Kenji Shiratori

Effects of fucoxanthin on lipopolysaccharide-induced inflammation in vitro and in vivo

The aim of the present study was to investigate the efficacy of fucoxanthin on endotoxin-induced uveitis (EIU) in rats. The effects of fucoxanthin on endotoxin-induced leucocyte and protein infiltration, nitric oxide (NO), prostaglandin (PG)-E2 and tumour necrosis factor (TNF)-alpha concentrations in rat aqueous humour, as well as on the cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) protein expression in a mouse macrophage cell line (RAW 264.7 cells) were studied. EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). Immediately after the LPS injection, either 0.1, 1 or 10mgkg(-1) of fucoxanthin was injected intravenously. The aqueous humour was collected 24hr later from both eyes, and both the number of cells infiltrating into the aqueous humour and the aqueous humour protein concentration were measured. The levels of PGE2, NO and TNF-alpha were determined by enzyme-linked immunosorbent assay. The RAW 264.7 cells were pretreated with various concentrations of fucoxanthin for 24hr and subsequently incubated with LPS for 24hr. COX-2 and iNOS protein expression was analysed by the Western blotting method. Levels of PGE2, NO and TNF-alpha production were determined. Fucoxanthin suppressed the development of EIU in a dose-dependent fashion. Treatment with fucoxanthin resulted in a reduction in PGE2, NO and TNF-alpha concentrations in the aqueous humour. The expression of COX and iNOS protein in the fucoxanthin treated RAW264.7 cells decreased significantly compared to that the LPS group. It also significantly reduced the concentration of PGE2, NO and TNF-alpha production in the medium of cells. The present result indicate fucoxanthin suppresses the inflammation of EIU by blocking the iNOS and COX-2 protein expression and its anti-inflammatory effect on eye is comparable with the effect of predinisolone used in similar doses.

Five New Genome Types of Adenovirus Type 37 Caused Epidemic Keratoconjunctivitis in Sapporo, Japan, for More Than 10 Years

ABSTRACT Human adenovirus type 37 (HAdV-37) is a major cause of epidemic keratoconjunctivitis and has recently been the largest causative agent of keratoconjunctivitis in Japan. To investigate the genetic characteristics of HAdV-37 strains isolated in Sapporo, we analyzed the genome types and genetic relationships of 51 strains isolated there from 1990 through 2001. By using DNA restriction analysis, eight genome types (HAdV-37/D1, HAdV-37/D3, and HAdV-37/D6 to HAdV-37/D11) were identified, including five new ones. The restriction fragments of these genome types shared more than 95% identity with those of the prototype strain. By DNA sequence analysis, five and three single nucleotide substitutions, respectively, were found in partial sequences of the hexon and fiber genes. The combinations of mutations resulted in four hexon and fiber types (hx1 to hx4 and f1 to f4) and six hexon/fiber pairs (hx1/f1, hx2/f1, hx1/f2, hx1/f3, hx3/f4, and hx4/f4). The six pairs correlated well with certain genome types. In all three epidemics of keratoconjunctivitis to strike Sapporo in the past 12 years, specific genome types and fiber types were usually isolated: in the first epidemic, HAdV-37/D1 (f1) and HAdV-37/D3 (f1); in the second, HAdV-37/D6 (f2) and HAdV-37/D8 (f3); and in the third, HAdV-37/D10 (f4) and HAdV-37/D11 (f4). We conclude that mutations in the adenovirus genome occurred chronologically and that certain mutations were correlated with the epidemics of adenoviral keratoconjunctivitis.

Inhibition of nuclear factor-kappa B activation attenuates hydrogen peroxide-induced cytotoxicity in human lens epithelial cells

Aims: Hydrogen peroxide (H2O2) is the major oxidant involved in cataract formation. Lens epithelial cells have been suggested to be the first site of oxidative damage. The authors investigated the relationship between H2O2-induced cytotoxicity and activation of nuclear factor kappa B (NF-κB) in human lens epithelial (HLE) cells. Methods: HLE B-3 cells were stimulated by various concentrations of H2O2 in the presence or absence of pyrrolidine dithiocarbamate (PDTC), a potent inhibitor of NF-κB. H2O2-induced cytotoxicity was measured by lactate dehydrogenase cytotoxicity assay. Translocation of NF-κB was examined by Western blot and immunocytochemistry using anti-p65 antibody. Results: H2O2-induced cytotoxicity increased in a concentration-dependent manner. PDTC treatment significantly suppressed the cytotoxicity induced by H2O2. After stimulated with H2O2, NF-κB was found translocated from cytoplasm into the nuclei. PDTC treatment also inhibited the translocation of NF-κB. Conclusions: NF-κB signal pathway may be important in the development of H2O2-induced damage in HLE cells that is involved in cataractogenesis.

Activation of nuclear factor-kappa B in the conjunctiva with the epithelial scraping of the mouse cornea and human epidemic keratoconjunctivitis

Aim: To examine the expression of p65, one of nuclear factor-kappa B (NF-κB), in the conjunctival epithelium of the C57Bl6 mouse and a patient with epidemic keratoconjunctivitis (EKC). Methods: Normal and epithelial scraped cornea obtained 6 hours after the injury were processed for paraffin section. Samples of a normal and an EKC conjunctival epithelium were obtained using impression cytology. Both samples were analysed by immunocytochemistry using anti-p65 antibody. Results: Immunocytochemistry with the anti-NF-κB p65 antibody revealed that p65 was localised in the cytoplasm of the conjunctival epithelium in the C57Bl6 mouse without the treatment. Six hours after the scraping of the cornea, p65 protein was expressed in the nuclei of the conjunctival epithelium. p65 was localised in the cytoplasm of the conjunctival epithelium in the human normal eye. p65 protein was expressed in the nuclei of the conjunctival epithelial cells in the EKC patient. Conclusion: These findings suggest that NF-κB was activated in the conjunctiva in the epithelial scraping of the mouse cornea and in human EKC.

Disappearance of p27(KIP1) and increase in proliferation of the lens cells after extraction of most of the fiber cells of the lens.

Purpose: Proliferation of the lens epithelial cells is involved in the fibrotic changes of lens capsules after cataract extraction. However, the mechanisms of the proliferation of the lens epithelial cells are largely unknown. The purpose of this study was to examine the correlation between the expression of p27(KIP1) and cell proliferation in the lens cells after the extraction of lens fiber cells. Methods: At embryonic days (E) 14 and 18, the C57Bl6 mice were anesthetized and the embryos were surgically removed. The eyes were dissected from these embryos and also from mice 12 weeks after birth. The 12-week-old mice were anesthetized, and then the lens fiber cells were extracted. Normal eyes at E14 and 18 and eyes fixed at 15 min and 48 hr after the extraction of the lens fiber cells were analyzed using immunohistochemistry with anti-p27(KIP1), p57(KIP2), and phosphorylated extracellular signal-regulated kinase (phospho-ERK) 1/2 antibodies, and cells in the S phase of the cell cycle were also examined using anti-bromodeoxyuridine (BrdU) antibody. Results: p27(KIP1) and p57(KIP2)-positive cells were present in the equatorial region of E14 mice. At E18, many lens fiber cells showed nuclear immunoreactivity for p27(KIP1), whereas a small number of cells were positive for p57(KIP2) in the equatorial region. At 12 weeks of age, all nuclei of the lens epithelial cells as well as lens fiber cells showed nuclear immunoreactivity for p27(KIP1). In contrast, p57(KIP2) and phospho-ERK1/2 were not expressed in the lens cells. At 15 min after the extraction of lens fiber cells, phospho-ERK1/2 as well as p27(KIP1) were detected in the lens cells. At 48 hr after the extraction of the lens fiber cells, a few p27(KIP1)-positive nuclei were observed in the equatorial region of the lens capsule. In contrast, many lens cells showed nuclear immunoreactivity for BrdU. Conclusions: These findings suggest that degradation of p27(KIP1) mediated by phosphorylation of ERK 1/2 is correlated with proliferation of the epithelial cells after the extraction of the lens fiber cells.

Phosphorylation of p27(KIP1) in the mitotic cells of the corneal epithelium.

Purpose: The mechanism in regulation of the cell cycle and proliferation of corneal epithelium in the homeostatic ocular surface remains unclear. The aim of this study is to examine the expression of p27(KIP1) and its phosphorylation in corneal epithelium. Methods: The eyes of C57BL/6 mice (7 weeks old) were enucleated. Formalin-fixed and paraffin-embedded tissue sections were examined using immunohistochemistry with anti-p27(KIP1), threonine 187 phosphorylated p27(KIP1) (T187-phospho-p27), and phosphorylated Histon H3 (pHiston H3) antibodies. Anti-T187-phospho-p27 and anti-pHiston H3 polyclonal antibodies were used for parallel immunofluorescent staining. Results: pHiston H3–immunopositive cells were noted in basal cells of the corneal epithelium. At high magnification of DAPI nuclear staining, mitotic and non-mitotic cells were observed in corneal basal layer. p27(KIP1)-positive nuclei were detected in corneal basal cells, where non-mitotic basal cells were located. In contrast, mitotic cells showed under detectable level on p27(KIP1) immunoreactivity. Immunoreactivity for T187-phospho-p27 was detected in basal cells of the corneal epithelium. At high magnification, it was confirmed that the immunopositive cells were mitotic cells. Immunoreactivity of T187-phospho-p27 as well as pHiston H3 was localized in the same corneal basal cells using double-staining immunohistochemistry. Conclusions: These results suggested that degradation of p27(KIP1) regulates progression into mitosis in corneal basal cells.

Expression of thymidine phosphorylase in choroidal malignant melanoma associated with neovascular glaucoma.

Reported herein is a case of 62‐year‐old man who complained of blurred vision and ocular pain in his right eye. The patient was diagnosed with choroidal melanoma complicated by neovascular glaucoma (NVG) and total retinal detachment, and he underwent enucleation of the eye. The isolated tumor was 2.5 × 2.5 cm in size. It was accompanied by intratumoral calcification, and consisted of epithelioid and spindle melanoma cells. There were a variety of microvessels in the stroma of the iris. The expression of thymidine phosphorylase (dThdPase), an angiogenic factor, was examined immunohistochemically. Cytoplasmic immunoreactivity for dThdPase was more prominent in the epithelioid cells than in spindle tumor cells. Another case of choroidal melanoma without NVG had less marked immunoreactivity. These results suggest that the production of dThdPase by melanoma cells correlates with the pathogenesis of NVG.