Koki Matsumoto

The Serratial 56K Protease as a Major Pathogenic Factor in Serratial Keratitis: Clinical and Experimental Study

A possible cause and the difference in clinical severity of serratial keratitis were investigated. Two strains of Serratia marcescens were isolated: one from a patient with severe liquefactive keratitis, who had diabetes mellitus, and one from a patient with mild superficial keratitis, but who had no underlying disease. When the same numbers of bacteria were injected separately into corneas of the same rabbits or guinea pigs, the strain from the first patient elicited severe corneal destruction, remarkable intracorneal edema; and liquefactive necrosis, but the strain from the second caused mild keratitis with erosion or intracorneal abscess. The keratitis induced by the former strain required a longer time to heal, and the prognosis was poorer than that for the other keratitis. Therefore, the difference in severity between the two cases of experimentally induced keratitis paralleled that of the clinical cases. Thus, the severity of the serratial keratitis might be attributed more to the virulence of the bacteria than the condition of the host. The virulence factor seemed to be a heat-labile metabolic product (or products) of the bacteria. To clarify this virulence factor, the major secretory protease (56K protease) produced by these two strains of bacteria was compared by using in vitro and in vivo systems. The virulent strain produced about ten times more protease during culture than the less virulent strain. When injected into the corneas of experimental animals, the 56K protease from the virulent strain induced severe lesions similar to those caused by the living virulent strain of bacteria. These results indicated that one of the major factors causing the virulence was correlated with the tissue destructive 56K protease produced by S. marcescens.

Lactoferrin Glu561Asp facilitates secondary amyloidosis in the cornea

Aim: To elucidate the pathogenic mechanism of amyloid formation in corneal amyloidosis with trichiasis. Methods: Ophthalmological examination was performed in nine patients to determine secondary corneal amyloidosis with trichiasis. Congo red staining and immunohistochemistry using anti-human lactoferrin antibody were used for biopsied corneal samples. For genetic analyses, single strand conformation polymorphism (SSCP), direct DNA sequence analysis, and polymerase chain reaction (PCR) induced mutation restriction analysis (IMRA) were employed to detect lactoferrin gene polymorphism. Results: All patients had had trichiasis at least for 1 year, and all amyloid-like deposits were found in one eye with trichiasis. Ophthalmological examination revealed that eight patients showed gelatinous type of amyloid deposition and one showed lattice type of amyloid deposition. Studies of biopsied corneal samples with Congo red stain revealed positive staining just under the corneal epithelial cells. Immunoreactivity of anti-human lactoferrin antibodies was recognised in all tissues with positive Congo red staining. Lactoferrin gene analysis revealed that seven patients were heterozygotic and two were homozygotic for lactoferrin Glu561Asp. The frequency of the polymorphism in the patients was significantly different from that in 56 healthy control subjects. Conclusion: Lactoferrin Glu561Asp is a key polymorphism related to facilitating amyloid formation in corneal amyloidosis with trichiasis.

A Novel Localized Amyloidosis Associated with Lactoferrin in the Cornea

We report a novel localized amyloidosis associated with lactoferrin. To elucidate the precursor protein of corneal amyloidosis associated with trichiasis, we analyzed amyloid deposits from three patients by histopathology and biochemistry. Amyloid deposits showed immunoreactivity, confirmed by electron microscopy, for only anti-human lactoferrin antibody. Electrophoresis of amyloid fibrils revealed lactoferrin with and without sugar chains; N-terminal sequence analysis revealed full-length lactoferrin and a truncated tripeptide of N-terminal amino acids, Gly-Arg-Arg. Carboxymethylated wild-type lactoferrin formed amyloid fibrils in vitro. Lactoferrin gene analysis in the three patients revealed a Glu561Asp mutation in all of the patients and a compound heterozygote of Ala11Thr and Glu561Asp mutations in one patient. A heterozygotic Glu561Asp mutation appeared in 44.8% of healthy Japanese volunteers, suggesting that the mutation may not be an essential mutation for amyloid formation (p = 0.104). Results thus suggest that lactoferrin is this precursor protein.

Role of cytokines and chemokines in pseudomonal keratitis.

Pseudomonal keratitis usually progresses rapidly, often resulting in corneal perforation and blindness. Remarkable events in pseudomonal keratitis include massive polymorphonuclear leukocyte infiltration in the cornea and various degrees of tissue destruction. With regard to initiation of these inflammatory events, various inflammatory cytokines and chemokines appear to be key substances and have been the subject of several studies. Inflammatory cytokines and chemokines believed to be important in pseudomonal keratitis include interleukin (IL)-1β, IL-6, macrophage inflammatory protein (MIP)-2 (homologous to human IL-8), macrophage inhibitory factor (MIF), IL-12, IL-18, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α. In this article, current concepts related to the role of inflammatory cytokines and chemokines in pseudomonal keratitis are reviewed.

Inhibitory effects of ovomacroglobulin on bacterial keratitis in rabbits

We studied the inhibitory effects of chicken egg-white ovomacroglobulin (ovoM) on keratitis induced by 56000-Da protease (56 KP) of Serratia marcescens and by elastase (PE) and alkaline protease (PAP) of Pseudomonas aeruginosa. The effects of ovoM on the serratial and pseudomonal keratitis in rabbits were also elucidated. In one model, four drops of 56 KP, PE, or PAP (1 mg/ml) were applied to wounded corneas of eight eyes. Thereafter, 80 μl ovoM (10 mg/ml) was dropped into four eyes and 0.01 M phosphate-buffed 0.15 M saline (pH 7.4) into the other eyes as a control. The other in vivo test system involved intrastromal injection of S. marcescens or P. aeruginosa, by which each sample (105–107 colony-forming units) mixed with ovoM was injected into one cornea and the other cornea received organisms without ovoM. OvoM completely inhibited the activity of these bacterial proteases in vitro and reduced corneal destruction in experimental keratitis in rabbits. In addition, greatly accelerated wound healing was observed.

Characteristics of Pseudomonas corneal infection related to orthokeratology.

Purpose: To describe a Pseudomonas aeruginosa corneal infection resulting from orthokeratology. Methods: Case report. Results: A 17-year-old boy wearing orthokeratology (OK) lenses was referred to our clinic because of redness in his right eye in spite of his usage of ofloxacin (OFLX) eye drops. An excavated paracentral corneal ulcer with an immune ring and hypopyon was observed. It was positioned under the paracentral steeper portion of the optic of the OK lens. Culture of the lens solution revealed P. aeruginosa. The patient was treated with topical OFLX and cefmenoxime (CMX) plus intravenous and subconjunctival injections of cefozopran (CZOP), successfully. The antibiotic susceptibility of P. aeruginosa by the disk diffusion susceptibility test was reduced under moderately hypoxic conditions. Glycocalyx slime was formed on the OK lens in vitro by P. aeruginosa isolated from the case. Conclusions: Changes in P. aeruginosa susceptibility to antibiotics under moderately hypoxic conditions and glycocalyx slime formation might affect the features of OK lens-associated infections.

Proteases in Bacterial Keratitis

Purpose. To investigate the proteases that may contribute to corneal ulceration in bacterial keratitis, especially those caused by Pseudomonas aeruginosa. Methods. Culture supernatants of several bacteria were examined for protease by using gelatin and casein zymography. A minimum amount (1 &mgr;g) of highly purified pseudomonal elastase and alkaline protease was injected into rabbit corneas and the corneal lesions were evaluated. In pseudomonal keratitis models in rats, intracorneal proteases were analyzed with gelatin and casein zymography 12 to 24 hours after infection. Proteases derived from rat corneas and peritoneal exudate cells (mostly polymorphonuclear leukocytes [PMNs]) were similarly analyzed. Results. All tested strains of P. aeruginosa and Serratia marcescens produced at least one protease, whereas Staphylococcus aureus strains did not. Pseudomonal proteases elicited liquefactive necrosis with remarkable stromal swelling similar to that caused by serratial 56K protease. Intracorneal proteases produced during infection with P. aeruginosa included matrix metalloproteinase (MMP)-2, MMP-9, the activated forms of MMP-2 and MMP-9, and gelatinases with molecular weights of >200 kd, as well as caseinase with molecular weight of 25 kd. Conclusion. Proteases that may contribute to ulceration in the early stages of corneal infection with P. aeruginosa could be of bacterial or corneal cell origin. However, in the later stages when numerous PMNs accumulate in the cornea, PMN-derived proteases predominate as the pathogenic factor in tissue destruction.

The role of Pseudomonas aeruginosa elastase in corneal ring abscess formation in pseudomonal keratitis

In order to identify the causative factors of ring abscess, which is the characteristic feature of pseudomonal keratitis, pseudomonal endotoxin, exotoxin A, and elastase were each separately injected into guinea pig cornea. There was no formation of ring abscess. Injection of livingPseudomonas aeruginosa strains IFO3455 and Takamatsu which produce all three molecules, clearly induced ring abscess. In contrast, when heat-killed bacteria strain IFO3455 or living bacteria of the non-elastase-producing strain PA103 were injected, ring abscess was not induced. Furthermore, when living bacteria strain IFO3455 were injected with anti-elastase antibody or a protease inhibitor, ovomacroglobulin, ring abscess formation was significantly inhibited. Histological examination demonstrated that the ring abscess was a dense accumulation and aggregation of polymorphonuclear leukocytes (PMN) with debris of cells and lamellae in the deep stroma at the corneal margins, suggesting prevention of PMN migration to the central lesion. The presence of anti-elastase antibody or a specific elastase inhibitor facilitated PMN migration towards living bacteria strain IFO3455 in an in vitro model. These results indicate that pseudomonal elastase is a necessary but not sufficient factor in the formation of ring abscess in pseudomonal keratitis.

Therapeutic intervention with chicken egg white ovomacroglobulin and a new quinolone on experimental Pseudomonas keratitis

Abstract⊎ Background: Chicken egg white ovomacroglobulin (ovoM) is a potent protease inhibitor with broad-spectrum activity against various proteases. The combined effects of ovoM and the new quinolone, ofloxacin (OFLX) on experimental Pseudomonas aeruginosa keratitis were investigated. ⊎ Methods: The in vitro inhibitory effects of ovoM on protease activity in culture fluid of clinically isolated P. aeruginosa and on activity of human neutrophil elastase and cathepsin G were assayed using azo-casein as substrate. Albino rabbits received intrastromal injection of the isolated Pseudomonas strain (1 × 105 colony-forming units). At 16 h after inoculation, three treatment groups -0.1% ovoM alone, 0.3% OFLX alone, and a combination of both —and a non-treatment control group were tested. ⊎ Results: Protease activity in the culture solution and human neutrophil elastase was inhibited by ovoM, whereas cathepsin G was not inhibited effectively. In vivo additive therapeutic effects of ovoM and OFLX were observed at 96 h (P < 0.05 compared with OFLX alone). ⊎ Conclusion: The results indicate that inhibition of proteolytic activity with ovoM is useful in preventing stromal degradation in P. aeruginosa keratitis.

Clinico-histopathological and biochemical analyses of corneal amyloidosis in gelatinous drop-like corneal dystrophy

Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan, Department of Immunology and Hematology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan, Department of Ophthalmology, Kumamoto Shinto General Hospital, Kumamoto, Japan, Department of Ophthalmology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan, Department of Morphological and Physiological Sciences, Graduate School of Health Sciences, Kumamoto University, Kumamoto, Japan, and Diagnostic Unit for Amyloidosis, Department of Neurology, Kumamoto University Hospital, Kumamoto, Japan