Kaoru Araki-Sasaki

Substance P-induced cadherin expression and its signal transduction in a cloned human corneal epithelial cell line

Although the absence of Substance P (SP), a neurotransmitter in the trigeminal nerve, has been speculated as a cause for developing neurotrophic keratitis, its exact pathogenesis is still not clarified. In a previous report, we showed with electron microscopic examination that epithelial cell attachment was weakened in denervated corneas. In this study, SV40‐transformed human corneal epithelial cells (HCE‐Ts) were used to explore the molecular mechanisms responsible for mediating regulation of E‐cadherin expression in response to Substance P receptor stimulation. Expression of the mRNAs for specific SP receptors, neurokinin (NK)‐1R, NK‐2R, and NK‐3R, was demonstrated with RT‐PCR. The cells were treated with various concentrations of SP in vitro, and the expression of an adhesion molecule E‐cadherin was analyzed by immunofluorescence, immunoblotting, and enzyme‐linked immunosorbent assay (ELISA) using an anti‐E‐cadherin antibody. E‐cadherin expression was increased by SP in a dose‐dependent manner both in the cytosolic fraction and in the cell membrane fraction. This increase in E‐cadherin expression was completely inhibited by Calphostin C (PKC inhibitor) and KN‐62 (CaMK inhibitor), but not by H‐89 (PKA inhibitor), indicating that SP‐induced E‐cadherin expression involves the activation of protein kinase C (PKC) and calmodulin kinase (CaMK). SP did not affect cell proliferation at all. All these findings indicate that SP induced E‐cadherin expression through PKC and CaMK activation and suggest that a lack of SP may account in part for the pathogenesis of neurotrophic keratitis. J. Cell. Physiol. 182:189–195, 2000.

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.

Dynamic Expression of Chemokines and the Infiltration of Inflammatory Cells in the HSV-Infected Cornea and its Associated Tissues

Background: The chemotactic signals regulating cell trafficking in the herpes simplex virus type 1 (HSV-1) infected cornea are well documented, however, those in the cornea-associated tissues, such as the trigeminal ganglion (TG) and draining lymph nodes (LNs), are largely unknown. Objectives: To examine chemokine expression and subsequent cell infiltration in the HSV-1 infected cornea and its associated tissues. Study design: Eight-week-old female BALB/c mice were infected with 10 μ l HSV-1 (CHR3 strain: 5 × 106 PFU/ml) by corneal scarification. Total RNAs were extracted from the corneas, TGs, and LNs at pre-inoculation, 3 days post-inoculation (P.I.) and 7 days P.I. The mRNA for 28 different chemokines in the extracts was amplified by RT-PCR. Infiltrating cells were identified by immunohistochemistry. Result: After the HSV-1 infection, the corneal stroma became edematous by infiltrated cells under the eroded epithelium. The TG and LNs were markedly swollen. The cornea was infiltrated with granulocytes and CD11b+ cells at 3 days P.I., followed by CD4+ and CD8+ T cells at 12 days P.I. In the TG, CD11b+ cells, but no granulocytes, infiltrated throughout the observation period. T cells migrated into the TG earlier than into the cornea. Gene expressions of neutrophil-attracting chemokines (CXCL1, 2, 3, and 5) increased in the cornea, but they did not enhance in the TG or LNs. On the other hand, gene expressions of chemokines which attract CD11b+ cells such as CCL2, 8, 7, 12, CCL3, 4, and CCL5, increased in the cornea and TG with its peak at 3 days P.I. Gene expressions of chemokines those work on T cells and B cells, such as CCL19, CCL21, CXCL9, CXCL13, CXCL10, XCL1, and CXCL16, were up-regulated and peaked at 3 days P.I. in the cornea and in the TG. Thus, pattern of chemokine gene expression was similar in the cornea and in the TG. On the contrary, gene expressions of chemokines in the draining LNs affecting CD11b+ cells and T cells were temporarily down-regulated. Conclusion: Upon HSV-1 infection, dynamic gene expression of chemokines was observed not only in the inoculated cornea but also in its associated tissues.

Neutrophil chemotaxis induced by corneal epithelial cells after herpes simplex virus type 1 infection

PURPOSE Neutrophil invasion is a primary event in the development of herpetic keratitis. It has been reported that HSV-1 infection of keratocytes induces the synthesis of IL-8, a potent neutrophil chemoattractant, while corneal epithelium does not. Nevertheless, little is known about the correlation between neutrophil migration and the production of chemotactic factors by HSV-1-infected corneal cells, especially in epithelial cells which form an initial barrier of the ocular surface. We examined whether human corneal epithelial cells as well as keratocytes could induce neutrophil chemotaxis in response to HSV-1 infection. METHODS Human corneal epithelial cells immortalized with SV40 (HCE) and human keratocytes were infected with HSV-1. The culture fluids collected at 4, 12, 24 h after infection were assayed for human neutrophil chemotaxis using a modified Boyden chamber method. IL-8 levels in these supernatants were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS The chemotactic activity induced by HCE and keratocytes after MP strain of HSV-1 infection peaked as early as 4 h postinfection, then declined. Chemotactic activity induced by HSV-1-infected HCE and IL-8 levels on these supernatants paralleled with the infectious virus titer. It was inhibited by monoclonal anti-IL-8 antibody. UV-inactivation of MP strain abrogated neither the induction of chemotactic activity nor IL-8 secretion of infected HCE. CONCLUSIONS At the early phase of HSV-1 infection, corneal epithelial cells play an important role in inducing neutrophil chemotaxis, which was mediated by IL-8.

Clinical Characteristics of Keratitis Due to Colletotrichum gloeosporioides

PURPOSE To determine the characteristics of the keratitis due to Colletotrichum gloeosporioides. METHODS The medical records of 3 cases of fungal keratitis caused by C. gloeosporioides were reviewed to determine the clinical characteristics. The minimal inhibitory concentrations of different antifungal drugs for all 3 isolates were determined. All 3 isolates were grown on Sabouraud dextrose agar at 25°C, 35°C, and 37°C to determine the temperature-sensitive growth. RESULTS All 3 patients lived in the southwestern part of Japan and had an ocular trauma involving organic materials. The infectious foci were localized in the anterior stroma, and they did not extend deep into the stroma in all cases. The keratitis was treated with antifungal medications including topical voriconazole and natamycin eye ointment, and was resolved in 2-3 weeks. All of the isolated strains grew well at 25°C but poorly at 35°C and 37°C. All isolated strains had similar drug-sensitivity profiles; they were sensitive to amphotericin B, itraconazole, miconazole, micafungin, and voriconazole, and relatively resistant to flucytosine, fluconazole, and natamycin. CONCLUSIONS All 3 cases of C. gloeosporioides keratitis had similar clinical features. The similarities in the drug-sensitivity profiles should be helpful in treating C. gloeosporioides keratitis.

Detection of cytomegalovirus DNA from cytomegalovirus corneal endotheliitis after penetrating keratoplasty.

Purpose: To report the detection of cytomegalovirus (CMV) DNA in the cornea of a CMV endotheliitis patient after penetrating keratoplasty (PKP). Case: A 71-year-old man without immunodeficiency developed corneal endotheliitis in the right eye. The patient had previously received PKP several times. Polymerase chain reaction (PCR) detected CMV-DNA in the aqueous humor in his affected eye, and we started administration of ganciclovir. There was resolution of the inflammation; however, bullous keratopathy was subsequently noted in the cornea. Additional PKP was performed with perioperative intravenous administration of ganciclovir. The failed graft obtained during the additional PKP was subjected to PCR analysis and histopathological examination. PCR analysis showed CMV-DNA in the failed graft. Little inflammatory change was noted in either the epithelial or stromal layers of the failed graft. With continued ganciclovir treatment, the graft remained clear and no recurrence or rejection occurred until 12 months after the last PKP. Conclusion: Our PCR analysis showed the presence of CMV-DNA within the cornea of the patient with corneal endotheliitis.

Human Hepatocyte Growth Factor (HGF) in the aqueous humor

Using an enzyme-linked immunosorbent assay, we measured the concentration of hepatocyte growth factor (HGF) in human aqueous humor and serum from 36 eyes of 33 patients and analyzed the relationship between HGF and several parameters of corneal endothelial cells. Aqueous HGF concentrations ranged from 0.020-0.194 ng/mL (average: 0.101 +/- 0.044 ng/mL) and was correlated positively with corneal endothelial cell density (r = 0.343, P = 0.04). The aqueous HGF level did not correlate with other corneal endothelial cell parameters or the serum HGF level. The HGF receptor, c-Met, was found in the corneal endothelial cell membranes. This study suggests that the aqueous humor HGF is a factor which maintains an integrity of corneal endothelial cells.

Morphological changes of corneal subepithelial nerve plexus in different types of herpetic keratitis

PurposeWe investigated by in vivo confocal microscopy alterations in the subepithelial nerve plexus in different types of herpes simplex keratitis (HSK).MethodsSeventeen patients (seven women and ten men, mean age 63.9 years) with a history of HSK were classified into three groups according to the classification of Herpetic Keratitis Infection Research Group. Slit-lamp examinations, corneal sensitivity measurements, and corneal in vivo confocal microscopy examinations [Rostock Corneal Module attached to the Heidelberg Retina Tomograph II (HRT II-RCM)] were performed.ResultsAmong the 17 cases, three were classified as epithelial type, ten as stromal type, and four as endothelial type HSK. The average corneal sensitivities were 11.41 ± 9.46 mm in the affected eyes and 43.24 ± 12.2 mm in controls. Decreases in three parameters in the affected eyes (long nerve-fiber density, nerve-branch density, nerve thickness) were statistically significant compared with controls. Decreases in the three parameters were more remarkable in the epithelial and stromal types than in the endothelial type.ConclusionThe morphology of the corneal subepithelial nerve plexus may get gradually destroyed along with recurrent episodes of epithelial and stromal HSK. However, the destruction does not seem remarkable in the endothelial type of HSK, suggesting the possibility of a different route of virus recruitment in this type.

Role of periostin and interleukin-4 in recurrence of pterygia.

PURPOSE To identify the candidate genes for pterygia recurrence from a pterygia transcriptome and to analyze their transcriptional regulation and functional relationships. METHODS Transcriptional networks for pterygia recurrence were constructed using network analysis that was applied to 184 genes that showed a significant twofold change in the whole genome. Of the identified recurrence-related candidate genes in the major networks, periostin and IL-4 were analyzed for transcriptional relationships using pterygia-derived fibroblasts. Immunohistochemical analysis was used to study pterygia tissue. Effector candidate molecule for recurrence periostin was analyzed for cell adhesive function. RESULTS The pterygia transcriptome was divided into four major biological networks with high significance scores (P < 10(-17)). The classifier with the highest accuracy using the support vector machine algorithm was periostin, which was successfully linked to the network of cell cycle, connective tissue development and function, and cell morphology. Analyses using pterygia-derived fibroblasts showed that periostin was required for cell adhesion that was mediated by a presumed pterygia-related extracellular matrix protein, fibronectin. Periostin was found to be transcriptionally induced by IL-4. The IL-4-stimulated periostin induction was suppressed by MAP kinase/ERK kinase 1 inhibitor, indicating an involvement of the MAP kinase pathway. Pathologically, IL-4 was transcriptionally elevated in recurrent pterygia tissue and was localized to perivascular tissues and endothelial cells in the stroma of the subconjunctiva of pterygia. CONCLUSIONS Periostin is induced by IL-4 and is involved in the fibronectin-mediated pterygia fibroblast adhesion. These findings indicate that periostin probably promotes the recurrence of pterygia.

Classification of secondary corneal amyloidosis and involvement of lactoferrin.

PURPOSE To classify secondary corneal amyloidosis (SCA) by its clinical appearance, to analyze the demographics of the patients, and to determine the involvement of lactoferrin. DESIGN Retrospective, observational, noncomparative, multicenter study. PARTICIPANTS Twenty-nine eyes of 29 patients diagnosed with SCA by corneal specialists at 9 ophthalmologic institutions in Japan were studied. METHODS The clinical appearance of SCA was determined by slit-lamp biomicroscopy and was classified into 3 types. The demographics of the patients, for example, age, gender, and the duration of the basic disease (trichiasis, keratoconus, and unknown), were determined for each clinical type. Surgically excised tissues were stained with Congo red and antilactoferrin antibody. The postoperative prognosis also was determined. MAIN OUTCOME MEASURES Clinical appearance of the 3 types of SCA, along with the gender, age, and duration of the basic diseases were determined. RESULTS Classification of SCA into 3 types based on clinical appearance found 21 cases with gelatinous drop-like dystrophy (GDLD)-like appearance (GDLD type), 3 cases with lattice corneal dystrophy (LCD)-like appearance (LCD type), and 5 cases with the combined type. Patients with the GDLD type were younger (average age: 40.9 years for the GDLD type, 74.3 years for the LCD type, and 46.8 years for the combined type), predominantly women (85.7% for the GDLD type, 33.3% for the LCD type, and 60% for the combined type), and had the basic disease over a longer time (average duration: 22.1 years for the GDLD type, 14.0 for the LCD type, and 11.4 for the combined type). The distribution of the basic diseases (trichiasis vs. keratoconus vs. unknown) was not significantly different for each type. Surgical treatments, for example, phototherapeutic keratectomy, lamellar keratoplasty, and simple keratectomy, resulted in a good resolution in all surgically treated cases. One subject dropped out of the study. Spontaneous resolution was seen in one subject after epilation of the cilia. Amorphous materials in the excised tissues showed positive staining results by Congo red and by antilactoferrin antibody. CONCLUSIONS Secondary corneal amyloidosis can be classified into 3 clinical types based on its clinical appearance. Larger numbers of females and lactoferrin expression were seen in all 3 types. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.