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Tryptic peptides of citraconylated fragment alpha1-CB3 and chymotryptic peptides of fragment alpha1-CB3 of bovine corneal collagen were prepared, isolated and characterized. Their amino acid compositions were consistent with the amino acid sequence of fragment alpha1-CB3 from calf skin collagen. Two glycoside sites were identified in bovine corneal fragment alpha1-CB3, one of them being the first located in the overlap region of collagen. The results are related to the uniformly narrow collagen fibres found in cornea and essential for its transparency.

Six CNBr-cleaved fragments of insoluble collagen from bovine cornea were isolated, characterized and examined for hydroxylysine glycosides. Thus the general distribution of most of the glycoside along the collagen molecule was determined. Collagen from bovine stroma is almost entirely type I. This work forms a basis for the pinpointing of glycoside-attachment sites along the collagen molecules of bovine cornea.

Acanthamoeba keratitis is a vision-threatening corneal infection. The mannose-binding protein of Acanthamoeba is thought to mediate adhesion of parasites to host cells. We characterized the amoeba lectin with respect to its carbohydrate binding properties and the role in amoeba-induced cytopathic effect (CPE). Sugar inhibition assays revealed that the amoeba lectin has the highest affinity for alpha-Man and Man(alpha1-3)Man units. In vitro cytopathic assays indicated that mannose-based saccharides which inhibit amoeba adhesion to corneal epithelial cells were also potent inhibitors of amoeba-induced CPE. Another major finding was that N-acetyl-D-glucosamine (GlcNAc) which does not inhibit adhesion of amoeba to host cells is also an inhibitor of amoeba-induced CPE. The Acanthamoebae are thought to produce CPE by secreting cytotoxic proteinases. By zymography, one metalloproteinase and three serine proteinases were detected in the conditioned media obtained after incubating amoebae with the host cells. The addition of free alpha-Man and GlcNAc to the co-culture media inhibited the secretion of the metalloproteinase and serine proteinases, respectively. In summary, we have shown that the lectin-mediated adhesion of the Acanthamoeba to host cells is a prerequisite for the amoeba-induced cytolysis of target cells and have implicated a contact-dependent metalloproteinase in the cytopathogenic mechanisms of Acanthamoeba.

Cell migration is a fundamental process of wound repair in biological systems. In an attempt to identify plasma membrane glycoconjugates which mediate cell migration, migrating and nonmigrating rabbit corneal epithelia were analyzed for reactivity with monoclonal antibodies (mAbs) specific for unsubstituted N-acetyl-lactosamine (mAb 1B2), Le(x) (mAbs 7A and MMA), and sialyl Le(x) (mAb CSLEX1) carbohydrate chains of neolactoglycoconjugates. Immunohistochemical analysis indicated that regardless of whether the epithelia analyzed were from corneas of animals in vivo, corneas in organ culture, or cells in tissue culture, migrating cells stained intensely with mAb 1B2, whereas nonmigrating cells either did not stain or stained only weakly. mAbs MMA and 7A stained migrating epithelium as well as basal and middle cell layers of normal, nonmigrating epithelium. mAb CSLEX1 did not stain wounded corneas but stained the superficial cell layer of normal corneal epithelium. Biochemical analyses by TLC immunostaining revealed the presence of three mAb 1B2-reactive glycosphingolipids (GSL), neolactotetraosyl-(nLc4, paragloboside), neolactohexaosyl- (nLc6), and neolacto-octaosylceramide (nLc8) in migrating epithelia. In contrast, nonmigrating epithelia contained only trace amounts of these glycolipids. Exogenous addition of nLc4, but not various other GSLs including a Le(x)-GSL (SSEA-1), stimulated re-epithelialization of wounds in an experimental model of corneal epithelial wound healing. Moreover, re-epithelialization of wounds was significantly inhibited by mAb 1B2 but not by mAb MMA. The data suggest that neolacto-GSLs of corneal epithelium may be among the molecules which mediate healing of corneal epithelial wounds by influencing cell migration.

ABSTRACTAcanthamoeba castellaniimannose-binding protein (MBP) mediates adhesion of the amoebae to corneal epithelial cells, a key first step in the pathogenesis of Acanthamoebakeratitis (AK), a devastating corneal infection. In the present study, we demonstrate that oral immunization with recombinant MBP ameliorates AK in a hamster animal model and that this protection is associated with the presence of elevated levels of anti-MBP immunoglobulin A in the tear fluid of the immunized animals.

Background. Evaluation of Ki-67 index in lung carcinoid tumors (LCTs) has been of interest in order to identify high risk subsets. Our objectives are (1) to evaluate the usefulness of Ki-67 index, mitoses, and tumor size in predicting metastasis and (2) to compare the Manual Conventional Method (MCM) and the Computer Assisted Image Analysis Method (CIAM) for Ki-67 calculation. Methods. We studied 48 patients with LCTs from two academic centres in Canada. For Ki-67 calculation, digital images of 5000 cells were counted using an image processing software and 2000 cells by MCM. Mitoses/10 HPF was counted. Results. We had 37 typical carcinoids (TCs) and 11 atypical carcinoids (ACs). 7/48 patients developed metastasis. There was a positive relationship between metastasis and carcinoid type (P=0.039) and metastasis and mitoses (≥2) (P=0.017). Although not statistically significant, the mean Ki-67 index for ACs was higher than for TCs (0.95% versus 0.72%, CIAM, P=0.299). Similarly, although not statistically significant, the mean Ki-67 index for metastatic group (MG) was higher than for nonmetastatic group (NMG) (1.01% versus 0.71% by CIAM, P=0.281). However when Ki-67 index data was categorized at various levels, there is suggestion of a useful cutoff (≥0.50%) to predict metastasis (P=0.106, CIAM). A significantly higher proportion of patients with mitosis ≥2 and Ki-67 index ≥0.50% had metastasis (P=0.033) compared to other patients. Similarly patients with tumor size ≥3 cm and Ki-67 ≥0.50% had a greater percentage of metastases than others (P=0.039). Although there was a strong correlation between two (MCM versus CIAM) counting methods (r=0.929, P=0.001), overall the calculated Ki-67 index was slightly higher by MCM (range 0 to 6.4, mean 1.5) compared to CIAM (range 0 to 2.9, mean 0.75). Conclusion. This study confirms that mitoses ≥2 is a powerful predictor of metastasis in LCTs. Although this is a small sample size, there is suggestion that analysis of Ki-67 index along with mitoses and tumor size may be a useful adjunct for predicting metastasis in LCTs.

Near-monosized and uniformly distributed cubic Fe core-shell nanoparticles (with an average side length of 56nm) were electrodeposited on H-terminated Si(100)in a FeCl2solution. X-ray photoelectron spectroscopy analysis indicates chemical states of the Fe nanocubes characteristic of predominant ferromagnetic metallic Fe cores covered by Fe2O3shells. Glancing-incidence X-ray diffraction analysis reveals a body-centered cubic (bcc) lattice structure. A nanometer-thick film consisting of 2–3 layers of cuboidal nanoparticles of the same bcc Fe core and Fe2O3shell can be obtained when the amount of charge transfer is increased. The prominent magnetic domains of these nanocubes can be illustrated by magnetic force microscopy.