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Purpose: To assess the effects of histocompatibility Y (H-Y) antigen matching on the rate of corneal allograft rejection after penetrating keratoplasty (PKP)., Methods: We retrospectively investigated the graft survival rate and rejection-free graft survival rate after PKP in 396 eyes. The compatible combinations of H-Y antigen included male donors and male recipients (n = 135), female donors and male recipients (n = 107), and female donors and female recipients (n = 60). Incompatible combination was from male donors and female recipients (n = 94). The eyes were classified into two groups--high-risk (168 eyes) and low-risk (228 eyes)--depending on the degree of vascularisation in the recipient corneas or a history of previous allograft rejection. Data were analysed using the Kaplan-Meier life table method, the log-rank test and the Cox proportional hazards model., Results: In both the high-risk and low-risk groups, the graft survival and rejection-free graft survival rates were not affected by the H-Y compatibility. The graft survival (p < 0.001) and rejection-free graft survival (p < 0.001) rates were higher in the low-risk group than in the high-risk group. High-risk PKP was associated with greater risk of graft failure (risk ratio, 2.33) and rejection (risk ratio, 2.05) than low-risk PKP., Conclusion: H-Y antigen matching does not influence the rate of allograft rejection after PKP.

Purpose: To survey the changes in indications for penetrating keratoplasty (PKP) and re-evaluate the risk factors for allograft rejection and graft failure., Methods: We evaluated the records of 396 eyes of 335 patients who had undergone PKP at the Tokyo University Hospital between 1987 and 1997. Clinical results were analyzed by the Kaplan-Meier life table method and the log-rank test., Results: The overall rates of graft survival and rejection-free graft survival at 10 years were 72.2% and 76.8%, respectively. The rates of graft survival and rejection-free graft survival were 98.8% and 86.6% in keratoconus, 87.0% and 56.5% in herpetic keratitis, 76.9% and 73.1% in corneal dystrophy and degeneration, 69.4% and 80.6% in nonherpetic keratitis, 62.5% and 75.0% in chemical burns, 61.8% and 72.1% in regrafting, and 51.1% and 79.8% in bullous keratopathy, respectively. The graft survival rates were statistically higher in the PKP alone group than in the combined operation group. The graft survival and rejection-free graft survival rates were statistically higher in the first operation group than in the regrafted group, and in the avascular cornea group than in the vascular cornea group., Conclusions: We recognized changes in indications for PKP. Combined operation, reoperation, and vascularization of recipient cornea were risk factors for graft failure.

Purpose: To investigate the effect of anti-inflammatory agents on conjunctival inflammation and corneal haze formation after excimer laser keratectomy., Setting: Department of Ophthalmology, University of Tokyo Faculty of Medicine, Tokyo, Japan., Methods: After excimer laser keratectomy was performed in 21 rabbits (42 eyes), saline, betamethasone 0.1%, or diclofenac 0.1% was topically applied 6 times a day for 4 weeks and then 3 times a day for 8 weeks. The degree of conjunctival inflammation was determined 1, 2, 3, and 7 days after the keratectomy. The degree of corneal haze was quantitatively measured using a digital analyzer before and once a week after the keratectomy. The expression of type IV collagen in the corneas at baseline and 4 and 12 weeks after the keratectomy was examined immunohistochemically., Results: Compared with saline, betamethasone and diclofenac significantly decreased early-phase conjunctival inflammation. Betamethasone significantly inhibited corneal haze formation compared with saline at 3 to 5 and 8 to 12 weeks. Diclofenac did not inhibit corneal haze formation significantly. Although betamethasone tended to be more effective in inhibiting corneal haze formation and deposition of type IV collagen than diclofenac, there was no statistical difference between the 2 anti-inflammatory agents., Conclusion: Topically applied betamethasone effectively suppressed corneal haze formation after excimer laser keratectomy. Diclofenac was not statistically effective in inhibiting corneal haze formation.

Two children with Landau-Kleffner syndrome were successfully treated with antiepileptic drugs and a high-dose intravenous corticosteroid. A combination of valproate and a benzodiazepine (clonazepam or diazepam) ameliorated epileptic seizures and electroencephalographic spikes and waves, but speech disturbances persisted. Both patients were treated with an intravenous infusion of high-dose methylprednisolone sodium succinate (20 mg/kg daily) for 3 consecutive days. This infusion was repeated three times with a 4-day interval between treatments, which resulted in a rapid improvement in speech ability. After intravenous therapy, prednisolone was given orally (2 mg/kg daily for 1 month, then gradually withdrawn), which maintained the clinical improvement in speech.

Purpose: To evaluate long-term follow-up results of excimer laser phototherapeutic keratectomy (PTK) in a Japanese population., Methods: Twenty-six patients (31 eyes) with corneal opacity were treated with excimer laser PTK. Preoperative diagnoses included 16 eyes with band keratopathy, 10 with granular dystrophy, and 5 with corneal scar. Mean postoperative follow-up was 27 months., Results: Corneal opacity was reduced in all patients. At postoperative month 12, best spectacle-corrected visual acuity (BSCVA) improved from the preoperative level in 22 eyes of 28 eyes, did not change in 3 eyes, and declined in 3 eyes. BSCVA at month 24 was better than the preoperative acuity in 17 eyes of 23 eyes, similar in 1 eye, and worse in 5 eyes. Eyes with granular dystrophy showed significantly better BSCVA improvement than those with band keratopathy. A hyperopic shift of +1.0 diopter or more occurred in 14 eyes of 28 eyes at month 12 and in 12 eyes of 23 eyes at month 24. No serious adverse effects were encountered during the 3-year follow-up period., Conclusions: Excimer laser PTK is a safe and effective procedure for the treatment of Japanese patients with superficial corneal opacity.

Purpose: To evaluate effects of ABO antigen blood-group matching on the rates of corneal allograft rejection after penetrating keratoplasty., Methods: We retrospectively studied clinical results of penetrating keratoplasties in terms of graft survival rates and rejection-free graft survival rates. Penetrating keratoplasties were done on 95 eyes between 1993 and 1997. Clinical results were analyzed using the Kaplan-Meier life table method and log-rank test. The corneal transplantations were classified into 2 groups, high-risk (35 eyes) and low-risk (60 eyes) transplantations. High-risk transplantation was defined as significant vascularization in the recipient corneas or a history of graft failure. The remaining transplantations were defined as low-risk., Results: The respective graft survival (p = 0.031) and rejection-free graft survival (p = 0.0097) rates were higher in the low-risk than in the high-risk group. In the high-risk group, the rejection-free graft survival was 68.9% for the ABO-compatible subgroup and 36.4% for the ABO-incompatible subgroup (p = 0.007)., Conclusion: ABO matching is effective in reducing the risk of allograft rejection in high-risk corneal transplantations.

Purpose: To evaluate the immunologic effect of anterior synechiae (AS) in a murine model of corneal transplantation., Methods: Orthotopic penetrating keratoplasty with 12 interrupted sutures was performed on C57BL/6 donor mice and BALB/c recipient mice without AS (AS- group). In contrast to suturing in the AS- group, 3 of the 12 sutures were placed to create AS (AS+ group). The average graft opacity scores and rejection rates of both groups were compared. Cytotoxic T-lymphocyte (CTL) reactions and delayed hypersensitivity (DH) were evaluated 3 weeks after transplantation. Corneal cytokine expression was evaluated., Results: The opacity scores of the AS+ group were consistently greater than those of the AS- group, and the rejection rate of the AS+ group was significantly greater than that of the AS- group (86% versus 54%, P = 0.03). The AS+ group had significantly higher CTL activity compared with the AS- group. There was no significant difference in DH between the two groups. The cytokine expression pattern in the AS+ group became similar to that of the AS- group in which the grafts were rejected., Conclusions: These findings indicate that AS impairs ocular immune privilege by mediating CTL activity, but without intensifying the DH response. Therefore, AS is a critical risk factor in allograft rejection in a murine model of corneal transplantation.

Bone mineral density is reduced in stroke patients on the hemiplegic and contralateral sides, reflecting a degree of paralysis and vitamin D deficiency. Because the deficiency of vitamin K, a factor essential for site-specific carboxylation of bone Gla protein, is also associated with reduced bone mineral density, an additional contribution of vitamin K to bone changes was assessed in 168 elderly patients with long-standing post-stroke hemiplegia and hypovitaminosis D. Sera were analyzed to relate vitamin K1 concentrations to bone-related biochemical indexes and bone mineral density measured by radiodensitometry of the second metacarpal. Bone mineral density was lower on both sides in patients than in the 56 controls (P < 0.02). Serum vitamin K1 concentrations, which correlated positively with bone Gla protein concentrations (P < 0.0001), were lower in patients (0.48 +/- 0.47 nmol/L) than controls (1.33 +/- 0.49; P < 0.0001). Serum bone Gla protein and 25-hydroxyvitamin D concentrations were lower in patients than controls (P < 0.0001), whereas ionized Ca concentrations were higher in patients (1.277 +/- 0.041 mmol/L) than controls (1.210 +/- 0.049; P < 0.0001), correlating with the Barthel index. Multivariate linear regression identified vitamin K1, bone Gla protein, 25-hydroxyvitamin D, ionized calcium, and the Barthel index as independent bone mineral density determinants on the hemiplegic side and 25-hydroxyvitamin D, calcium, and the Barthel index on the intact side. Immobilization and vitamin K deficiency had stronger osteopenic effects on the hemiplegic side than contralaterally.

Purpose: To evaluate the long-term outcome of 2% topical cyclosporine A (CsA) treatment as an adjunct to topical corticosteroid in 86 eyes after penetrating keratoplasty (PK)., Material and Methods: The subjects were 86 eyes of 83 patients who had undergone PK and received topical CsA treatments. Ninety-seven eyes of 95 patients who had undergone PK and received similar postoperative treatments except for topical CsA treatments served as control: The clinical outcome of PK was evaluated by rates of graft survival and rejection-free graft survival using Kaplan-Meier's method and compared with the log-rank test. The patients were subdivided into high-risk and low-risk groups. The high-risk patients were those who had corneal vascularization in 2 or more quadrants of the cornea preoperatively or who received regrafting. All other patients were assigned to the low-risk group. Thirty-six eyes of the CsA group and 50 eyes of the control group were high-risk cases., Results: In the high-risk patients, the rejection-free graft survival rate was 69.7% in the CsA group and 45.4% in the control group (p = 0.030). However, there was no significant difference in the graft survival rate between the two groups. In the low-risk patients, there was no significant difference in the rates of rejection-free graft survival and graft survival between the CsA and the control group., Conclusion: 2% topical cyclosporine is effective in reducing the risk of allograft rejection in high-risk recipients.

Purpose: Transforming growth factor-beta (TGF-beta) superfamily consists of many multifunctional cytokines including TGF-beta, activins, and bone morphogenetic proteins (BMPs). Previous reports show that these factors are expressed in ocular tissue, and exert their effects through type I and type II serine/threonine type receptors. The purpose of this study is to observe the expression of these receptor families in normal rat eyes as the first step in investigating the functions of TGF-beta superfamily members in ocular tissue., Methods: The expression of receptors was examined immunohistochemically in one eye each of 6 Lewis rats. The type I receptors examined were the TGF-beta type I receptor (TbetaR-I), activin type I receptor (ActR-I), activin type IB receptor (ActR-IB), bone BMP type IA receptor (BMPR-IA), BMP type IB receptor (BMPR-IB), and activin receptor-like kinase-1 (ALK-1). The type II receptors examined were TGF-beta type II receptor (TbetaR-II), activin type II receptor (ActR-II), and BMP type II receptor (BMPR-II)., Results: All 6 type I receptors, ActR-II and the alternative spliced long version of BMPR-II were expressed in the following cells: corneal and conjunctival epithelium, corneal keratocytes, corneal endothelium, epithelium of iris and ciliary body, epithelium of lens, sensory retina excluding the outer segments of photoreceptors, retinal pigment epithelium (RPE), and vascular cells. TbetaR-II and the short version of BMPR-II (truncated form) were expressed in the outer segments of the photoreceptors in addition to the above. The results of staining were similar in all the sections examined., Conclusions: The cornea, ciliary body, iris, lens, retinal cells, RPE and vascular cells expressed the receptors for TGF-beta superfamily at protein level.

Prenatal screening of oculo-cerebro-renal syndrome of Lowe (OCRL; McKusick 309000) was performed using cultured amniocytes. Following identification of defective mRNA expression in the OCRL; gene of the proband's fibroblasts, the mRNA size and quantity of the cultured amniocytes were compared. Based on this analysis, the fetus was diagnosed as being normal and was subsequently delivered as a healthy boy. This is the first reported successful prenatal screening of OCRL using a comparison with defective mRNA of OCRL1 from affected subjects.

Background: Anti-alphabeta T cell receptor monoclonal antibody (R73) has been reported to be a potent immunosuppressant. The suppressive effects of this antibody on allograft rejection after corneal transplantation are unknown., Methods: Orthotopic rat penetrating keratoplasty was performed using Lewis rats as recipients and Brown Norway and Fisher rats as donors. The treated groups received R73 intraperitoneally until day 12 after the transplantation. In grafted rats with or without R73 treatment, cytokine expression of the aqueous humor, corneal-infiltrating cells, draining lymph nodes, and splenocytes was determined. Delayed-type hypersensitivity (DTH) responses were compared., Results: All allografts in the untreated controls of Fisher-to-Lewis or BN-to-Lewis rat combinations were rejected within 14 days. In contrast, indefinite survival rates of the postoperative R73-treated group increased to 86% in the Fisher-to-Lewis and 23% in the Brown Norway-to-Lewis combinations, respectively. Interferon-y, interleukin (IL)-2 (T helper [Th]1), and IL-10 (Th2), but not IL-4 (Th2), expression of the eye and DTH responses in the control group were suppressed in the R73-treated group. Both IL-2 and IL-10 expression after mixed lymphocyte culture in the R73-treated group were significantly lower than those of the naive and untreated control group., Conclusions: alphabeta T cell receptor-targeted therapy prevents allograft rejection in rat corneal transplantation as evidenced by suppression of DTH responses. The cytokine profile after R73 treatment was characterized by low interferon-gamma, IL-2, and IL-10, and high IL-4 expression.

Background and Purpose: Stroke-induced hemiparesis involving the arm and hand results in regular, repeated overuse of the opposite hand and wrist. Because repetitive hand and wrist movement is a common cause of carpal tunnel syndrome (CTS), we examined the nonparetic upper limb in stroke patients for evidence of CTS., Methods: We measured bilaterally sensory nerve conduction velocity (SNCV), motor nerve conduction velocity (MNCV), sensory nerve action potentials (SNAP) at the wrist, palm-to-wrist distal sensory latency (DSL), palm-to-wrist SNAP, compound motor action potentials (CMAP), and distal motor latency (DML) in stroke patients and control subjects. Controls were right-handed, >/=65 years old, lucid, independent in their activities of daily living, and had no disease known to cause CTS. Stroke patients were divided into a functioning hand group (n=61) and a disused hand group (n=71). All patients had hemiplegia., Results: Tinel's sign was observed on the nonparetic side in 57.7% of patients with a disused hand and in 31.1% of those with a functioning hand. All electrophysiological indices were significantly more abnormal on the nonparetic side than on the hemiparetic side or in controls. Patients with a disused hand showed greater abnormality on the nonparetic side in SNCV, SNAP, palm-to-wrist DSL, DML, and CMAP than patients with a functioning hand., Conclusions: Overuse of the nonparetic hand and wrist of the nonparetic side may result in CTS in stroke patients, especially when the paretic hand is not functional. Wrist splinting or other prophylactic treatments beginning soon after stroke might help to prevent CTS.

We report a case of dermatomyositis (DM) associated with invasive thymoma in a 22-year-old woman who was admitted to our hospital complaining of dyspnea which required ventilation support. The reddened elevated scaly eruptions were prominent over the extensor surfaces. Chest X-ray and computed tomography showed mediastinal masses, which were diagnosed as mixed type thymoma. Muscle and skin biopsy specimens were compatible with DM. She was treated with methylprednisolone pulse therapy followed by extended removal of the anterior mediastinal tumor and subsequent radiotherapy. She has had a good clinical course without recurrence of thymoma or DM for more than 3 years. The role of thymoma in the development of DM is discussed.

We report here a 9-year-old girl with ophthalmoplegic migraine. At the age of 2 years and 6 months she first developed left ptosis and ophthalmoparesis that resolved gradually within 2 weeks. She experienced similar episodes repeatedly. After 5 years of age, left periorbital pulsatile pain preceded ptosis and ophthalmoparesis, and after 7 years, she showed permanent left third nerve paresis even between the attacks. On cranial MRI the left oculomotor nerve showed swelling and contrast enhancement, the latter being more prominent in the ictal than interictal images. Ophthalmoplegic migraine should be considered in the differential diagnosis of opthalmoplegia in children even in the absence of headache. The diagnosis is strongly suspected when MRI demonstrates swelling and enhancement of the oculomotor nerve.

Background: Cytokine profile is a key in understanding the mechanisms of allograft rejection. Cytokine expression in the aqueous humor and the correlation between the aqueous humor cells and corneal infiltrating cells are not fully understood in corneal transplantation., Methods: Orthotopic mouse corneal transplantation was performed using BALB/c (H2d) mice as recipients, and C3H/He (H2k) and BALB/c mice as donors for allografts and isografts, respectively. Immunocytochemistry was performed on aqueous humor cells. Corneal graft was studied immunohistochemically. Cytokine gene expressions of the cells infiltrating the aqueous humor and corneal grafts were determined by the semiquantitative reverse transcription and polymerase chain reaction method., Results: Interferon-gamma, interleukin (IL)-2, IL-4, and IL-10 were detected in the cells infiltrating the aqueous humor and corneal grafts at both the protein and gene expression levels. T helper 1 (Th1) cytokine expressions at the protein level, however, were consistently predominant in the rejected allografts compared to those of Th2 cytokines. The cytokine and surface marker profiles of the cells in the aqueous humor corresponded well to those of the cells infiltrating the corneal grafts. Cytokine protein and mRNA expression levels in the aqueous humor decreased rapidly., Conclusions: Allorejection in corneal transplantation is Th1 cytokine-predominant. Infiltrating cells do not express Th2 cytokine so much in allograft rejection, as compared with Th1 cytokine. The cell infiltration patterns of the aqueous humor were well correlated with those of the cornea.

Purpose: To investigate the structural changes in corneal stromal collagen fibrils after excimer laser keratectomy in relation to the degree of corneal haze., Setting: University of Tokyo Hospital, Tokyo, Japan., Methods: Corneal haze was quantitatively measured by analyzing the light scattering in Scheimpflug images of the corneas of white rabbits after excimer laser keratectomy. Collagen fibril structure was examined using scanning electron microscopy after chemical digestion with sodium hydroxide solution; the same specimens were examined by transmission electron microscopy after re-embedding., Results: Corneal haze reached a peak 4 weeks after excimer laser keratectomy and then gradually decreased. The collagen fibrils of the normal cornea were regularly arranged parallel to the surface of the cornea, with small interfibrillar distances. After excimer laser keratectomy, the arrangement was highly disordered, with increased interfibrillar distances. These structural changes were most prominent 4 weeks after excimer laser keratectomy., Conclusion: The structural changes in the collagen fibrils of the corneal stroma, especially the increase in interfibrillar distances and the disordered arrangement, were associated with corneal haze after excimer laser keratectomy.

Purpose: Production of extracellular matrix (ECM) by corneal endothelial cells is related to physiologic functions and pathologic conditions and is regulated by many cytokines, including transforming growth factor-beta (TGF-beta). In this study, the molecular mechanism of ECM production regulation by TGF-beta was investigated in cultured corneal endothelial cells., Methods: The production of ECM components (laminin and fibronectin) was detected in cultured corneal endothelial cells by western blot analysis. To determine the signal transduction pathways, mutant TGF-beta type I receptor (TbetaR-I) and/or Smad protein family members (intracellular signal transducers in TGF-beta signaling) were overexpressed by transfecting their cDNA into the cultured cells, and the effects on ECM production were observed., Results: The production of laminin and fibronectin was stimulated by treatment with TGF-beta1 or TGF-beta2. After transient transfection of cDNA of the constitutively active (CA) mutant of TbetaR-I, the production of laminin and fibronectin was stimulated even in the absence of TGF-beta. The transfection of the dominant negative mutant of TbetaR-I counteracted the effects of TGF-beta. These results confirm that TGF-beta directly stimulates ECM production from corneal endothelial cells through TbetaR-I. The ECM production stimulation by TGF-beta or CA TbetaR-I was accelerated by the overexpression of Smad2, Smad3, and/or Smad4 and inhibited by that of Smad7. These results show that TGF-beta signals connected to ECM production are regulated by Smad family members, located downstream of TbetaR-I., Conclusions: The results of this study show that TGF-beta stimulates ECM production from corneal endothelial cells through TbetaR-I and Smad family transducers.

To examine contamination of donor eye preservation media with methicillin-resistant Staphylococcus aureus (MRSA), we studied the media to detect specific genes for MRSA by use of the polymerase chain reaction (PCR) method. The materials were 36 samples of donor eye preservation media (EP-II) in which donor eyes had been stored for keratoplasty. Polymerase chain reaction procedures were carried out to simultaneously detect the spa gene, which codes protein A of S. aureus, and the mecA gene, which codes penicillin-binding protein-2' contributing resistance to methicillin. Along with PCR analyses, the preservation media was examined by conventional culture methods to determine bacteriologic contamination. The PCR analyses of the 36 samples revealed that both the spa and the mecA genes were positive in five samples, only the spa gene was positive in two, and only the mecA gene was positive in two. The conventional culture of the media showed positive for MRSA in 5 samples, methicillin-susceptible S. aureus (MSSA) in 2, methicillin-susceptible coagulase-negative staphylococci (MS-CNS) in 4, and methicillin-resistant coagulase-negative staphylococci (MR-CNS) in 2 of the 36 samples. The results of PCR coincided well with those of conventional bacteriologic culture. Polymerase chain reaction analysis for spa and mecA genes is useful in detecting contamination of donor eye preservation media by MRSA, MSSA, MR-CNS, or MS-CNS in a shorter time than by conventional culture.

MR imaging of a patient with cortical blindness revealed areas of abnormal signal bilaterally in the lateral putamen and the striate cortices. The coronal T2-weighted image clearly demonstrated the selective involvement of the bilateral striate cortices. The differential diagnosis and the vulnerability of the striate cortex are discussed.

Although Acanthamoeba keratitis has been recognized as one of the important infectious diseases of the cornea, especially in contact lens wearers in recent years, its taxonomy has not been well established. We carried out mitochondrial DNA (mtDNA) analysis of the Acanthamoeba organisms isolated from corneal lesions in four eyes of three patients who had suffered from keratitis. The mtDNA was analyzed by restriction fragment length polymorphism (RFLP) using restriction enzymes Bg/II and EcoRI. The RFLP analyses revealed that the DNA phenotypes of the Acanthamoeba organisms were identical to those of the Ma strain in two patients and to the Castellani strain in one patient.

The enzyme oligosaccharyltransferase (dolichyl-diphosphooligosaccharide-protein glycosyltransferase; EC 2. 4.1.119) (DDOST) catalyzes the transfer of a high-mannose oligosaccharide (GlcNac2Man9Glc3) from a dolichol-linked oligosaccharide donor (dolichol-P-GlcNac2Man9Glc3) onto the asparagine acceptor site within an Asn-X-Ser/Thr consensus motif in nascent polypeptide chains across the membrane of the endoplasmic reticulum. We isolated mouse and human DDOST cDNAs from retinoic acid-treated mouse P19 EC cells and human NT-2 cells, respectively. DDOST mRNA is expressed intensely in heart and pancreas, but at lower levels in brain. Here we show that the human DDOST 48-kDa subunit gene (HGMW-approved symbol DDOST) is organized into 11 exons expanding about 9 kb. This DDOST subunit gene is localized on chromosome 1p36.1 by fluorescence in situ hybridization analysis., (Copyright 1997 Academic Press.)

Background: The expression of Fas ligand (FasL) in the eye has been proposed to be an important component of ocular immune privilege. Since the unusually favorable outcome of corneal transplantation is thought to result from the immune privilege of the eye, examination of the function of FasL on corneal allografts would be a test of that hypothesis., Methods: To investigate the role of Fas-FasL interaction in corneal allografts, orthotopic corneal transplantation was performed using C57BL/6 (B6, FasL+) and B6-gld (FasL-) mice as cornea donors and BALB/c mice as recipients. The rejection rate of B6-gld grafts (FasL- group) was compared with that of normal B6 control corneas., Results: The rejection rate at the final observation (8 weeks) in the FasL- group (89%) was significantly higher than in the FasL+ control group (47%). FasL expression was found on the corneal endothelium by staining with anti-FasL monoclonal antibodies. The TdT-mediated dUTP nick-end labeling assay revealed that apoptotic cells were attached to the endothelium in the control group but not in the FasL- groups., Conclusions: Apoptosis of infiltrating cells on the corneal endothelium resulting from Fas-FasL interaction plays an important role in the high success rate of corneal transplantation.

The inoculation of antigens into the anterior chamber (AC) of the eye induces an antigen-specific immune response that inhibits delayed-type hypersensitivity (DTH). This regulatory response is known as anterior chamber-associated immune deviation (ACAID). The ACAID response appears to be complex, as it can be elicited by a wide variety of soluble and cell-associated antigens, including foreign, self, tumor, and alloantigens. To evaluate the contribution of Fas/Fas ligand (FasL) interaction to the induction of ACAID to alloantigens, gld and lpr mutant mice were used in conjunction with normal C3H, MRL, and BALB/c mice. ACAID was induced by inoculation of non-irradiated splenocytes from donor mice into the AC of various recipients. After 1 week, recipients were primed intradermally with donor splenocytes. One week later DTH was measured by ear swelling. C3Hgld mutants lacking functional FasL did not develop ACAID after the AC inoculation of BALB/c splenocytes. Conversely, the AC inoculation sensitized these mutants. MRL/pr mutants, which lack Fas, developed ACAID following inoculation of BALB/c cells. AC inoculation of lpr splenocytes did not induce ACAID, but sensitized C3H recipients. Treatment of the AC inoculum with an anti-Fas antibody blocked ACAID induction in a transient manner, as the recipients developed ACAID later. These results show that interaction of the Fas and FasL is required to induce ACAID to allogeneic cells. In the absence of Fas expression on donor splenocytes, or FasL expression by the recipient, AC inoculation primes for a DTH response rather than inducing ACAID.

Background: The mechanisms of corneal xenogeneic immunoreaction, as well as the potential role of immunosuppressive therapy in the suppression of corneal xenograft rejection, have not been thoroughly explored., Methods: BALB/c mice who received orthotopic corneal transplants (Lewis rats donors) were administered intraperitoneally anti-leukocyte function associated antigen-1 (LFA-1) monoclonal antibody (mAb) or FK506 (3 mg/kg/day) or both of these immunosuppressants during a 12-day postoperative period. Histological (hematoxylin-eosin stain) and immunohistochemical evaluations of enucleated eyes were performed. Humoral immune response and delayed-type hypersensitivity (ear-swelling assay) were evaluated., Results: The mean (+/-SD) graft survival time in the untreated control, FK506-treated, anti-LFA-1 mAb-treated, and combined-treatment groups was 5.8+/-0.8, 9.4+/-4.0, 8.7+/-5.0, and 67.7+/-16.4 days, respectively. In the untreated control group, mouse IgG, IgM, and C3 were expressed on the rat corneal grafts during the early postoperative phase. Flow cytometry studies revealed high titers of xenoreactive IgG and IgM antibodies. T helper 1 cytokines were expressed on xenografted corneal beds, and delayed-type hypersensitivity was induced. However, local expression of IgM, C3 and T helper 1 cytokines, serum antibodies of IgG and IgM, and delayed-type hypersensitivity were suppressed in the anti-LFA-1 mAb- plus FK506-treated group., Conclusions: Both humoral and cell-mediated immune reaction play an important role in the initial rejection in rat-to-mouse corneal xenotransplantation. The treatment with anti-LFA-1 mAb in combination with FK506 synergistically suppresses concordant corneal xenogeneic reaction.

It has been reported that allograft rejection is mediated by a variety of adhesion molecules. Using a corneal allograft model in mice, we studied the role of very late antigen (VLA)-4 and leukocyte function-associated antigen (LFA)-1 adhesion molecules in corneal allograft rejection and the effects of monoclonal antibodies (mAbs) to them in suppressing corneal rejection. C3H/He donor corneas were transplanted into BALB/c corneal beds. The allografted mice were treated with a control mAb (M18/2), mAbs to VLA-4, or LFA-1 or their combination by i.p. injection until day 7. The expression of VLA-4, LFA-1, major histocompatibility complex (MHC) class II antigens, interleukin (IL)-2, IL-2 receptor and interferon gamma (IFNgamma) in the grafted cornea were studied immunohistochemically. Cytotoxic T lymphocyte (CTL) responses to donor alloantigens were assessed. The skins from a syngeneic donor or a third-part strain were transplanted 8 weeks after the initial keratoplasty onto the mice treated with anti-LFA-1 plus anti-VLA-4 mAbs. Fourteen of 16 allografts in non-treated mice and control mAb-treated mice became opaque by 2 weeks after transplantation. At 2 weeks, non-treated allografts showed expression of MHC class II antigens on keratocytes and mononuclear cells at the host-graft junction. Also, mononuclear cells expressing VLA-4, LFA-1, IL-2, IL-2 receptor and IFNgammawere present in the stroma at the host-graft junction. The allografts treated with either anti-VLA-4 or anti-LFA-1 alone, or anti-VLA-4 plus anti-LFA-1 remained transparent for more than 2 weeks, and the survival rates at 14 weeks was 0%, 16.7%, and 75.0%, respectively. The combined use of anti-VLA-4 and anti-LFA-1 mAbs prolonged graft survival significantly (P<0.05) at 14 weeks as compared with anti-LFA-1 mAb alone. At 3 weeks, CTL responses to donor alloantigens were depressed in mice treated with either anti-LFA-1 alone or anti-LFA-1 plus anti-VLA-4. Specific prolongation of donor-syngeneic skin was observed after treatment with the combination of these two mAbs. These results indicate that VLA-4 and LFA-1 have important roles in rejection process of corneal allografts, and that the combined use of mAbs to these molecules has remarkable effects on inducing alloantigen-specific immunosuppression in corneal transplantation.

To examine the effectiveness of topical corticosteroids in decreasing subepithelial haze induced by excimer laser keratectomy, we performed excimer laser keratectomy on 24 eyes of 12 albino rabbits using a scanning excimer laser system (EC-5000, NIDEK, Japan). Topical dexamethasone 0.1% was applied topically to 8 corneas after surgery (steroid group), but not to the others (control group). Slit-lamp biomicroscopic examination and objective measurement of scattered light from subepithelial haze were performed before and 2, 4, 8 and 12 weeks after surgery, the rabbits were killed after 2 or 12 weeks and the eyes were examined histologically. In both groups, densitometric values of scattered light intensity increased up to 4 weeks after surgery, and then decreased gradually. However, the values of the steroid group were larger than those of the control group at all stages, and the values of the steroid group at 12 weeks after surgery were similar to preoperative values. There was statistically significant difference in scattered light intensity between the two groups (p < 0.005). Two weeks after surgery, the corneas of the control group showed epithelial hyperplasia and a remarkable increase in activated keratocytes and subepithelial fibrous tissues. However, in the steroid group these light microscopic findings could not be observed. At 12 weeks after surgery, the corneas of the control group retained a small amount of activated keratocytes and fibrous tissues, and those of the steroid group were normal. This study suggests that a short course of topical corticosteroids may be very effective in decreasing subepithelial haze after excimer laser corneal surgery.

Tissue factor (TF) is an integral membrane glycoprotein that serves as a cofactor for blood coagulation factor VIIa. The induction of TF on the surface of endothelial cells is initiated by various stimuli including lipopolysaccharide, interleukin-1 beta, and tumor necrosis factor alpha. We have demonstrated that recombinant human C5a induces TF activity in a dose-dependent fashion in human umbilical vein endothelial cells (HUVEC). Peak activity (4.9-fold increase) was obtained 3-6 h after treatment with 10 microM C5a. TF mRNA as assessed by RT-PCR method was also significantly increased (3.75-fold) after 3 h incubation with C5a, suggesting that C5a induces TF activity on HUVEC, at least in part, by enhancing the level of TF mRNA. The increase in TF activity by C5a was inhibited by methylprednisolone. The induction of TF on endothelial cells by C5a may represent one of many potential interrelationships between the inflammatory and coagulation schemes.

We clarified the preoperative risk factors for graft failure in penetrating keratoplasty with fresh donor corneas. This analysis covered 698 consecutive keratoplasty cases in a single center between 1971 and 1992, and was designed to assess the risk factors. The risk factors in penetrating keratoplasty were determined by the Cox multiple regression model which considers the follow-up periods and multiple factors simultaneously. The factors which worsen the prognosis of keratoplasty significantly were found to be preoperative endothelial dysfunction (hazard ratio = 2.3), prior glaucoma/ocular hypertension (hazard ratio = 2.0), preoperative corneal vascularization (hazard ratio for within one quadrant = 1.3, hazard ratio for 2 or more quadrants = 1.6), anterior synechiae of iris (hazard ratio = 1.5), aphakia or pseudophakia (hazard ratio = 1.4), and older donor age (hazard ratio = 1.3 in proportion to the increase of each 10-year period of age). Surgeons must take these risk factors into consideration to obtain a better prognosis for keratoplasty.

Background: It is known that the osmolarity of tears increases in keratoconjunctivitis sicca (KCS) patients and therefore could be a sensitive and specific indicator for the diagnosis of KCS. However, owing to the difficulties in using the current methods of tear fluid measurement these procedures have not been employed in clinical practice. A newly devised flexible conductimetric sensor fabricated using microelectronic techniques is small and flexible enough to be placed on the ocular surface to measure the electrical conductivity can be considered as an indirect function of electrolyte activity, or osmolarity. Therefore, we applied this new sensor to measure the tear fluid conductivity in KCS patients and healthy volunteers., Methods: A flexible conductimetric sensor, consisting of a hydrophilic polytetrafluoroethylene membrane placed between two gold-coated layers, was placed directly into the temporal conjunctival cul-de-sac. The tear fluid conductivity was monitored graphically on a computer display. The sodium chloride concentration of tear fluids was calculated from the calibration curve and converted to the equivalent electrolyte concentration., Results: The electrolyte concentrations were 324.8 +/- 41.0 mEq/1 in KCS patients (29 samples obtained from 16 KCS patients) and 296.4 +/- 30.1 mEq/1 in healthy persons (33 samples obtained from 17 healthy persons). The difference was significant (P < 0.01). A positive correlation was found between the electrolyte concentrations in KCS and the rose bengal score (coefficient = 0.36)., Conclusion: The tear fluid conductivity in healthy persons and KCS patients could be monitored without ocular damage, and the measured values were consistent with previous reports. This method will be a new diagnostic tool for detecting tear abnormalities.

Corneal transplantation was performed by grafting C3H/He donor corneas into BALB/c corneal beds. The allografted mice were injected either with 0.5 mg/day of anti-very late antigen (VLA)-4 antibody, anti-leukocyte function-associated antigen (LFA)-1 antibody, or 0.25 mg/day each of both antibodies on days -2, 0, 1, 3, 5, and 7. After 3 weeks, cytotoxic T lymphocyte (CTL) responses to donor alloantigens were assessed. Splenocytes in the allografted mice without treatment demonstrated greater CTL responses that those in naive mice. CTL responses were depressed in mice treated with either anti-LFA-1 alone or a combination of anti-LFA-1 and anti-VLA-4 antibodies as compared with splenocytes from allografted mice without treatment. These data suggest that CTLs play significant roles in eliciting immune response to corneal allografts.

Background: It has been reported that adhesion molecules play an important role in immunological rejection after organ transplantation. In the present study, we examined the role of ICAM-1/ LFA-1 adhesion molecules in corneal allograft rejection and evaluated the immunological specificity of monoclonal antibodies (mAbs) in preventing allograft rejection in mice., Methods: The allografted mice were intraperitoneally injected with 100 micrograms/day of the following mAbs: a control mAb, anti-ICAM-1 mAb, anti-LFA-1 mAb, or a mixture of anti-ICAM-1 and anti-LFA-1 mAbs from 1 day before to 7 days after surgery. The expression of ICAM-1 and LFA-1 molecules in the grafted cornea was studied immunohistochemically. The corneas from a syngeneic donor or a third-party strain were transplanted 4 weeks after the initial keratoplasty onto the mice treated with both anti-ICAM-1 and anti-LFA-1 mAbs., Results: The allografts treated with anti-LFA-1 mAb alone or both anti-ICAM-1 and anti-LFA-1 mAbs remained transparent for more than 2 weeks, and the survival rate at 8 weeks was 40% in both groups. ICAM-1 was expressed on the mononuclear cells, keratocytes and endothelial cells in the allografts without treatment. The second corneal grafts syngeneic to the initial donor remained transparent at 2 weeks, whereas those from the third party were rejected., Conclusions: ICAM-1 and LFA-1 adhesion molecules play a crucial role in the pathophysiology of corneal transplant rejection. The immunosuppressive effects of anti-ICAM-1 and anti-LFA-1 mAbs are highly allospecific. The administration of mAbs to the adhesion molecules represents a new means of suppressing allograft rejection after penetrating keratoplasty.

The fourth component of complement (C4) in urine was measured in 19 patients with systemic lupus erythematosus (SLE). Urinary C4 was detectable in all SLE patients using an enzyme linked immunosorbent assay. In 11 of 13 patients whose urinary C4 was serially measured, a decrease of urinary C4 was found in parallel with a decrease of disease activity of SLE. In the remaining 2 patients, the amount of C4 increased preceding a flare-up of the disease. The measurement of urinary C4 may be useful in evaluating the disease activity of SLE in individual patients and sometimes in predicting the flare-up of the disease. The specific hemolytic activity of excreted C4 and Western blotting analysis showed that urinary C4 consisted mainly of degraded fragments of C4. In two cases, camostat, a serine protease inhibitor, rapidly decreased the urinary C4 excretion, suggesting that the complement activation occurred in the glomerulus in lupus nephritis.

Accurate calibration of ablation rate by excimer laser prerequisite for precise photorefractive keratectomy (PRK). When a polymethylmethacrylate (PMMA) plate is ablated by excimer laser, debris is generated, which may affect homogeneity of the laser beam and energy density, and change the ablation rate. In order to clarify the effects of the debris on the ablation rate, we studied the changes in the ablation rate when the debris was removed by blowing or aspirating over the ablation area during PRK.PMMA plates were ablated using a scanning excimer laser system (EC-5000, NIDEK, Japan) in PRK mode under the following conditions: (1) with air blowing over the ablation area, (2) with aspiration of the debris, and (3) without treatment. The ablation rates were determined by measuring the refractive power of PMMA plates with a lens meter. The ablated surface was observed by scanning electron microscope (SEM). The ablation rate with blowing was the highest among the three conditions, that with aspiration was the second, and that without treatment was the lowest. The ablation rates with blowing showed no significant change when the ablation rates were changed. However, the ablation rates with aspiration or without treatment decreased as the pulse rate increased. The surface ablated during blowing was the smoothest in SEM photographs. We concluded that calibration of the ablation rate using PMMA plates must be done with appropriate air blowing.

Tear fluid conductivity was measured by a non-invasive method. A flexible 3 mm-wide conductimetric sensor was placed inside the human subjects temporal lower cul-de-sac (similar to Schirmer test strip), and used to evaluate electrolyte concentration and turnover rate in tear for normal, healthy subjects aged between 30 to 85 years in a normal, light indoor environment. The tear electrolyte concentration was calculated from tear conductivity to give a mean value of 297 mEq/l (S.D. 30 mEq/l; n = 29) which was consistently with previously reported values. Tear turnover rate was calculated by a single exponential equation of tear conductivity change, following the application to the eye-drops of 40.0 milligrams sodium chloride solution. The mean turnover rate was 44.2% per minute (S.D. = 13.3% per minute; n = 30), being in agreement with previously reported values.

The existence and pathophysiological role of glycosaminoglycans in the tear fluid in humans was investigated using quantitative analyses of hyaluronic acid and chondroitin sulfate in the tear fluid. The subjects were 42 eyes of 31 normal controls, 9 eyes of 9 patients with superficial punctate keratitis (SPK), and 13 eyes of 13 patients with epithelial defect. After an instillation of 100 microliters saline solution in the conjunctival sac, as much tear fluid as possible was collected from the lower cul-de-sac. The glycosaminoglycans in the tears were then treated with chondroitinase ABC to make fractions of unsaturated disaccharides. The quantities of disaccharides were determined by high-performance liquid chromatography. Concentrations were expressed as nanomoles of unsaturated disaccharides per protein in the tears. The concentrations of hyaluronic acid and chondroitin sulfate in the normal controls were 0.07 +/- 0.12(n mol/mg protein) and 6.91 +/- 3.63 (n mol/mg protein), respectively. The mean concentration of hyaluronic acid was significantly higher in patients with epithelial erosion than in normal controls, whereas the mean concentration of chondroitin sulfate was significantly lower in patients with epithelial erosion than in normal controls. There was no significant difference in the concentration of glycosaminoglycans between the patients with SPK and normal controls. The results of our study suggest that glycosaminoglycans are synthesized and endogenously secreted into the tear fluids and, especially in the case of hyaluronic acid, may play an important role in corneal epithelial wound healing in patients with epithelial erosion.

To evaluate the influence of alignment errors in focusing on the results of measurements in corneal topography, we experimented using polymethyl-methacrylate balls with definite radii of curvature as test objects. We used 3 different corneal topographic systems with different working distances to clarify the relationship between the degree of errors and the working distance. We determined the refractive power of the test balls by the topographic systems and studied changes in the values as the balls were slightly displaced from the alignment position axially, or horizontally. The test balls were set on the stage with a micrometer so that the distance of displacement could be determined precisely. When the test balls were placed precisely in alignment, there was no significant relationship in the magnitude of measurement errors with working distance of the topographical systems. When the test balls were displaced axially, the measurement values decreased as the balls come near from the alignment position and increased as they moved away from the alignment position. The magnitude of the measurement errors caused by back and forth displacement of the object became larger as the working distance of the topographic system became shorter. There was no specific relationship between the degree of measurement errors and the working distance when the balls were displaced horizontally.

We performed quantitative analyses of glycosaminoglycans in the tear fluids in a rabbit wound healing model. We ablated rabbit corneal epithelium with trephine and spatula, and sampled tear fluids before the epithelial ablation, and at 3, 24, 48, and 72 hours after it. After an instillation of 200 microliters saline solution in the conjunctival sac, as much tear fluid as possible was collected from the lower cul-del-sac. The glycosaminoglycans in the tears were then treated with chondoroitinase ABC to make fractions of disaccharides. The quantities of disaccharides were determined with high-performance liquid chromatography as weight per unit protein in the tears. The concentrations of delta Di-HA in the tear fluids at 3 and 24 hours were significantly higher than those before the treatment and returned to the initial value at 72 hours after making the epithelial wound. Among the disaccharides of chondroitin sulfate, delta Di-0S and delta Di-6S showed a significant increase at 3 hours after the treatment but delta Di-4S did not show any significant variation. The results suggest that the glycosaminoglycans in the rabbit tear fluids may play an important role in the corneal epithelial wound healing process.

In order to evaluate preoperative risk factors that affect the incidence of endothelial rejection after penetrating keratoplasty, we performed statistical analysis on 571 consecutive cases who had been followed at the University of Tokyo Hospital for at least one year. We applied a logistic regression model (multiple variate analysis) to determine the significance of the association between the preoperative factors and the incidence of rejection. We identified four preoperative risk factors of endothelial rejection in a year as follows: the extent of corneal vascularization, failed previous graft continuous sutures, and preoperative corneal endothelial damage. When the rejection cases were divided into an early group that suffered rejection within 3 months and a late group that suffered rejection after 6 to 12 months, young patients were likely to suffer rejection in the early postoperative period.