Immunomodulatory Effect of Human Lactoferrin on Toll-like Receptors 2 Expression as Therapeutic Approach for Keratoconus.

Keratoconus (KC) is a corneal disorder whose etiology shares a close relationship with Lactoferrin (LTF) dysregulation and Toll-like Receptors 2 (TLR2) overexpression. This study shows how these two important biomarkers are clinically and molecularly interrelated, increasing knowledge about KC pathophysiology, and opening the door to future therapies. In this prospective clinical study, serum and tear LTF concentrations were quantified in 90 KC patients and 60 controls. A correlation analysis with multiple blood and tear immunoinflammatory mediators, and KC-associated tomographic parameters, was performed. An in vitro study using HEK-BlueTMhTLR2 cell cultures was also conducted to determine the expression and functionality of TLR2 under the influence of LTF treatment. As a result, a LTF decreased was observed in KC patients compared to controls (p < 0.0001), evidencing the strong correlation with TLR2 overexpression at systemic and ocular surface level, with inflammatory mediator upregulation and with KC severity. In stimulated cell cultures, TLR2 expression was decreased using 2 mg/mL of LTF. The levels of secreted embryonic alkaline phosphatase (SEAP) and interleukin-8 (IL-8) were also reduced in supernatants after LTF treatment. As conclusions, the dysregulation of LTF and TLR2 in the ocular surface of KC patients contributes to KC severity by maintaining a detrimental chronic immune−inflammatory state. The immunomodulatory properties of LTF on TLR2 expression suggest its potential as a therapeutic approach for KC.

Shotgun Proteomics for the Identification and Profiling of the Tear Proteome of Keratoconus Patients.

Purpose: The qualitative approach followed in this study aims to obtain an extensive view of the keratoconus (KC) tear proteome, which could highlight proteins previously undetected and enlarge our knowledge of the disease's pathophysiology. Methods: Twenty-five patients diagnosed with KC and 25 control subjects were studied in a prospective, cross-sectional study. KC screening examinations, including clinical and tomographic examinations, were performed on all participants. Tear samples were collected using Schirmer strips and analyzed by liquid chromatography-tandem mass spectrometry in a data-dependent workflow. A spectral count was used as a semiquantification tool. The tear proteomes of both groups were identified and profiled, and the functional interactions and biological characterization of differential proteins were analyzed using in silico tools. Results: We identified a total of 232 proteins, of whom 133 were expressed in both groups' samples; 41 were observed only in control samples and 58 were identified just in tears of patients with KC. A semiquantitative analysis showed the dysregulation of 17 proteins in the KC samples. An in silico analysis linked proteins only expressed in KC samples to oxidative stress, skin development, and apoptosis. The dysregulation of proteins involved in iron transport, inflammation, oxidative stress, and protease inhibition was observed in the semiquantitative results. Conclusions: A shotgun analysis showed that the tear proteome of patients with KC differed from controls by more than one-third of the total proteins identified, highlighting the relationship of the proteins only expressed in KC tears with processes of cell death, oxidative damage, and inflammation. The underexpression of proteins involved in iron pathways might support the iron imbalance as a contributing factor to cellular damage and death in KC disease.

Subconjunctival PRGF Fibrin Membrane as an Adjuvant to Nonpenetrating Deep Sclerectomy: A 2-Year Pilot Study.

PURPOSE: To evaluate the potential role of the autologous PRGF (plasma rich in growth factors) fibrin membrane in tissue regeneration after glaucoma filtering surgery. MATERIALS AND METHODS: Ten patients with medically uncontrolled primary open-angle glaucoma underwent nonpenetrating deep sclerectomy and were treated with PRGF fibrin membrane as adjuvant. Intraocular pressure reduction was the primary outcome. This variable was measured preoperatively and also at each follow-up visit. Secondary outcomes included the number of antiglaucoma medications, anterior segment optical coherence tomography bleb examination, photographic bleb evaluation, and subjective clinical symptomatology evaluation. RESULTS: The surgical technique showed a significant reduction (p < 0.05) in intraocular pressure in relation to preoperative values at each time of the study, decreasing from 23.3 ± 6.4 to 15.2 ± 4.6 mm Hg at 2 years. Furthermore, the number of antiglaucoma medications consumed showed a significant reduction at the end point of the study compared with the preoperative situation. Optical coherence tomography and photographic filtering bleb variables experienced a progressive reduction during the follow-up. Subjective symptoms showed a reduction from 8.3 ± 4.5 to 4.2 ± 5.3 at 2 years. CONCLUSIONS: PRGF-Endoret treatment could promote ocular surface regeneration after glaucoma surgery, enhancing the surgery success rates and reducing the need for postoperative medications. It is important to highlight that this is a preliminary study and some large clinical studies are necessary to verify these results.

Optical coherence tomography findings in recurrent corneal erosion syndrome.

PURPOSE: To report the findings of optical coherence tomography (OCT) in the corneas of patients with recurrent corneal erosion syndrome (RCES). METHODS: Ten normal subjects and 25 patients with RCES were recruited for the study. Ten patients with RCES suffered from an acute episode of pain and 15 patients complained of typical chronic symptoms of RCES. All eyes were scanned with the anterior segment 5 line raster acquisition protocol of the Cirrus HD-OCT platform (Zeiss). The etiology of RCES was investigated and treatment was provided following a standard clinical protocol. Scans were obtained at different stages of the pathology and each patient was followed-up after treatment. RESULTS: Tear film, epithelium, basement membrane, Bowman layer, stroma, and Descemet-endothelium complex were identified by OCT in all normal corneas. The same structures were identified in nonaffected areas of RCES corneas. The findings on OCT from patients suffering from acute RCES were as follows: anterior stromal hyper-reflectivity (100%), epithelial edema (100%), and irregular breaks in the epithelium (90%). Undetected epithelial basement membrane (100%), intraepithelial basement membrane (60%), intraepithelial inclusions (60%), and anterior stromal hyper-reflectivity (40%) were seen in patients with chronic RCES. These findings correlated well with the clinical symptoms and with previously described histological reports of RCES. CONCLUSIONS: Corneal OCT can be useful to study and manage RCES. Specific OCT findings may clarify etiology, confirm a suspected diagnosis, and give advice on the decision to treat patients.

Clinical presentation and causes of recurrent corneal erosion syndrome: review of 100 patients.

PURPOSE: To study the clinical features and etiology of recurrent corneal erosion syndrome (RCES). METHODS: We examined a total of 100 patients (117 eyes) with the diagnosis of RCES who presented at our institution (Instituto Clinico Quirúrgico de Oftalmología, Bilbao, Spain). Studied data included demography, etiology, corneal location, and association with meibomian gland dysfunction (MGD). RESULTS: The mean age of patients was 44.5 (range, 14-80) years. Attributed causes of RCES were previous minor trauma (46 eyes, 39.3%), epithelial basement membrane corneal dystrophy (20 eyes, 17.1%), photorefractive keratectomy (20 eyes, 17.1%), laser-assisted in situ keratomileusis (9 eyes, 7.7%), and of unknown origin (22 eyes, 18.8%). The most frequent site of RCES was the inferior paracentral cornea (68.4%), followed by the upper cornea (21.3%) and widespread location (21.3%). An association with MGD was found in 59% of patients. CONCLUSIONS: RCES has various etiologies, which explains the variety in the clinical presentation of the disorder. Interestingly, a significant number of RCES patients begin to manifest the syndrome after keratorefractive surgery, and a high percentage of patients also present with MGD.

Proteomic analysis of the tear film in patients with keratoconus.

PURPOSE: To identify proteins differentially expressed between the tear film of keratoconus (KC) patients and control subjects using two dimensional electrophoresis (2-DE) and mass spectrometry-based techniques. METHODS: Twenty two patients (44 eyes) diagnosed with bilateral KC and 22 control subjects (44 eyes) were studied in a prospective case-control study. Keratoconus screening programs and Orbscan II topographies were performed on all participants. Tear samples were collected by the Schirmer I method using filter paper. Proteins were extracted from the Schirmer strips and separated by 2-DE. Comparison of protein patterns was performed using PDQuest Software and protein differences were identified by mass spectrometry. Finally, results were validated by western-blot. RESULTS: Four spots were identified to be differentially expressed between KC patients and control subjects. Three of them were more expressed in healthy subjects and they were identified as zinc-α2-glycoprotein (ZAG), lactoferrin, and IGKC (immunoglobulin kappa chain). The other spot was more expressed in KC patients and it was identified as ZAG. Differences in ZAG seem controversial in two different spots because different posttranslational modifications, however, analysis of both spots revealed that globally, ZAG is overexpressed in healthy subjects. Founded differences in ZAG, lactoferrin, and IGKC expression were subsequently validated by western blot. CONCLUSIONS: IGKC protein, ZAG, and lactoferrin are under-expressed in the tears of patients diagnosed with bilateral KC compared with healthy subjects. These differences could contribute to the knowledge of the pathophysiology of this disease.

Videokeratoscopic indices in relation to epidemiological exposure to keratoconus.

BACKGROUND: To establish the relationship between videokeratoscopic indices and the degree of epidemiological exposure to keratoconus in three groups of clinically normal subjects. METHODS: Cross-sectional study in which 75 subjects lacking clinical signs of keratoconus were divided into three groups according to epidemiological exposure to the condition: 25 fellow eyes of subjects with clinical signs on the contralateral eye only (the "fellow eye" group), to be compared to 25 first-degree relatives of patients with keratoconus (the "relatives" group) and 25 controls without a family history of the disease (the "control" group). Qualitative patterns and quantitative parameters describing curvature (central curvature), irregularity (root mean square of the higher-order corneal wavefront aberration), and asymmetry (inferior-superior dioptric asymmetry, Zernike vertical coma) obtained from videokeratoscopy were used for comparison between groups. RESULTS: Members of the fellow eye group featured a greater number of asymmetric curvature patterns and increased values in indices describing asymmetry and irregularity than subjects included in both control and relatives groups. Control and relatives groups were not significantly different. Despite significant differences in the distribution of values between the groups, no single index was able to effectively discriminate between groups using ROC curve analysis. CONCLUSIONS: A prior threefold classification of clinically normal subjects according to epidemiological exposure to keratoconus was not sustained by significant differences in videokeratoscopic indices when comparing between groups.

Unilateral keratoconus: videokeratography and Orbscan study--optical correction.

OBJECTIVES: To assess the frequency of unilateral keratoconus (KC) and vision correction with a contact lens (CL) in the KC eye. METHODS: Transverse study in which we revised the clinical records of 385 KC patients. Of these, 35 (9%) showed clinical unilateral KC. Corneal topography was evaluated by a Placido disk and an elevation topographer. We also determined distance and near visual acuity before and after correction with spectacles or CL in KC eye. RESULTS: Mean central corneal curvature (K) of the KC eyes was 51.45 +/- 6.65 D, versus 43.58 +/- 1.71 D for the fellow eye (P<0.001). Mean maximum curvature of the inferior cornea was 54.62 +/- 5.91 D in the KC eye and 44.66 +/- 2.38 in the fellow eye (P<0.001). Mean of the thinnest point pachymetry was 411 +/- 55 microm in the KC eye and 477 +/- 42 microm in the contralateral eye (P<0.0001). In most of the 29 patients in whom CLs were fitted, aspheric rigid gas-permeable CLs were used, corrected visual acuities were significantly better (P<0.0001) than without correction. CONCLUSIONS: All unilateral KC patients showed higher corneal curvature in the affected eye. Corneal thickness was likewise clearly reduced in the KC eye, and reduced with respect to normal values in the contralateral eye. Aspheric rigid gas-permeable CL were the most effective for improving distance and near visual acuity in patients with adequate tolerance.

Inflammatory response to contact lenses in patients with keratoconus compared with myopic subjects.

PURPOSE: To determine the levels of inflammatory molecules in the tears of patients who wore rigid gas permeable (RGP) contact lenses (CLs) and who had either keratoconus or myopia. METHODS: A prospective, case-control study with 4 groups enrolled 20 RGP CL keratoconus wearers and 28 keratoconus non-lens wearers, 20 myopic CL wearers, and 20 subjects with myopia that were non-lens wearers (1 eye per patient). Fifteen microliters of tears were collected by capillary flow. The concentration of cytokines (interleukin-6 [IL-6], IL-10, and tumor necrosis factor [TNF]-alpha), cell adhesion molecules (intercellular adhesion molecule 1 [ICAM-1] and vascular cell adhesion molecule 1 [VCAM-1]), and matrix metalloproteinase (MMP-9) was measured by enzyme-linked immunosorbent assay. RESULTS: The most significant differences associated with the wearing of RGP CLs in patients with keratoconus were seen in increased levels of proinflammatory cytokines (IL-6, 23.7 vs. 6.4 pg/mL, P = 0.001; TNF-alpha, 21.3 vs. 3.8 pg/mL, P = 0.028) and cell adhesion molecules (ICAM-1, 32.8 vs. 7.7 ng/mL, P < 0.0001; VCAM-1, 57.4 vs. 29.3 ng/mL, P < 0.0001). In patients with myopia, increased levels of TNF-alpha (4.2 vs. 1.8 pg/mL, P < 0.0001) and MMP-9 (12.9 vs. 6.1 ng/mL, P < 0.0001) were associated with the wearing of RGP CLs. CONCLUSIONS: Wearing RGP CLs induces overexpression of IL-6, TNF-alpha, ICAM-1, and VCAM-1 in the tears of patients with keratoconus. These increased levels are higher in cases with severe keratoconus.

Confocal microscopy and histopathological examination of diffuse lamellar keratitis in an experimental animal model.

PURPOSE: To investigate the cell populations and structural alterations of the cornea in an experimental model of diffuse lamellar keratitis (DLK) using confocal microscopy and histopathology. METHODS: A corneal flap was cut in 22 eyes of 11 New Zealand rabbits and the stromal interface was exposed to balanced salt solution (BSS, BSS group) and Pseudomonas aeruginosa lipopolysaccharide (LPS) endotoxin (5 mg/mL) (LPS 5 mg/mL group) and (3.5 mg/mL) (LPS 3.5 mg/mL group). Postoperatively, eyes were examined with a slit-lamp microscope (DLK grading) and confocal microscopy. Animals were sacrificed on day 3 (BSS group and LPS 5 mg/mL group) and day 4 (LPS 3.5 mg/mL group). Corneoscleral buttons were excised and processed for histopathologic examination. RESULTS: Seven eyes were excluded. Slit-lamp microscopy revealed no cellular infiltration in the BSS group (five eyes). In the LPS groups, all eyes developed DLK, with iritis only observed in grade III eyes. In the LPS 5 mg/mL group, four eyes had DLK grade III, with iritis in three eyes. In the LPS 3.5 mg/mL group, three eyes had grade II and three eyes had grade III with iritis. On confocal microscopy, the BSS group had no cellular infiltration. Dense accumulation of inflammatory cells at the interface was noted in both LPS groups. Histopathology in the BSS group had a normal appearance. In the LPS groups, an inflammatory infiltrate was present at the interface that consisted of three cell populations--eosinophils, neutrophils, and lymphocytes. CONCLUSIONS: Lipopolysaccharide endotoxin induced DLK in all exposed eyes, with iritis in a considerable proportion of eyes. The infiltrate consisted of three cell populations. Confocal microscopy showed the infiltrate in all affected eyes. Histopathological and confocal microscopic findings correlated well with the clinical appearance.

Prophylactic perioperative antiviral therapy for LASIK in patients with inactive herpetic keratitis.

PURPOSE: To report the outcome of LASIK in patients with inactive herpetic keratitis in which perioperative antiviral prophylaxis was used to prevent the recurrence of ocular herpes. METHODS: We report an uncontrolled series of five patients with inactive herpetic keratitis for at least 1 year before surgery in whom LASIK was successfully performed. All patients showed normal topography, pachymetry, and corneal sensitivity with no central corneal scarring. Perioperative prophylaxis was used in each case with oral valacyclovir and topical acyclovir ointment. RESULTS: None of the eyes developed reactivation of herpetic keratitis during follow-up. CONCLUSIONS: This study suggests that perioperative antiviral prophylaxis may protect the cornea from herpes simplex virus reactivation after LASIK.

Confocal microscopy of stage 4 diffuse lamellar keratitis with spontaneous resolution.

PURPOSE: To report confocal microscopic findings at the onset of stage 4 diffuse lamellar keratitis and after its resolution. Stage 4 is the most severe form of diffuse lamellar keratitis. Its incidence is approximately 1 in 5000 and is associated with stromal melting, deep flap folds, central haze, hyperopic shift, irregular astigmatism, and severe decrease in visual acuity. METHODS: A 22-year-old woman underwent bilateral uncomplicated laser in situ keratomileusis (LASIK) for myopia. Postoperative course in the right eye was uneventful; however, in the left eye, stage 4 diffuse lamellar keratitis developed. Confocal microscopy examination was performed in both eyes at the onset of the syndrome and after its resolution. Findings in the eye with diffuse lamellar keratitis (left eye) were compared with the uninvolved (right) eye. RESULTS: The condition improved spontaneously and 2 years later, slit-lamp microscopy showed resolution of the folds and haze with subsequent improvement of visual acuity. However, confocal microscopic examination in the left eye revealed a persistent stromal subclinical haze on both sides of the lamellar cut and prominent folds that extended into the anterior stroma. CONCLUSION: Confocal microscopy revealed that in spite of normal appearance on slit-lamp microscopy, micromorphological alterations persisted after spontaneous resolution of stage 4 diffuse lamellar keratitis.

Confocal microscopy in late-onset diffuse lamellar keratitis after laser in situ keratomileusis.

This article reports a case of diffuse lamellar keratitis, without exposure of the flap interface, that developed in a patient who underwent intraepithelial photorefractive keratectomy 1 year after bilateral LASIK. Confocal microscopy was performed in both eyes at the onset of the diffuse lamellar keratitis and after its resolution. In the eye with diffuse lamellar keratitis, abundant round structures (inflammatory cells) were present at the interface; these structures disappeared after the keratitis resolved and were not present in the contralateral eye at any time. These confocal microscopic findings further support the hypothesis that diffuse lamellar keratitis is a nonspecific inflammatory response in corneas with a lamellar interface.