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.

[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.

Expression of matrix metalloproteinases 2 and 9 in experimental corneal injury and fungal keratitis.

PURPOSE: Levels of matrix metalloproteinases (MMPs) can be modulated during corneal infection, but little is known about MMP profiles during fungal keratitis. The purpose of this study was to determine the effect of corneal trauma and immunosuppressive treatment on the expression kinetics of MMP-2 and MMP-9 during experimental keratomycosis. METHODS: Corneas of immunocompetent and cyclophosphamide-treated adult BALB/c mice were topically inoculated with 1 x 10 culturable units of Fusarium solani or mock-inoculated with or without superficial corneal scarification. Eyes were scored daily for disease severity and processed for zymography after 1.5 hours, 6 hours, 1 day, 4 days, or 8 days. Gelatinase activity was densitometrically quantitated and normalized to MMP-2 and MMP-9 controls. RESULTS: MMP-9 levels in nontraumatized eyes transiently increased at 6 hours after fungal exposure, but this increase was inhibited by cyclophosphamide treatment. Corneal injury significantly induced early MMP-9 expression that returned to baseline levels within 4 days. Cyclophosphamide pretreatment reduced and delayed MMP-9 after scarification. Fusarium exposure dampened the MMP-9 response to corneal trauma in immunocompetent and cyclophosphamide-treated animals. Ocular levels of MMP-2 were not affected by scarification, fungal exposure, or immunosuppressive treatment. CONCLUSIONS: Ocular MMP-9 levels, but not MMP-2 levels, increased soon after corneal injury. A similar, although muted, MMP-9 response occurs during early filamentous fungal keratitis, with a kinetic profile similar to corneal disease progression. The early stage of ulcerative keratitis may involve selective regulation of corneal matrix metalloproteinases, suggesting an initial opportunity for therapeutic intervention.

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.

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.