus corneas. The ratio of MMP/TIMP was also higher in the fungal keratitis corneas. CONCLUSIONS: Infiltrating PMNs in the cornea of patients with fungal keratitis contributed to the increased activities of MMP-8 and MMP-9, thereby enhancing tissue destruction and derangement.

Roles of adherence and matrix metalloproteinases in growth patterns of fungal pathogens in cornea.

PURPOSE: To investigate the roles of adherence and matrix metalloproteinases (MMPs) in growth patterns of major fungal pathogens in cornea. METHODS: Ninety-six eyes in 96 rabbits were equally divided into four groups receiving inoculation of fungal conidia of Aspergillus fumigatus, Candida albicans, Fusarium solani, and Penicillium citreo-viride, respectively, to induce fungal keratitis. Corneas in each group were obtained at 2, 8, 16 hr, and 1, 2, 3, 5, and 8 days after inoculation and were subjected to scanning electron microscopy, histopathological examination, and gelatin zymography. Eight saline-inoculated eyes in another eight rabbits served as controls. RESULTS: All eyes in the fungus-inoculated groups developed fungal keratitis. The binding of conidia to corneal epithelial basement membrane was initiated earlier in the A. fumigatus and C. albicans groups than in the F. solani and P. citreo-viride groups. Destruction of basement membrane began at 1 to 3 days. Histopathologically, infiltration of inflammatory cells was more evident in the A. fumigatus and C. albicans groups than the F. solani and P. citreo-viride groups at 3 days. The hyphae of A. fumigatus and C. albicans traversed the cornea in a plane perpendicular to the stromal lamellae, whereas the hyphae of F. solani and P. citreo-viride lay parallel to the corneal lamellae. MMP-9 and MMP-2 were found in all infected corneas. At 3 days, proteolysis was most active; the level of MMP-9 was higher in the A. fumigatus and C. albicans groups than in the F. solani and P. citreo-viride groups. There were positive correlations among the number of binding conidia, degree of inflammation, and level of MMP-9 (p < 0.05). CONCLUSIONS: The adherence ability, chemotaxis to neutrophils, and MMP-9 expression level differ in eyes with different fungal pathogens, which may contribute to the different growth patterns of fungi in cornea.

Group IIA phospholipase A(2) concentration of tears in patients with ocular rosacea.

PURPOSE: To determine the concentration of group IIA phospholipase A(2) (GIIAPLA(2)) in tears of patients with ocular rosacea, and to compare it with GIIAPLA(2) concentration in tears of age-matched healthy controls. METHODS: The GIIAPLA(2) concentration in tears was measured with a time-resolved fluoroimmunoassay in 21 patients with ocular rosacea (mean age 55.6+/-9.2 years) and in 21 normal subjects (mean age 53.4+/-8.2 years). Conjunctival brush cytology was carried out and eosinophils, neutrophils, lymphocytes, squamous epithelial cells, columnar epithelial cells, metaplastic changes and goblet cells were calculated separately. RESULTS: The GIIAPLA (2) concentration in tears was statistically significantly lower in patients with ocular rosacea (31.0+/-18.4 microg/ml, p=0.0099) and, more specifically, in patients who had dry eye (25.8+/-15.1 microg/ml, p=0.0034), compared to that in normal controls. There was no correlation between the GIIAPLA (2) content of tears and the conjunctival cells collected by the brush cytology. CONCLUSION: The tears of patients with dry eye symptoms due to ocular rosacea have decreased GIIAPLA (2) content. The pathogenic importance of this finding is discussed.