Changes in lysyl oxidase (LOX) distribution and its decreased activity in keratoconus corneas.

Inadequate cross-linking between collagen lamellae is a characteristic feature of keratoconus corneas. The formation of covalent bonds between collagen and elastin fibrils, which maintain the biomechanical properties of the cornea, is mediated by the cuproenzyme lysyl oxidase and four lysyl oxidase-like enzymes. The aim of this study was to determine the distribution of lysyl oxidase and the total lysyl oxidase activity (lysyl oxidase and the four lysyl oxidase-like enzymes) in control and keratoconic corneas. Seven control and eight keratoconic corneas were used for the imunohistochemical detection of lysyl oxidase in corneal cryosections using two different antibodies. The total lysyl oxidase activity in the culture medium of corneal fibroblasts from six explanted keratoconic and four control corneas was measured using a fluorometric assay in the presence and absence of the lysyl oxidase inhibitor beta-aminopropionitrile and determined as the production of H(2)O(2) in nM per µg of total protein. In the control tissue, the most intense signal for lysyl oxidase was present in the corneal epithelium, in which perinuclear dots brightly projecting from more or less homogenous cytoplasmic staining may represent the lysyl oxidase propeptide. Less intense staining was present in keratocytes, the extracellular matrix and in the corneal endothelium. The epithelium of the limbus and the perilimbal conjunctiva showed intense to very intense staining. The distribution of lysyl oxidase was clearly decreased in at least five of the eight keratoconic specimens. The most marked signal reduction was observed in the stromal matrix and in keratocytes. Moreover, the signal in pathological specimens revealed a more irregular pattern, including the presence of intra- and extracellular clumps in the epithelium. Interestingly, endothelial cells showed no or very weak staining in areas just beneath negative stromal tissue. The mean activity of total lysyl oxidase in the keratoconic samples (2.60 ± 2.23 nM H(2)O(2)/µg of total protein) was more than 2.5-fold lower than in control tissue (6.83 ± 2.53 nM H(2)O(2)/µg of total protein), and the decrease was statistically significant (p = 0.0178). The location of lysyl oxidase in the healthy cornea, limbus and perilimbal conjunctiva was described. We hypothesize that the restricted lysyl oxidase distribution in keratoconic corneas, and particularly the decrease of total lysyl oxidase activity in cultured keratoconic fibroblasts, is one potential reason for the inadequate collagen cross-linking that is a hallmark of this disease.

Aberrant HLA-DR expression in the conjunctival epithelium after autologous serum treatment in patients with graft-versus-host disease or Sjögren's syndrome.

The aim of this study was to determine the effect of autologous serum (AS) eye drops on the density of human leucocyte antigen (HLA)-DR-positive epithelial cells and Langerhans cells on the ocular surface of patients with bilateral severe dry eye disease (DED) due to graft-versus-host disease (GvHD) or Sjögren's syndrome (SS). The study was conducted on 24 patients (48 eyes). AS was applied 6-10 times daily for 3 months together with regular artificial tear therapy. HLA-DR-positive cells were detected by direct immunocytochemistry on upper bulbar conjunctiva imprints obtained before and after treatment. The application of AS drops led to a statistically significant increase in the mean density of aberrant HLA-DR-positive conjunctival epithelial cells (p < 0.05) and HLA-DR-positive Langerhans cells (p < 0.05) in the GvHD group. Aberrant HLA-DR-positive epithelial cells in the SS group were decreased non-significantly. All patients reported a significant decrease in the Ocular Surface Disease Index (p < 0.01), which indicates improvement of the patient's subjective feelings after therapy. There was an expected but non-significant decrease of aberrant HLA-DR-positive conjunctival epithelial cells in the SS group only. However, the increased density of HLA-DR-positive cells, indicating slight subclinical inflammation, does not outweigh the positive effect of AS in patients with DED from GvHD.

Paraproteinemic keratopathy associated with monoclonal gammopathy of undetermined significance (MGUS): clinical findings in twelve patients including recurrence after keratoplasty.

PURPOSE: To describe the ocular findings of 12 subjects with paraproteinemic keratopathy associated with monoclonal gammopathy of undetermined significance (MGUS). METHODS: Ocular examination included corneal spectral domain optical coherence tomography. In three individuals with an initial diagnosis of a lattice or Thiel-Behnke corneal dystrophy, the TGFBI gene was screened by conventional Sanger sequencing. RESULTS: We confirmed a diagnosis of MGUS by systemic examination and serum protein electrophoresis in 12 individuals (9 males and 3 females), with a mean age at presentation of 52.2 years (range 24-63 years) and mean follow-up 6.4 years (range 0-17 years). The best-corrected visual acuity (BCVA) at presentation ranged from 1.25 to 0.32. In all individuals, the corneal opacities were bilateral. The appearances were diverse and included superficial reticular opacities and nummular lesions, diffuse posterior stromal opacity, stromal lattice lines, superficial and stromal crystalline deposits, superficial haze and a superficial ring of hypertrophic tissue. In one individual, with opacities first recorded at 24 years of age, we documented the progression of corneal disease over the subsequent 17 years. In another individual, despite systemic treatment for MGUS, recurrence of deposits was noted following bilateral penetrating keratoplasties. The three individuals initially diagnosed with inherited corneal dystrophy were negative for TGFBI mutations by direct sequencing. CONCLUSION: A diagnosis of MGUS should be considered in patients with bilateral corneal opacities. The appearance can mimic corneal dystrophies or cystinosis. In our experience, systemic treatment of MGUS did not prevent recurrence of paraproteinemic keratopathy following keratoplasty.

Using corneal confocal microscopy to track changes in the corneal layers of dry eye patients after autologous serum treatment.

BACKROUND: In vivo corneal confocal microscopy allows the examination of each layer of the cornea in detail and the identification of pathological changes at the cellular level. The purpose of this study was to identify the possible effects of a three-month treatment with autologous serum eye-drops in different corneal layers of patients with severe dry eye disease using corneal confocal microscopy. METHODS: Twenty-six patients with dry eye disease were included in the study. Corneal fluorescein staining was performed. The corneas of the right eyes were examined using in vivo corneal confocal microscopy before and after a three-month treatment with autologous serum drops. The densities of superficial and basal epithelial cells, Langerhans cells, the keratocytes and activated keratocytes, the density of endothelial cells and the status of the sub-basal nerve plexus fibres were evaluated. RESULTS: A significant decrease in corneal fluorescein staining was found after the three-month autologous serum treatment (p = 0.0006). The basal epithelial cell density decreased significantly (p = 0.001), while the density of superficial epithelial cells did not change significantly (p = 0.473) nor did the number of Langerhans cells or activated keratocytes (p = 0.223; p = 0.307, respectively). There were no differences in the other corneal cell layers or in the status of the nerve fibres. CONCLUSIONS: The results demonstrate the ability of corneal confocal microscopy to evaluate an improvement in the basal epithelial cell layer of the cornea after autologous serum treatment in patients with dry eye disease. More studies with longer follow-up periods are needed to elucidate the suitability of corneal confocal microscopy to follow the effect of autologous serum treatment on nerve fibres or other corneal layers in dry eye disease patients.

The presence of lysyl oxidase-like enzymes in human control and keratoconic corneas.

PURPOSE: Lysyl oxidases, a family comprising lysyl oxidase (LOX) and four LOX-like enzymes (LOXL1-4), catalyse the cross-linking of elastin and collagen fibrils. Keratoconus (KC) is characterized by progressive thinning leading to irregular astigmatism, resulting in significant visual impairment. Although the pathogenesis of KC remains unclear, one of the current hypotheses is based on alterations in the organization and structure of collagen fibrils. To extend existing general knowledge about cross-linking enzymes in the human cornea, in the present study we have focused on the detection of LOXL enzymes. METHOD: The localization and distribution of LOXL1-4 were assessed in cryosections of 7 control donors (three males and three females; 25-68 years; mean age 46±17.6 years) and 8 KC corneas (5 males and 3 females; 25-46 years; mean age 31.3±7.5 years) using indirect fluorescent immunohistochemistry (IHC). The specimens were examined using an Olympus BX51 microscope (Olympus Co., Tokyo, Japan) at a magnification of 200-1000x. Western blot analysis of 4 control and 4 KC corneas was performed for all tested enzymes. RESULTS: All four LOX-like enzymes were present in all layers of control corneas as well as in the limbus and conjunctiva. Almost no differences between control and pathological specimens were found for LOXL1. A lower staining intensity of LOXL2 was found using IHC and Western blot analysis in KC specimens. Decreases of the signal and small irregularities in the staining were found in the epithelium, keratocytes and extracellular matrix, where a gradual anterior-posterior weakening of the signal was observed. LOXL3 IHC staining was lower in the corneal stromal extracellular matrix and keratocytes of KC samples. No prominent differences were detected using IHC for LOXL4, but a slight decrease was observed in KC corneas using Western blot analysis. CONCLUSION: We presume that the decrease of LOXL2 in KC corneas is more likely a consequence of the associated pathological processes (activation of stromal cells due to tissue weakening and consequent structural changes) than a direct cause leading to KC development. At this time, we are unable to provide a coherent explanation for the observed decrease of LOXL3 and LOXL4 in KC corneas.

Validation of rs2956540:G>C and rs3735520:G>A association with keratoconus in a population of European descent.

Corneal ectasias, among which keratoconus (KC) is the single most common entity, are one of the most frequent reasons for corneal grafting in developed countries and a threatening complication of laser in situ keratomileusis. Genome-wide association studies have previously found lysyl oxidase (LOX) and hepatocyte growth factor (HGF) associated with susceptibility to KC development. The aim of our study was to validate the effects of seven single-nucleotide polymorphisms (SNPs) within LOX and HGF over KC. Unrelated Czech cases with KC of European descent (108 males and 57 females, 165 cases in total) and 193 population and gender-matched controls were genotyped using Kompetitive Allele Specific PCR assays. Fisher's exact tests were used to assess the strength of associations. Evidence for association was found for both of the tested loci. It was strongest for rs3735520:G>A near HGF (allelic test odds ratio (OR)=1.45; 95% confidence interval (CI), 1.06-1.98; P=0.018) with A allele being a risk factor and rs2956540:G>C (OR=0.69; 95% CI, 0.50-0.96; P=0.024) within LOX with C allele having a protective effect. This first independent association validation of rs2956540:G>C and rs3735520:G>A suggests that these SNPs may serve as genetic risk markers for KC in individuals of European descent.

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.

Further genetic and clinical insights of posterior polymorphous corneal dystrophy 3.

IMPORTANCE: Posterior polymorphous corneal dystrophy (PPCD) is a very rare disorder characterized by primary changes of the posterior corneal layers. Sequence variants in 3 genes are associated with the development of PPCD, including ZEB1 that is responsible for PPCD3. Evidence suggests at least 1 more gene remains to be identified. OBJECTIVE: To determine the molecular genetic cause of PPCD3. DESIGN: We performed extensive ophthalmological examination, including rotating Scheimpflug imaging technology and specular microscopy, and direct sequencing of the ZEB1 coding region. Comprehensive review of published PPCD3-causing variants was undertaken. SETTING: Ophthalmology department of a university hospital. PARTICIPANTS: Four Czech probands. MAIN OUTCOMES AND MEASURES: Results of ophthalmological examination and direct sequencing of the ZEB1 coding region. RESULTS: The following 2 novel frameshift mutations within ZEB1 were identified: c.2617dup in exon 8 in a 22-year-old woman, considered to be most likely de novo in origin, and c.698dup in exon 6 in a 20-year-old man. The first patient had mild changes consistent with PPCD and bilateral best-corrected visual acuity of 1.00. The corneal phenotype of the patient in the second case was more severe, with best-corrected visual acuity of 0.40 OD and 0.05 OS. Corneas of both probands were abnormally steep (keratometry readings, flat ≥ 47.4 diopters [D] and steep ≥ 49.2 D) with increased pachymetry values but no pattern indicative of keratoconus. Specular microscopy in both patients revealed reduced endothelial cell density (range, 1055/mm² to 1655/mm²). Both probands had a history of surgery for inguinal hernia; the male patient also reported hydrocele. CONCLUSIONS AND RELEVANCE: Nucleotide changes within the coding region of ZEB1 underlie the pathogenesis of PPCD in 4 of 23 Czech probands (17%). The cumulative de novo ZEB1 mutation rate is at least 14%. Possible involvement of ZEB1 sequence variants not readily identified by direct sequencing of coding regions needs to be further investigated. Our findings also have implications for patient counseling.

Large proteoglycan complexes and disturbed collagen architecture in the corneal extracellular matrix of mucopolysaccharidosis type VII (Sly syndrome).

PURPOSE: Deficiencies in enzymes involved in proteoglycan (PG) turnover underlie a number of rare mucopolysaccharidoses (MPS), investigations of which can considerably aid understanding of the roles of PGs in corneal matrix biology. Here, the authors analyze novel pathologic changes in MPS VII (Sly syndrome) to determine the nature of PG-collagen associations in stromal ultrastructure. METHODS: Transmission electron microscopy and electron tomography were used to investigate PG-collagen architectures and interactions in a cornea obtained at keratoplasty from a 22-year-old man with MPS VII, which was caused by a compound heterozygous mutation in the GUSB gene. RESULTS: Transmission electron microscopy showed atypical morphology of the epithelial basement membrane and Bowman's layer in MPS VII. Keratocytes were packed with cytoplasmic vacuoles containing abnormal glycosaminoglycan (GAG) material, and collagen fibrils were thinner than in normal cornea and varied considerably throughout anterior (14-32 nm), mid (13-42 nm), and posterior (17-39 nm) regions of the MPS VII stroma. PGs viewed in three dimensions were striking in appearance in that they were significantly larger than PGs in normal cornea and formed highly extended linkages with multiple collagen fibrils. CONCLUSIONS: Cellular changes in the MPS VII cornea resemble those in other MPS. However, the wide range of collagen fibril diameters throughout the stroma and the extensive matrix presence of supranormal-sized PG structures appear to be unique features of this disorder. The findings suggest that the accumulation of stromal chondroitin-, dermatan-, and heparan-sulfate glycosaminoglycans in the absence of ß-glucuronidase-mediated degradation can modulate collagen fibrillogenesis.