Ectopic GRHL2 Expression Due to Non-coding Mutations Promotes Cell State Transition and Causes Posterior Polymorphous Corneal Dystrophy 4.

In a large family of Czech origin, we mapped a locus for an autosomal-dominant corneal endothelial dystrophy, posterior polymorphous corneal dystrophy 4 (PPCD4), to 8q22.3-q24.12. Whole-genome sequencing identified a unique variant (c.20+544G>T) in this locus, within an intronic regulatory region of GRHL2. Targeted sequencing identified the same variant in three additional previously unsolved PPCD-affected families, including a de novo occurrence that suggests this is a recurrent mutation. Two further unique variants were identified in intron 1 of GRHL2 (c.20+257delT and c.20+133delA) in unrelated PPCD-affected families. GRHL2 is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT) and is a direct transcriptional repressor of ZEB1. ZEB1 mutations leading to haploinsufficiency cause PPCD3. We previously identified promoter mutations in OVOL2, a gene not normally expressed in the corneal endothelium, as the cause of PPCD1. OVOL2 drives mesenchymal-to-epithelial transition (MET) by directly inhibiting EMT-inducing transcription factors, such as ZEB1. Here, we demonstrate that the GRHL2 regulatory variants identified in PPCD4-affected individuals induce increased transcriptional activity in vitro. Furthermore, although GRHL2 is not expressed in corneal endothelial cells in control tissue, we detected GRHL2 in the corneal "endothelium" in PPCD4 tissue. These cells were also positive for epithelial markers E-Cadherin and Cytokeratin 7, indicating they have transitioned to an epithelial-like cell type. We suggest that mutations inducing MET within the corneal endothelium are a convergent pathogenic mechanism leading to dysfunction of the endothelial barrier and disease.

Autosomal-Dominant Corneal Endothelial Dystrophies CHED1 and PPCD1 Are Allelic Disorders Caused by Non-coding Mutations in the Promoter of OVOL2.

Congenital hereditary endothelial dystrophy 1 (CHED1) and posterior polymorphous corneal dystrophy 1 (PPCD1) are autosomal-dominant corneal endothelial dystrophies that have been genetically mapped to overlapping loci on the short arm of chromosome 20. We combined genetic and genomic approaches to identify the cause of disease in extensive pedigrees comprising over 100 affected individuals. After exclusion of pathogenic coding, splice-site, and copy-number variations, a parallel approach using targeted and whole-genome sequencing facilitated the identification of pathogenic variants in a conserved region of the OVOL2 proximal promoter sequence in the index families (c.-339_361dup for CHED1 and c.-370T>C for PPCD1). Direct sequencing of the OVOL2 promoter in other unrelated affected individuals identified two additional mutations within the conserved proximal promoter sequence (c.-274T>G and c.-307T>C). OVOL2 encodes ovo-like zinc finger 2, a C2H2 zinc-finger transcription factor that regulates mesenchymal-to-epithelial transition and acts as a direct transcriptional repressor of the established PPCD-associated gene ZEB1. Interestingly, we did not detect OVOL2 expression in the normal corneal endothelium. Our in vitro data demonstrate that all four mutated OVOL2 promoters exhibited more transcriptional activity than the corresponding wild-type promoter, and we postulate that the mutations identified create cryptic cis-acting regulatory sequence binding sites that drive aberrant OVOL2 expression during endothelial cell development. Our data establish CHED1 and PPCD1 as allelic conditions and show that CHED1 represents the extreme of what can be considered a disease spectrum. They also implicate transcriptional dysregulation of OVOL2 as a common cause of dominantly inherited corneal endothelial dystrophies.

Identification of six novel mutations in ZEB1 and description of the associated phenotypes in patients with posterior polymorphous corneal dystrophy 3.

Posterior polymorphous corneal dystrophy 3 (PPCD3) is a rare autosomal dominant disorder caused by mutations in ZEB1. To date all identified disease-causing variants were unique to the studied families, except for c.1576dup. We have detected six novel ZEB1 mutations; c.1749_1750del; p.(Pro584*) and c.1717_1718del; p.(Val573Phefs*12) in two Czech families, c.1176dup; p.(Ala393Serfs*19), c.1100C>A; p.(Ser367*), c.627del; p.(Phe209Leufs*11) in three British families and a splice site mutation, c.685-2A>G, in a patient of Sri Lankan origin. An additional British proband had the c.1576dup; p.(Val526Glyfs*3) mutation previously reported in other populations. Clinical findings were variable and included bilateral congenital corneal opacity in one proband, development of opacity before the age of 2 years in another individual and bilateral iris flocculi in yet another subject. The majority of eyes examined by corneal topography (10 out of 16) had an abnormally steep cornea (flat keratometry 46.5-52.7 diopters, steep keratometry 48.1-54.0 diopters). One proband underwent surgery for cryptorchidism. Our study further demonstrates that PPCD3 can present as corneal edema in early childhood, and that an abnormally steep keratometry is a common feature of this condition. As cryptorchidism has been previously observed in two other PPCD3 cases, its association with the disease warrants further investigation.

Correction of moderate to high hyperopia with an implantable collamer lens: medium-term results.

PURPOSE: To evaluate the medium-term results of phakic posterior chamber implantable collamer lens implantation to correct moderate and high hyperopia. METHODS: In this retrospective study, patients were treated for hyperopia with the Visian Implantable Collamer Lens (ICH model V3; STAAR Surgical AG, Nidau, Switzerland). Examined parameters were manifest refraction spherical equivalent, uncorrected distance visual acuity, corrected distance visual acuity, vault, anterior chamber depth, anterior chamber angle width, endothelial cell density, intraocular pressure, patient satisfaction, and complications. RESULTS: The mean age of 15 patients (28 eyes) was 28 years (range: 18 to 36 years), with a mean follow-up period of 3.6 years (range: 3 to 6 years). The mean manifest refraction spherical equivalent decreased from +6.30 ± 1.42 diopters (D) (range: +4.25 to +8.50 D) preoperatively to -0.37 ± 0.56 D (range: -1.25 to +1.00 D) at 3 years postoperatively. The mean uncorrected distance visual acuity improved from 0.77 ± 0.38 logMAR (range: 0.16 to 1.30 logMAR) to 0.20 ± 0.17 logMAR (range: 0.00 to 0.48 logMAR) at the 3-year follow-up. Postoperatively, 62% of eyes gained one line of corrected distance visual acuity or remained unchanged. The mean vault reduced from 367.1 ± 253.6 µm (range: 70.0 to 1,190.0 µm) at 1 month postoperatively to 283.6 ± 210.0 µm (range: 75.0 to 915.0 µm) at the last follow-up visit (P = .005). The mean preoperative anterior chamber depth and anterior chamber angle width also decreased at the last follow-up visit (P = .037 and < .0001, respectively). The mean endothelial cell loss was 4.91% (P = .089). No serious complications occurred. Thirteen (87%) patients were satisfied with the outcomes and no patient was dissatisfied. CONCLUSIONS: Implantation of a posterior chamber implantable collamer lens is a safe, effective, predictable, and stable method for the correction of moderate and high hyperopia in highly selected patients. No case of cataract or anterior subcapsular opacities formation was recorded in relation to the decrease of vault over the studied period and low vault in some eyes.

Recurrence of posterior polymorphous corneal dystrophy is caused by the overgrowth of the original diseased host endothelium.

Posterior polymorphous corneal dystrophy (PPCD) is a rare, bilateral autosomal dominant disorder affecting primarily the corneal endothelium and descemet membrane (DM). The aim of this study was to establish the origin of abnormal endothelium in a patient with PPCD exhibiting cornea graft failure after keratoplasty surgery. A sex-mismatched graft obtained from a patient with PPCD who underwent repeat penetrating keratoplasty and the patient's original cornea were investigated. Combined fluorescent immunohistochemistry for cytokeratin (CK) 19 (a marker of aberrant PPCD endothelium) with fluorescence in situ hybridization (FISH) of the sex chromosomes were used in order to characterize the cells on the posterior graft surface. The pathological endothelium of the failed PPCD cornea revealed strong positivity for CK19 using fluorescent immunohistochemistry. In all the CK19-positive cells, both X and Y chromosomes were simultaneously detected using FISH. The results clearly showed the original cells of the patient (XY), within 3.5 years, almost totally overgrown the posterior corneal surface of the graft (XX). Moreover, an abnormal posterior collagenous layer populated by fibroblast-like cells was observed between DM and the endothelium in the failed graft, but its exact origin could not be established due to the low number of cells. Simultaneous detection of CK19 using fluorescent immunohistochemistry together with the detection of gonosomes using FISH was performed for the first time in the cornea and allowed us to prove that the recurrence of PPCD was caused by pathological abnormal proliferation and migration of recipient cells into donor graft.

Matrix metalloproteinases in recurrent corneal melting associated with primary Sjörgen's syndrome.

PURPOSE: To investigate the contribution of matrix metalloproteinases (MMPs) to recurrent corneal melting in keratoconjunctivitis sicca associated with primary Sjörgen's syndrome (pSS). METHODS: One native melted cornea and ten melted corneal grafts from two patients with severe pSS were used. The presence of MMPs (1, 2, 3, 7, 8, 9, and 13) was detected using indirect enzyme immunohistochemistry. The active forms of MMP 2 and 9 and MMP 3 and 7 were examined by gelatin and casein zymography, respectively. The concentrations of active MMP 1 were measured using an activity assay. Eleven unaffected corneas served as controls. RESULTS: The average values of the staining intensity revealed very intense MMP 1, intense MMP 2, 7, and 9 and moderate MMP 3 and 8 positivity, in the corneal epithelium of melted corneas. Intense MMP 1 and 9 staining, moderate MMP 2, 3, and 8 staining, and weak MMP 7 staining were found in the anterior stroma. The posterior stroma revealed intense MMP 1, moderate MMP 3 and 9, and weak MMP 2, 7, and 8 positivity. Immunostaining of the endothelium was moderate for MMP 9 and weak for MMP 1, 2, 3, 7, and 8. MMP 13 was negative in all but four melted specimens, where weak-to-moderate staining was found in the epithelium and stroma. Control corneas revealed moderate MMP 1 and 2 and weak MMP 8 staining in the epithelium, weak MMP 2 staining in the anterior stroma, and weak MMP 1 and 8 staining in the endothelium. Significantly elevated MMP 1 activity and extremely elevated MMP 9 activity were found in most of the tested pathological specimens, compared to healthy controls, where no activity of the two enzymes was present. Markedly elevated MMP 2 activity was found in 2 of 11 specimens, compared to normal tissue. The inactive form of MMP 3 was detected in half of the tested specimens, and inactive MMP 7 in all melted corneas. Active MMP 3 and 7 were found in one melted sample. Neither of these MMPs was found in any of the control specimens. CONCLUSIONS: The increased expression and elevated activity of a wide range of MMPs in melted cornea samples from two patients diagnosed with pSS confirm that these enzymes contribute to the tissue destruction, leading to serious consequences such as corneal perforation and loss of vision.

Phenotype associated with the H626P mutation and other changes in the TGFBI gene in Czech families.

AIMS: To evaluate mutations in the transforming-growth-factor-beta-induced (TGFBI) gene in patients of Czech origin with autosomal dominant corneal dystrophies. METHODS: The coding sequence of the TGFBI gene was analysed in 22 affected Czech individuals from 7 apparently unrelated families. Comparison of phenotype to genotype was performed. RESULTS: A H626P mutation, previously only described in a family with a variant of lattice corneal dystrophy (LCD), was detected in one family with superficial geographic corneal opacities. Light microscopy of 2 samples obtained following either a prior superficial keratectomy or keratoplasty showed amyloid but no fuchsinophilic deposits. In a family with LCD type I, an R124C mutation was identified. The R124L mutation was shown to be causative of Reis-Bucklers corneal dystrophy in 2 families. A family with Thiel-Behnke corneal dystrophy exhibited an R555Q mutation. In 2 families with granular corneal dystrophy type I, the typical R555W change was identified. CONCLUSION: The phenotype of the family with the H626P mutation differed from the phenotype previously reported for this change.

Novel mutations in the ZEB1 gene identified in Czech and British patients with posterior polymorphous corneal dystrophy.

We describe the search for mutations in six unrelated Czech and four unrelated British families with posterior polymorphous corneal dystrophy (PPCD); a relatively rare eye disorder. Coding exons and intron/exon boundaries of all three genes (VSX1, COL8A2, and ZEB1/TCF8) previously reported to be implicated in the pathogenesis of this disorder were screened by DNA sequencing. Four novel pathogenic mutations were identified in four families; two deletions, one nonsense, and one duplication within exon 7 in the ZEB1 gene located at 10p11.2. We also genotyped the Czech patients to test for a founder haplotype and lack of disease segregation with the 20p11.2 locus we previously described. Although a systematic clinical examination was not performed, our investigation does not support an association between ZEB1 changes and self reported non-ocular anomalies. In the remaining six families no disease causing mutations were identified thereby indicating that as yet unidentified gene(s) are likely to be responsible for PPCD.

A new model of corneal transplantation in the miniature pig: efficacy of immunosuppressive treatment.

Corneal allograft rejection is frequently studied in small rodent or rabbit models. To study mechanisms of rejection in a model that more closely mimics transplantation in humans, we performed orthotopic corneal transplantation in the miniature pig using a 7-mm diameter donor graft. Four groups of recipients were studied: 1) untreated naive, 2) untreated vascularized (high risk), 3) high-risk grafts treated by topical application of prednisolone, or 4) high-risk grafts treated with a combined systemic immunosuppression regime of oral prednisone, cyclosporine A, and mycophenolate mofetil. Both the clinical features and histological assessment of corneal graft rejection showed close similarities to graft rejection in humans. Interestingly, preliminary results indicated that topical steroid treatment was superior to systemic immunosuppression in significantly promoting graft survival. Thus, corneal transplantation in the pig represents an animal model most closely resembling corneal grafting in humans, and offers possibilities for testing various clinically applicable immunosuppressive treatments.

Mucolipidosis IV: report of a case with ocular restricted phenotype caused by leaky splice mutation.

PURPOSE: To confirm and define a molecular basis for a case of mucolipidosis type IV (ML IV) with an extremely atypical phenotype pattern. DESIGN: Observational case report of a patient with ML IV with disease progression restricted to ocular symptoms. METHODS: Complete ophthalmologic and neurologic examination. Ultrastructural examination of white blood cells, skin, conjunctiva, and corneal epithelium. The MCOLN1 gene was sequenced from cDNA and the proportion of splicing variants were assessed by quantitative allele-specific polymerase chain reaction. RESULTS: Absence of any neurological abnormalities. Retinal pathologic features were the main cause of visual disability: low visual acuity and cloudy corneas since 2 years of age, progressive decrease in visual acuity since the age of 9 years. Ultrastructural examination showed storage lysosomes filled with either concentric membranes or lucent precipitate in corneal and conjunctive epithelia and in vascular endothelium. Cultured fibroblasts were free of any autofluorescence. Sequencing of the MCOLN1 gene identified compound heterozygosity for D362Y and A-->T transition leading to the creation of a novel donor splicing site and a 4-bp deletion from exon 13 at the mRNA level. Both normal and pathologic splice forms were detected in skin fibroblasts and leukocytes, with the normal form being more abundant. CONCLUSIONS: The case of this patient with ML IV is unique and is characterized by a curious lack of generalized symptoms. In this patient, the disorder was limited to the eyes and appeared without the usual psychomotor deterioration. The resulting phenotype is the mildest seen to date.

Effect of suturing technique on corneal xenograft survival.

PURPOSE: Understanding xenograft rejection is crucial for the potential introduction of xenotransplantation into clinical practice. Small-animal models play an essential role in this context and substantially contribute to our knowledge about mechanisms of xenograft rejection. METHODS: Rat-to-mouse corneal xenografts were performed by using 2 suturing techniques. Sutures were left either as long or as short as possible to limit the extent of a nonspecific inflammatory response. Cyclosporine A (CsA), monoclonal antibody anti-T cells, and a specific inhibitor of inducible NO synthase (alone or in a combination with CsA) were tested as immunosuppressants. RESULTS: Grafts with long sutures were rejected in 7.3 +/- 1.2 days, whereas those with short sutures were rejected after 11.8 +/- 1.0 days (P < 0.001). Similarly, long sutures induced more pronounced corneal neovascularization (P < 0.001). Although groups of recipients with long sutures all tested immunosuppressants significantly (P < 0.01-0.001) prolonged corneal graft survival, none of them showed a comparable efficacy in groups of recipients with short sutures. CONCLUSIONS: This study showed that suturing technique significantly affects the outcome of corneal concordant xenograft transplantation, influences the effectiveness of immunosuppressive regimens, and therefore must be taken into account when evaluating their efficacy.

Presence of snake-like chromatin in epithelial cells of keratoconjunctivitis sicca followed by a large number of micronuclei.

OBJECTIVE: To evaluate the number of micronuclei in snake-like chromatin (SLC) cells in the conjunctival epithelium of keratoconjunctivitis sicca (KCS) patients. To elucidate possible correlations between SLC cell numbers and KCS intensity. STUDY DESIGN: Impression cytology specimens from the bulbar conjunctiva of healthy controls and KCS patients were harvested and divided into 3 groups: group 1, controls; group 2, KCS SLC-negative; and group 3, KCS SLC-positive. The number of micronuclei (MNi) in SLC-negative and SLC-positive epithelial cells of each group was counted. RESULTS: The number of MNi in SLC-negative cells of groups 1 and 2 did not exceed 1 MNi/1,000 cells. A significant increase in the frequency of micronuclei in the upper bulbar conjunctiva was noted in SLC-positive (14.75 +/- 8.09 MNi/1,000 cells) as well as SLC-negative cells (4.0 +/- 3.83 MNi/1,000 cells) of group 3. CONCLUSION: We demonstrate here that the presence of MNi in the conjunctival epithelium of KCS patients could be a characteristic feature accompanying SLC cells. The fact that increased numbers of SLC cells correlates with impaired values in clinical test as well as decreased goblet and epithelial cell densities confirms that the presence of SLC cells correlates with KCS intensity.

Active transforming growth factor-ß2 in the aqueous humor of posterior polymorphous corneal dystrophy patients.

PURPOSE: Posterior polymorphous corneal dystrophy (PPCD) is characterized by abnormal proliferation of corneal endothelial cells. It was shown that TGF-ß2 present in aqueous humor (AH) could help maintaining the corneal endothelium in a G1-phase-arrest state. We wanted to determine whether the levels of this protein are changed in AH of PPCD patients. METHODS: We determined the concentrations of active TGF-ß2 in the AH of 29 PPCD patients (42 samples) and 40 cadaver controls (44 samples) by ELISA. For data analysis the PPCD patients were divided based on either the molecular genetic cause of their disease as PPCD1 (37 samples), PPCD3 (1 sample) and PPCDx (not linked to a known PPCD loci, 4 samples) or on the presence (17 samples) or absence (25 samples) of secondary glaucoma or on whether they had undergone penetrating keratoplasty (PK, 32 samples) or repeated PK (rePK, 7 samples). RESULTS: The level of active TGF-ß2 in the AH of all PPCD patients (mean ± SD; 386.98 ± 114.88 pg/ml) in comparison to the control group (260.95 ± 112.43 pg/ml) was significantly higher (P = 0.0001). Compared to the control group, a significantly higher level of active TGF-ß2 was found in the PPCD1 (P = 0.0005) and PPCDx (P = 0.0022) groups. Among patients the levels of active TGF-ß2 were not significantly affected by gender, age, secondary glaucoma or by the progression of dystrophy when one or repeated PK were performed. CONCLUSION: The levels of active TGF-ß2 in the AH of PPCD patients are significantly higher than control values, and thus the increased levels of TGF-ß2 could be a consequence of the PPCD phenotype and can be considered as another feature characterizing this disease.

FTY720 in corneal concordant xenotransplantation.

BACKGROUND: : Currently, there are no effective treatments for the control of corneal xenograft rejection. We evaluated the efficacy and mode of action of a novel immunosuppressant, FTY720, in a model of corneal xenograft transplantation. METHODS: : Rat-to-mouse corneal xenografts were performed and the effects of treatment with daily intraperitoneal injections of FTY720 (0.5 or 3.0 mg/kg/day) or saline from 2 days pretransplantation were assessed clinically. Immunohistochemical studies of the grafts and flow cytometry of the draining lymph node subpopulations were performed at the time of clinical rejection. RESULTS: : Treatment with FTY720 delayed the onset of corneal rejection, from 8 days postgraft in saline-treated mice to 12.0 +/- 0.89 days for low-dose FTY720 treatment and 15.6 +/- 3.1 days for high-dose FTY720 treatment (both P<0.001). Histologically, FTY-treated animals had a markedly reduced inflammatory response in the anterior chamber and cornea after replacement of the xenograft epithelium with normal healthy host epithelium. In contrast, saline-treated xenografts had persisting corneal epithelial defects and ulceration. In the draining lymph nodes, FTY720 not only inhibited the increase in the cell number observed in saline-treated recipients of xenografts, but also reduced the expression of activation markers on B cells (MHC class II and CD86). CONCLUSIONS: : FTY720 treatment significantly delayed rejection and decreased its severity in a dose-dependent manner in a rat-to-mouse model of corneal xenotransplantation. Since corneal xenograft rejection is mediated not by natural antibodies or CD8+ T cells directly, but by CD4+ T cells, the data from these experiments imply that FTY720 mediated its effect via CD4+ T cells.

Posterior polymorphous corneal dystrophy in Czech families maps to chromosome 20 and excludes the VSX1 gene.

PURPOSE: Posterior polymorphous corneal dystrophy (PPCD) is an autosomal dominant disorder, affecting both the corneal endothelium and Descemet's membrane. In the Czech Republic, PPCD is one of the most prevalent corneal dystrophies. The purpose of this study was to determine the chromosomal locus of PPCD in two large Czech families, by using linkage analysis. METHODS: Linkage analysis was performed on 52 members of two Czech families with PPCD and polymorphic microsatellite markers and lod scores were calculated. The candidate gene VSX1 was also screened for mutations. RESULTS: Significant lod scores were obtained with microsatellite markers on chromosome 20. Linkage analysis delineated the Czech PPCD locus to a 2.7-cM locus on chromosome 20, region p11.2, between flanking markers D20S48 and D20S139, which excluded VSX1 as the disease-causing gene in both families. In addition, the exclusion of VSX1 was confirmed by sequence analysis. CONCLUSIONS: This study reports the localization of PPCD in patients of Czech origin to chromosome 20 at p11.2. Linkage data and sequence analysis exclude VSX1 as causative of PPCD in two Czech families. This refined locus for PPCD overlaps the congenital hereditary endothelial dystrophy (CHED1) disease interval, and it is possible that these corneal dystrophies are allelic.

Susceptibility of corneal allografts and xenografts to antibody-mediated rejection.

The effects of passive transfer of antisera containing cytotoxic antibodies to allo- and xenoantigens on survival of corneal allografts and xenografts were evaluated in experimental models. Corneas from allogeneic B10 or xenogeneic rat Lewis donors were grafted orthotopically into BALB/c mice. Recipient mice were treated with donor-specific antisera administered at the period of grafting or at 2 weeks after transplantation. Rejection was determined by the severity of corneal opacity using a standard scoring system. Treatment of graft recipients with donor-specific antisera accelerated the onset of graft rejection and significantly shortened survival times of both corneal allografts and xenografts. Corneal xenografts, which had been accepted after treatment with anti-CD4 monoclonal antibody, were acutely rejected by the passive transfer of antiserum against xenoantigens. The results suggest that corneal grafts are vulnerable to antibody-dependent immunity and that cytotoxic antibodies against graft donor antigens can mediate rejection of both corneal allografts and xenografts.

The immune response to corneal allograft requires a site-specific draining lymph node.

BACKGROUND: Recent studies have shown that head-neck draining lymph nodes (DLN) are required for priming the immune response during corneal allograft rejection. In this study we have investigated further the role of the DLN and spleen in corneal graft rejection in mice. METHODS: Individual DLN (submandibular [SM]; superficial cervical [SC]; and internal jugular) or their combinations were removed in mice undergoing corneal allografting (C57BL/10, H2(b) to BALB/c, H2(d)). In some mice, DLN from syngeneic mice were retransplanted, whereas other mice underwent removal of the spleen before corneal allografting. In a high-risk group of mice, removal of the DLN before a second corneal graft procedure was performed. RESULTS AND CONCLUSIONS: The data show that a single specific lymph node, i.e., the SM node, is the major DLN involved in corneal graft rejection whereas its nearest neighbor, the SC DLN, not only cannot substitute for the SM node in priming the immune response but may be involved with the spleen in immune privilege. Retransplantation studies of syngeneic LN indicate that the site of the DLN is more important to the process of graft rejection than the specific DLN tissue. This applies to the DLN whether it contains naive or memory allospecific T cells as shown in experiments in which removal of the SM DLN from mice who had already been primed by a previous corneal graft, prevented rejection of a second corneal graft in the same strain combination.

Lymph node removal enhances corneal graft survival in mice at high risk of rejection.

BACKGROUND: As shown previously, the submandibular (SM) lymph node (LN) is required for priming the immune response during corneal graft rejection. In this study, we wished to determine whether corneal grafts at "high-risk" of rejection were also protected after selective SM LN removal and if so to investigate whether this improved corneal graft survival was due to induction of specific regulatory/suppressor cells or was due to immunological "ignorance". METHODS: Two sets of experiments were performed. (1) Adoptive transfer of possible regulatory splenocytes from mice with long-term accepted corneal graft after SM LN removal. (2) SM LN removal and corneal grafts in "high-risk" hosts, which had been (A) subjected to corneal trauma with vascularization or (B) allosensitized by previous corneal graft or (C) allosensitized by previous skin graft. RESULTS: Adoptive transfer of splenocytes from tolerant mice after SM LN removal did not enhance corneal graft survival in naive recipients (p > 0.05). SM LN removal in mice with corneal vascularization enhanced corneal allograft survival compared to grafted controls with/without vascularization (p < 0.0001). The removal of the SM LN in mice, who had already been allosensitized by a previous corneal graft, did not statistically prolong corneal graft survival (p > 0.05). SM LN removal procedure did not delay rejection of corneal grafts in mice allosensitized by a previous skin transplant with the same strain combination (p > 0.05). CONCLUSION: The results suggest that removal of the SM LN in "high-risk" mice prevents rejection by mechanisms involving immune "ignorance", since prior allosensitization prevents graft acceptance after LN removal. In allosensitized recipients the stronger the allosensitization (skin- vs. corneal graft-presensitization) the greater the possibility of priming for rejection at alternative draining LN sites.

The application of autologous serum eye drops in severe dry eye patients; subjective and objective parameters before and after treatment.

AIM: To assess the impact of autologous serum (AS) eye drops on the ocular surface of patients with bilateral severe dry eye and to draw a comparison between the clinical and laboratory examinations and the degree of subjective symptoms before and after serum treatment. MATERIALS AND METHODS: A three-month prospective study was conducted on 17 patients with severe dry eye. AS eye drops were applied a maximum of 12 times a day together with regular therapy. Dry eye status was evaluated by clinical examination (visual acuity, Schirmer test, tear film breakup time, vital staining, tear film debris and meniscus), conjunctival impression cytology (epithelial and goblet cell density, snake-like chromatin, HLA-DR-positive and apoptotic cells) and subjectively by the patients. RESULTS: The application of AS eye drops led to a significant improvement in the Schirmer test (p < 0.01) and tear film debris (p < 0.05). The densities of goblet (p < 0.0001) and epithelial cells (p < 0.05) were significantly increased, indicating a decrease of squamous metaplasia after AS treatment. A significant decrease (p < 0.05) was found in the number of apoptotic, HLA-DR-positive and snake-like chromatin cells on the ocular surface. A significant improvement was found in all evaluated subjective symptoms. Altogether, the clinical results were improved in 77%, the laboratory results in 75% and the subjective feelings in 63% of the eyes. CONCLUSIONS: We found that three-month AS treatment led especially to the improvement of ocular surface dryness and damage of the epithelium. The improvement of dry eye after AS treatment correlated well with the clinical, laboratory and subjective findings. From the patients' subjective point of view, the positive effect of AS decreased with time, but still persisted up to three months after the end of therapy.