Shozo Sakuragi

Tyrosinase Family Proteins Are Antigens Specific to Vogt-Koyanagi-Harada Disease

Vogt-Koyanagi-Harada (VKH) disease (and sympathetic ophthalmia) is an ocular inflammatory disease that is considered to be a cell-mediated autoimmune disease against melanocytes. The purpose of this study was to determine the Ags specific to VKH disease and to develop an animal model of VKH disease. We found that exposure of lymphocytes from patients with VKH disease to peptides (30-mer) derived from the tyrosinase family proteins led to significant proliferation of the lymphocytes. Immunization of these peptides into pigmented rats induced ocular and extraocular changes that highly resembled human VKH disease, and we suggest that an experimental VKH disease was induced in these rats. We conclude that VKH disease is an autoimmune disease against the tyrosinase family proteins.

Wistar Rat Palpebral Conjunctiva Contains More Slow-cycling Stem Cells That Have Larger Proliferative Capacity: Implication for Conjunctival Epithelial Homeostasis

PURPOSE To determine the location of conjunctival epithelial stem cells. METHODS Wistar rats received daily injection of 5-bromo-2-deoxyuridine (BrdU) at a dose of 5 mg/100 g for 2 weeks followed by a 1-month BrdU-free period before death. After the rats were sacrificed, the orbital contents and eyelids were exenterated en bloc, fixed in buffer formalin, and embedded in paraffin. To compare the proliferative capacity of ocular epithelial cells, 1.0% phorbol myristate (TPA) in petrolatum was topically applied once daily to both eyes of Wistar rats for 12 days. After 6, 12, 18, and 24 hours and 2, 4, 6, 8, 10, and 12 days of TPA treatment, rats were administered BrdU intraperitoneally 7 hours before they were sacrificed. The ocular epithelium was fixed and processed for immunochemistry, and the labeling index (LI) of every epithelial zone was determined. RESULTS Slow-cycling cells, detected as label-retaining cells (LRCs), were found in bulbar, fornical, and palpebral epithelia and mucocutaneous junctions, as well as in limbal epithelia. The greatest numbers of LRCs were identified in palpebral epithelium. Under normal situations, in conjunctiva the LI was lowest in palpebral epithelium (2.1 +/- 0.5) compared with bulbar (2.2 +/- 0.5), fornical (2.3 +/- 0.4) epithelia and mucocutaneous junction (3.4 +/- 0.9), respectively. In cornea, the LI was lowest in limbal epithelium (1.8 +/- 0.7) compared with central corneal epithelium (3.5 +/- 0.6). Twenty-four hours after TPA treatment, an 8.2-fold increase in the palpebral epithelial basal cell labeling index was noted compared with 4.7-fold, 5.7-fold, and 3.8-fold increases in bulbar, fornical, and mucocutaneous junction epithelial basal cell labeling indices, and a sevenfold increase in the limbal basal cell labeling indices compared with a 2.1-fold increase in the corneal basal cell labeling index, respectively. Limbal and palpebral epithelia maintained a significantly greater proliferative response (5.5-to 6.3-fold increase, respectively) during chronic stimulation than corneal, bulbar, fornical epithelia, and mucocutaneous junction (0.6- to 2.3-fold increase, respectively). CONCLUSIONS In Wistar rat conjunctiva, slow-cycling cells are primarily located in palpebral epithelium, which has greater proliferative capacity than other conjunctival epithelia. This finding means that, in the Wistar rat, the conjunctival epithelial stem cells are mainly located in palpebral epithelium. These data open new perspectives in ocular epithelial development and are relevant in conjunctival wound repair.

Application of revised diagnostic criteria for Vogt-Koyanagi-Harada disease in Japanese patients

PurposeTo determine whether the International Revised Diagnostic Criteria for Vogt-Koyanagi-Harada (VKH) disease is applicable to Japanese patients at the early stage of VKH disease.MethodsThe medical records of 49 patients with VKH disease were studied. The patients had been examined at the Akita University School of Medicine Hospital between 1989 and 2001, and their mean age was 47.6 years with a range of 15 to 69 years. In our study, the patients were divided into two groups; an Early Group, which consisted of those who were examined within 1 month of the onset of signs or symptoms, and a Late Group, which consisted of those who were examined more than 1 month after the onset of signs or symptoms. The initial diagnosis was based on findings by ophthalmoscopy, fluorescein angiography, pleocytosis of cerebrospinal fluid, and genotyping of human leukocyte antigen (HLA). The final diagnosis was based on the presence of these findings in addition to skin and internal ear manifestations of VKH disease.ResultsIn our retrospective study, 41 of the 49 patients were placed in the Early Group, and 8 were placed in the Late Group. When classified by the Revised Diagnostic Criteria, 39 of the 41 in the Early Group would have been diagnosed with incomplete VKH, and the other 2 patients would have been diagnosed as not having VKH disease. None of the patients would have been diagnosed as having complete VKH disease, even at 2 weeks after the onset of symptoms or signs. At the final examination, 35 patients would still have been classified as having incomplete VKH disease, and only 6 patients would have been classified as having complete VKH disease according to the Revised Diagnostic Criteria. In the Late Group, all of the patients would have been diagnosed as having incomplete VKH at 2 weeks after the onset of any signs or symptoms. At the final examination, two of eight patients would still have been diagnosed as having incomplete VKH, and the other six would have been diagnosed as having complete VKH. The skin manifestations always appeared later than the other alterations, with an average of 143.5 days from disease onset to detection.ConclusionsAlthough the Revised Diagnostic Criteria are effective for making the final diagnosis of VKH disease, they are not an effective tool for diagnosis at the very early stage of VKH disease in Japanese patients.

Analysis of the human, bovine and rat 33-kDa proteins and cDNA in retina and pineal gland.

A monoclonal antibody (mAb) was produced against a bovine retinal 33-kDa protein. Several clones of 33-kDa protein were isolated from each library of cDNA from human, bovine and rat retinas and rat pineal gland by mAb screening and by hybridization with cDNA probes. Each of the four cDNA sequences was determined and amino acid (aa) sequences were deduced from the nucleotide sequences. The latter were nearly identical in rat retina and rat pineal gland (99.6%) and were similar in human, bovine and rat retina (more than 87%). Each of these cDNAs had one long ORF and encoded 245 or 246 aa. The deduced aa sequences in rat retina and rat pineal gland were virtually identical and the sequences in human, bovine and rat retina were highly homologous (more than 88%). The predicted Mr for each of these proteins was 28,246 in the human, 28,176 in bovine, 28,143 in rat retina, and 28,129 in rat pineal gland. Each of the sequences has a putative site for phosphorylation by A kinase; we have confirmed that the putative site is Ser73. These results show that the 33-kDa proteins in the retina and pineal gland have the same sequences and the same phosphorylation site and suggest that the functional role of this protein is the same in the retina and pineal gland.

Decreased tear lactoferrin concentration in patients with chronic hepatitis C

BACKGROUND/AIMS Decreased tear volume in patients with chronic hepatitis C has been reported in the literature. Lactoferrin is abundantly present in human tears, the main source of which is the acini of the lacrimal glands. In this study tear lactoferrin levels were measured to investigate the dry eye condition of patients with chronic hepatitis C. METHODS Lactoferrin in tears/fluid was measured by a radial immunodiffusion assay in 42 patients with chronic hepatitis C. The rate of lacrimal secretion was determined by the cotton thread test. Rose bengal staining of the ocular surface was also performed. RESULTS Only three patients out of 42 complained of dry eye sensation and, in 31 patients, six showed positive results on the rose bengal staining test of the ocular surface. The lactoferrin concentration of tear fluid in the chronic hepatitis C group (1.42 (SD 0.56) mg/ml) was significantly lower than in the control group (1.90 (0.62) mg/ml; p <0.00048). The cotton thread test results in the chronic hepatitis C group (12.9 (5.5) mm) were significantly lower than in the control group (17.9 (5.3) mm; p<0.00048). Also, in the chronic hepatitis C group, tear lactoferrin concentration correlated with the results of the cotton thread test (r = 0.35, p<0.05). CONCLUSION Chronic hepatitis C patients showed both decreased tear volume, and decreased tear lactoferrin concentration. These findings suggest that there may be dysfunction of the lacrimal glands in patients with chronic hepatitis C, which may account for the mild dry eye.

Pathogenesis of Vogt-Koyanagi-Harada disease.

Vogt-Koyanagi-Harada (VKH) disease is an ocular inflammatory disease manifested as an aseptic meningitis with vitiligo and internal ear inflammation. The results of earlier studies indicated that VKH disease was probably a cell-mediated autoimmune disorder acting against melanocytes. The melanocytes of all organs are affected in VKH disease. The lymphocytes of patients with VKH disease attach to and attack the melanocytes. These lymphocytes also proliferate when challenged by antigens from melanocytes and are cytotoxic to the melanocytes in vitro. In human studies, total proteins from the melanocytes or crude soluble or insoluble fractions have been used as antigens. However, an analysis of the specific antigens for the disease has not been performed.

Suppression of Experimental Autoimmune Uveoretinitis by Dietary Calorie Restriction

To investigate the inhibitory effect of dietary calorie restriction on experimental autoimmune uveoretinitis (EAU) in rats, and its mechanism. Lewis rats were maintained on a 50% calorie-restricted diet for 2 months or 6 months. The control group was maintained on a 90% ad libitum intake for the same length of time. Experimental autoimmune uveoretinitis was elicited in both groups by immunization with an inter-photoreceptor retinoid-binding protein or its peptide. Rats in both groups were examined clinically, histopathologically, and immunologically. The severity of EAU was milder in the restricted diet group than in the control group. In EAU rats, production of interferon-gamma (IFN-gamma) in eyes and of IFN-gamma and tumor necrosis factor-alpha in draining lymph node cells was significantly lower in the restricted diet group than in the control group. Our results indicate that a calorie-restricted diet suppresses the development of EAU. The suppressed Th1-dependent immunological response is one of the reasons for the mildness of EAU in the calorie-restricted diet group of rats.

Intraocular pressure during pneumatic retinopexy.

BACKGROUND AND OBJECTIVE A simplified method of monitoring intraocular pressure (IOP) during pneumatic retinopexy was devised and used clinically to decrease the risk of complications associated with unnecessary IOP elevation. PATIENTS AND METHODS Intraoperative IOP and IOP immediately after sulfur hexafluoride (SF6) injection were monitored during pneumatic retinopexy in 22 eyes with rhegmatogenous retinal detachment. Intraoperative IOP was measured with a sphygmomanometer connected to the side of a gas-filled syringe. RESULTS Intraoperative IOP and IOP immediately after gas injection were 97.2 +/- 28.5 (mean +/- SD) and 46.5 +/- 19.6 mm Hg, respectively. Two eyes temporarily lost light perception immediately after the gas injection. The intraoperative IOP and the IOP immediately after the gas injection were 130 and 80 mm Hg, respectively, in one eye, and 180 and 90 mm Hg, respectively, in the other. CONCLUSIONS In general, to ensure effective and safe results of pneumatic retinopexy, the authors recommend that the gas be injected either to approximately 90 mm Hg of intraoperative IOP or to the maximum acceptable volume (2.0 to 2.5 ml of 100% SF6).

WYBURN-MASON SYNDROME: REPORT OF A RARE CASE WITH COMPUTED TOMOGRAPHY AND ANGIOGRAPHIC EVALUATIONS

A case of Wyburn-Mason syndrome comprising unilateral retinocephalic vascular malformations is reported in a 7-year-old girl presenting with proptosis of the left eye associated with injection of the bulbar conjunctiva. This rare congenital vascular disorder is reviewed in the literature, and in particular the neuroradiological findings are detailed in the presented case.

Analysis of uveitogenic sites in phosducin molecule.

PURPOSE Phosducin, a retinal photoreceptor protein, induces experimental autoimmune uveitis (EAU). In this study, we attempted to determine the numbers of uveitogenic sites in phosducin using synthetic peptides. METHODS Antigen peptides were synthesized according to the amino acid sequence of the rat-derived phosducin with a peptide-synthesizer and purified by reversed-phase HPLC. First, 13 peptides covering the entire sequence of phosducin were synthesized, and each was injected into the hind footpad of Lewis rats for immunization, and induction of EAU was examined clinically and histologically. Next, peptides that appeared to contain sequences of a uveitogenic site were newly synthesized and examined clinically and immunologically. RESULTS Of the 13 peptides used in the first immunization, 7 induced inflammation. Similar to other EAU antigens, clinical changes began with fibrin deposition in the anterior segment and posterior synechia, followed by posterior chamber hypopyon. Histologically, inflammation was observed mainly in the outer segment of photoreceptor cells and outer nuclear layer, and serous retinal detachment was found in cases of severe inflammation. Infiltration of inflammatory cells in the pineal gland was also observed. In experiments designed to further specify the uveitogenic sites, the presence of inflammation-inducing sequences was inferred for amino acid sequences 1-20, 23-37, 79-91, 127-142 and 198-212. The rats immunized with these peptides also exhibited high value on lymphocyte proliferation assay. CONCLUSION Phosducin has 5 uveitogenic sites. Among others, one of them has potent and others weak uveitogenicity.