Expression profiles of NOS isoforms in gingiva of nNOS knockout mice.

Nitric oxide is a gaseous molecule associated with many distinct physiological functions, and is derived from L-arginine catalyzed by nitric oxide synthase (NOS). Nitric oxide synthase has 3 isoforms: nNOS, iNOS and eNOS. Although these NOS isoforms are believed to play an important role in gingival tissue, little information is available on their morphological dynamics. The aim of this study was to investigate the profiles of NOS isoforms in deficiency of nNOS in gingiva of mice. Twelve male (6 normal (C57BL/6) and 6 nNOS knockout) mice were used. All mice were 5-week-old, weighing approximately 20-25 g each. After sacrifice, the jaws of the mice were removed by mechanical means and specimens analyzed by histology, in situ hybridization and immunohistochemistry. Immunohistochemical observation revealed positive staining for iNOS and eNOS, especially in lamina propria. Similar results in the mRNA expression levels were shown by in situ hybridization analysis. It may suggest that iNOS and eNOS compensated nNOS deficiency in the gingiva of nNOS knockout mice.

Pterygium and dry eye.

PURPOSE: To assess the correlation between dry eye and pterygium. METHODS: Tear breakup time (BUT), Schirmer test with/without anesthesia and tear function index were evaluated in both eyes of patients with unilateral pterygium. RESULTS: Tear BUT, was shortened significantly in the eye with pterygium. The Schirmer test with anesthesia was shortened and the tear function index was decreased in the eye with pterygium with marginal significance. CONCLUSION: There is a correlation between pterygium formation and shortened BUT. Unstable tear film may contribute to the initiation of pterygium.

Changes in ocular surface caused by antiglaucomatous eyedrops: prospective, randomised study for the comparison of 0.5% timolol v 0. 12% unoprostone.

AIM: To study changes induced in ocular surface epithelia and the tear film by antiglaucomatous eyedrops. A beta blocker (0.5% timolol) and a novel prostaglandin F(2alpha) metabolite related drug (0.12% unoprostone) were examined in a prospective, randomised fashion. METHODS: 40 patients were randomly assigned to use either 0. 5% timolol (timolol group) or 0.12% unoprostone eyedrops (unoprostone group) twice a day for 24 weeks. In addition to routine ocular examinations, corneal epithelial integrity (vital staining tests, tear film break up time (BUT), anterior fluorometry, specular microscopy) and tear function (Schirmer's test, cotton thread test, tear clearance test (TCT)) were examined before and after the treatment. RESULTS: Both eyedrops caused significant reduction in intraocular pressure from the baseline levels. No significant changes were noted in corneal integrity in both groups, except a decrease in BUT at 20 weeks in the timolol group. The timolol group demonstrated significant decreases in Schirmer's test, tear clearance test, and tear function index (Schirmer's test value multiplied by clearance test); however, no such changes were noted in the unoprostone group. CONCLUSION: While unoprostone eyedrops caused no adverse effects on the corneal epithelial integrity and tear function, timolol caused significant impairments in tear production and turnover.

Trabeculectomy with mitomycin C for post-keratoplasty glaucoma.

AIM: To investigate the effect of trabeculectomy with and without mitomycin C in post-keratoplasty glaucoma. METHODS: A retrospective study was performed on patients who underwent trabeculectomy for glaucoma after penetrating keratoplasty. 34 eyes of 32 patients were included in this study. 26 eyes received trabeculectomy with mitomycin C and eight eyes without mitomycin C. The procedure was deemed successful if the intraocular pressure was maintained below 21 mm Hg with or without use of additional antiglaucoma medication (mean follow up time 22.3 (SD 10.3) months). RESULTS: At the last examination trabeculectomy was successful in 19 of 26 eyes (73.0%) with mitomycin C (+) and two of eight (25.0%) without (p=0.0219). When the prognosis was analysed by Kaplan-Meier curve, the mitomycin C (+) group showed a better prognosis (p=0.0182). Mean intraocular pressure and average number of glaucoma medications improved in the group with mitomycin C without severe side effects on the graft. Graft rejection after trabeculectomy was seen in two eyes in the mitomycin C group. Final graft clarity rate was 69.2% (18/26) in the mitomycin C (+) group and 37.5% (3/8) in the mitomycin C (-) group. Complications such as persistent epithelial defect, cystoid macular oedema, choroidal detachment, leakage from bleb were seen in four eyes in the mitomycin C (+) group and in one eye in the mitomycin C (-) group. CONCLUSIONS: Trabeculectomy with mitomycin C showed better results for glaucoma following keratoplasty.

Telomerase activity and p53 expression in pterygia.

PURPOSE: To investigate tolomerase activity and p53 expression in pterygial tissue. METHODS: Pterygia tissue was obtained during excisional surgery fr om 35 eyes of 35 patients, and superior bulbar conjunctival tissue from the same eye was also sampled as control when possible. Fluorescence telomeric repeat amplification protocol was used to measure telomerase activity in whole pterygium samples from 9 cases and in the epithelium and stroma of pterygium from another 10 cases. p53 protein content was measured by enzyme-linked immunosorbent assay (ELISA) in tissues obtained from 7 eyes, as well as in epithelial cell suspensions collected by brush cytology in 8 eyes. Six samples were also analyzed for UV-specific mutations in the p53 gene by the single-strand conformation polymorphism technique and DNA sequencing. A conjunctival epithelial cell line was irradiated with sublethal levels of UV-B to investigate whether telomerase activity can be induced in vitro. RESULTS: In all, 63% of pterygia samples demonstrated telomerase activity, whereas all 10 paired conjunctival control samples were negative (P = 0.05, chi-square test). Of the 10 samples in which telomerase activity was measured separately in the epithelium and stroma of pterygia, 5 samples were positive in the epithelium, only 1 of which had activity in the stroma. Average telomerase activity in positive samples was 18.44 +/- 8.77 U/microg protein, compared with telomerase activity measured in a carcinoma in situ patient (33.73 U/microg), and in an immortalized conjunctival epithelial cell line (50.72 +/- 15.55 U/microg). Telomerase activity was not upregulated in this cell line by UV-B exposure. All 6 pterygia samples tested for p53 mutations did not reveal the UV-specific mutations in exons 5, 6, 7, or 8. No statistical significance was observed in the pterygium or conjunctiva p53 protein levels in epithelial cells collected by brush cytology, while p53 protein level was lower in pterygia when measured in whole tissue samples. CONCLUSIONS: Telomerase activity was detected in some pterygia, mostly in the epithelium. Pterygia was not associated with an increase in epithelial p53 protein content measured by ELISA.

Effect of cyclosporine on anterior chamber-associated immune deviation with retinal transplantation.

PURPOSE: To determine whether immunosuppression using cyclosporine interferes with anterior chamber associated immune deviation (ACAID) and can promote survival of retinal allografts in the anterior chamber. METHODS: Neonatal neural retinas of C57BL/6 mice or ovalbumin were injected into the anterior chamber of BALB/c adult mice. In the test group recipients were injected with cyclosporine (10 mg/kg per day) from day 0 to 11 or from day 11 to 34 after implantation. At 12 and 35 days after transplantation, lymphocytes from the test group were injected into naive BALB/c mice to assay for the presence of suppressor T cells (adoptive transfer). The fate of the retinal grafts was determined by histologic examination at day 12 and 35. To evaluate the potential neurotoxic effects of cyclosporine in the absence of immune rejection mechanisms, cyclosporine was given to SCID mice during days 11 to 34 after syngeneic neonatal neural retinal grafts were placed in the anterior chamber. RESULTS: At 12 days after transplantation, spleens of both cyclosporine-treated and control mice contained suppressor cells against donor alloantigens. The retinal grafts in the anterior chamber of both groups of mice were fully developed and well differentiated. The same duration of administration of cyclosporine did not interfere with the production of efferent suppressor cells after inoculation of ovalbumin into the anterior chamber. At 35 days after transplantation, only spleen cells from the cyclosporine-treated group showed the capacity to suppress donor-specific delayed hypersensitivity. However, allografts in the cyclosporine group had deteriorated by 35 days in a fashion similar to the control group. Syngeneic grafts in SCID mice showed differentiated retinal layers 35 days after transplantation. CONCLUSIONS: Cyclosporine treatment does not interfere with the ability of allogeneic neonatal retinal grafts to induce anterior chamber associated immune deviation when placed in the anterior chamber, nor does prolonged treatment with this drug interfere with the persistence of allospecific suppressor cells for 35 days after the graft. Because 35-day grafts of cyclosporine-treated mice display histologic evidence of graft failure similar to grafts placed in the anterior chamber of untreated mice, graft destruction is either the result of immune effector mechanisms not inhibited by cyclosporine, or the consequence of nonimmunologic factors.

Immunity and immune privilege elicited by cultured retinal pigment epithelial cell transplants.

PURPOSE: To determine whether cultured retinal pigment epithelial (RPE) cells implanted in the subconjunctival space induce an immune response against autoantigens and whether an active downregulation is achieved by RPE grafts placed in the anterior chamber and within the subretinal space. METHODS: Cultured RPE cells from eyes of newborn C57BL/6 mice were implanted in the subconjunctival space, the anterior chamber, or the subretinal space of eyes of adult C57BL/6 mice. At postimplantation day 12, the recipients were evaluated for RPE-specific delayed hypersensitivity and examined clinically and histologically for evidence of rejection. To facilitate their identification, RPE cells were labeled with 5-bromodeoxyuridine, before intraocular transplantation. RESULTS: Cultured RPE cells implanted in the subconjunctival space of syngeneic mice elicited an intense RPE-specific delayed hypersensitivity associated with a vehement cellular infiltration of the graft when examined at postimplantation day 12. By contrast, grafts in the anterior chamber and subretinal space displayed no evidence of rejection, and their recipients failed to display RPE-specific delayed hypersensitivity. Additionally, the spleens of these mice contained regulatory T cells that suppressed RPE-specific delayed hypersensitivity in naive syngeneic recipients. CONCLUSIONS: Cultured RPE cells can induce an immune response against autoantigens. Implantation of RPE cells in immune-privileged sites of the eye induces a deviant immune response that is associated with spleen cells that suppress RPE-specific delayed hypersensitivity and autoimmune rejection.

Suppression of actively induced experimental autoimmune uveoretinitis by CD4+ T cells.

BACKGROUND: Helper/inducer T cells that exert an inhibitory effect on disease induction have been recently found in many experimental models. In order to clarify the mechanisms of spontaneous remission of experimental autoimmune uveoretinitis (EAU), we investigated the inhibitory effect and the phenotype of the post-recovery suppressor cells. METHODS: In a series of experiments, we separated spleen cells of rats that had recovered from EAU. Three groups of spleen cells, CD4+ T, CD8+ T and B cells, were each adoptively transferred into naive syngeneic rats before active immunization with retinal soluble antigen (S-Ag) and Freund's complete adjuvant or passive immunization with uveitogenic T cells from donor rats. Inflammation was examined clinically and histologically. RESULTS: The development of EAU could be significantly prevented by adoptive transfer of CD4+ T cells, whereas CD8+ T cells could not suppress the onset. However, post-recovery CD4+ T cells failed to inhibit EAU induced by passive immunization with uveitogenic T cells. CONCLUSION: These findings indicate that CD4+ post-recovery (suppressor) T cells may play an important role in the remission of EAU.

Prevalence of HTLV-I-associated uveitis in the Kanto Plain, Japan.

BACKGROUND: Seroprevalence of antibody to human T-lymphotropic virus type I (HTLV-I) is high in the island of Kyushu, Japan. Reports on the etiological analysis of HTLV-I in patients with uveitis primarily document cases in this island. We studied the seroprevalence of HTLV-I at the Department of Ophthalmology in Yokohama City University Hospital and in Odawara Municipal Hospital, which are in the Kanto Plain on the island of Honshu, Japan. METHODS: The subjects were 741 patients who visited the two hospitals. The presence of serum antibodies against HTLV-I was assessed using the method of particle agglutination. RESULTS: Of 454 patients with nonuveitic ocular diseases, 9 (1.98%) were seropositive. Of 143 patients with definite diagnosis of uveitis, 1 (0.70%) was seropositive. Of 144 patients with non-specific uveitis (etiology undefined), 8 (5.56%) were seropositive. Thus, the prevalence of serum antibodies to HTLV-I was higher in patients with non-specific uveitis than in patients with specific uveitis or nonuveitic ocular diseases. Common ocular symptoms of 8 HTLV-I-infected patients with non-specific uveitis were compatible with the clinical features of uveitis described as HTLV-I-associated uveitis (HAU). CONCLUSION: It is important to suspect HAU in patients with uveitis of unknown etiology, even outside known areas of prevalence.

Unconventional rejection of neural retinal allografts implanted into the immunologically privileged site of the eye.

The unique feature of neural transplantation in the central nervous system is that the graft is derived from and implanted into an immunologically privileged site. The eye, as a part of the central nervous system, normally maintains an immunosuppressive microenvironment in which alloantigens induce an active down-regulation of specific delayed hypersensitivity. To determine whether neural retinal allografts are eventually rejected and, if so, what type of immunity is associated with rejection, we implanted allogeneic and syngeneic newborn neural retinal grafts into the anterior chamber of the eyes of immune-competent mice. In addition, similar allografts were implanted into severe combined immune-deficient (SCID) mice. The fate of these grafts was determined by clinical and histological examination. At post-implantation day 12, all allogeneic and syngeneic grafts survived comparably well with no evidence of inflammation. At post-implantation day 35, the syngeneic grafts in the immune-competent mice and the allogeneic grafts in the SCID mice continued to thrive, whereas the allografts in the immune-competent mice were remarkably reduced in size and had lost the organization of their retinal cell layers. Interestingly, these grafts' deterioration occurred with no obvious cellular infiltration. When systemic graft-specific immunity was examined, it was found that delayed hypersensitivity was impaired at post-implantation day 12 in allograft recipients. However, by post-implantation 35 day when deterioration was detected in these grafts, suppression of immunity was replaced by vigorous delayed hypersensitivity. These results suggest that intraocular retinal allografts eventually succumb to rejection and that rejection is correlated with the emergence of donor-specific delayed hypersensitivity. The possible relationships of atypical, chronic rejection of intraocular neural retinal allografts to emergent delayed hypersensitivity are discussed.

FK506 treatment of noninfectious uveitis.

PURPOSE: We studied the clinical effects of the immunosuppressive agent FK506 in patients with noninfectious uveitis. METHODS: This study was designed as a multicenter open clinical trial in Japan. Sixteen patients with noninfectious uveitis who had visited the Uveitis Survey Clinic of the Yokohama City University Hospital were given FK506. Eight had Behçet's disease; five, Vogt-Koyanagi-Harada syndrome; one, sympathetic ophthalmia; one, retinal vasculitis; and one, sarcoidosis. In patients with Behçet's disease, ocular attack score before and after therapy was compared to judge clinical status. For the other diseases, the ocular inflammatory symptoms were observed after the initiation of FK506 treatment. All patients underwent blood and urine examinations, electrocardiography, and chest x-rays before and after FK506 treatment. RESULTS: Of the patients with Behçet's disease, five improved, one remained unchanged, one deteriorated, and the status of one could not be determined. Of the patients with Vogt-Koyanagi-Harada syndrome, four improved, and one remained unchanged. The patient with sympathetic ophthalmia improved, the patient with retinal vasculitis remained unchanged, and the status of the patient with sarcoidosis could not be determined. Major adverse effects were sensations of warmth, hypomagnesemia, renal dysfunction, glucose intolerance, nausea, vomiting, and disorders of the central nervous system. All adverse effects disappeared or improved when FK506 was stopped or when the dosage was decreased. Renal dysfunction and glucose intolerance appeared when the blood level of FK506 was high. CONCLUSIONS: FK506 was effective in patients with uveitis, but it is important to monitor the occurrence of adverse effects.

The role of tumor necrosis factor-alpha in the induction of experimental autoimmune uveoretinitis in mice.

PURPOSE: To examine the role of tumor necrosis factor-alpha (TNF) in the induction of experimental autoimmune uveoretinitis (EAU), the authors compared in vivo TNF production in EAU-susceptible and EAU-resistant strains of congenic mice and attempted to determine whether TNF can enhance the inflammation in EAU by injection of TNF at the time of immunization. METHODS: The production of TNF after stimulation with lipopolysaccharide (LPS) in B10.A and B10.D2 mice was measured by bioassay with L929 cells. The incidence and severity of EAU was compared between the group immunized with conventional methods and the group that alternatively received additional subcutaneous injection of recombinant human TNF (rhTNF) at the time of immunization in both B10.A and B10.D2 mice. RESULTS: Serum concentration of TNF after stimulation with 50 micrograms of LPS was significantly higher in B10.A mice than in B10.D2 mice. The incidence of EAU in B10.A mice was 60%, but it was only 10% in B10.D2 mice using the conventional method. Extremely severe chorioretinitis and iridocyclitis occurred in B10.A mice with the injection of rhTNF at the time of immunization for EAU. The incidence of EAU in B10.A and B10.D2 rose to 100% and 40%, respectively. When administered alone, rhTNF did not cause any inflammatory change in the uvea. CONCLUSIONS: The rhTNF was found to enhance the immune response to interphotoreceptor retinoid-binding protein in mice. These results suggest that susceptibility to EAU is in some part mediated by the ability of mice to produce TNF.