Juan A. Durán

SUBCLINICAL KERATOCONUS AND INFLAMMATORY MOLECULES FROM TEARS.

Background/aims: Tissue degradation in corneal thinning disorders, such as keratoconus (KC), involves the expression of inflammatory mediators. The purpose of this study was to determine the levels of proinflammatory cytokines and matrix metalloproteinase 9 (MMP-9) in tears from both eyes of unilateral keratoconus (KC) patients. Methods: Thirty patients diagnosed as having asymmetrical KC (30 KC eyes, and 30 subclinical KC eyes) and 20 normal control subjects (one eye) were studied in a prospective, cross-sectional study. Keratoconus screening programmes were performed on these participants. Ten microlitres of tears was collected from each eye. The concentrations of cytokines (interleukin-6 (IL-6) and tumour necrosis factor α (TNF-α)) and MMP-9 were measured by ELISA. Results: Mean values for IL-6 levels were similar in KC and subclinical KC samples (5.5 (4.9 to 6.9) vs 5.7 (4.5 to 6.2) pg/ml, p = 0.131), but significantly higher in relation to the control group (2.2 (1.0 to 4.1) pg/ml, p<0.0001). Significant differences were found in TNF-α levels between KC and subclinical KC eyes (5.4 (4.1 to 6.8) vs 4.8 (4.2 to 6.0) pg/ml, p = 0.032) and control group (1.8 (1.5 to 2.3) pg/ml, p<0.0001). Increased values of MMP-9 were found in KC (59.4 (50.6 to 66.1) ng/ml) vs subclinical KC eye (7.0 (4.8 to 8.6) ng/ml) (p<0.0001). MMP-9 levels in the control group (6.1 (3.9 to 8.3) ng/ml) and subclinical KC were similar (p = 0.203). Conclusions: IL-6 and TNF-α are overexpressed in the tears of subclinical and KC eyes. Increased MMP-9 levels were found only in the KC eye. These results indicate that the pathogenesis of KC may involve chronic inflammatory events.

Inflammatory Markers in the Tears of Patients with Ocular Surface Disease

Purpose: To determine the concentration of interleukins (IL-1β and -6) and matrix metalloproteinase 9 (pro-MMP-9) in the tears of patients with different ocular surface diseases and to examine the possible relationship between the disorders and molecular inflammation. Methods: 77 patients diagnosed as having different ocular surface disorders and 18 normal control subjects were studied. Patients were routinely examined and separated into 5 groups: (1) control, (2) blepharitis, (3) ocular allergic disease, (4) dry eye and (5) conjunctivochalasis. Ten microliters of tears were collected by a Weck cell sponge. The concentrations of IL-1β, IL-6 and pro-MMP-9 were measured by enzyme-linked immunosorbent assay, and the MMP-9 activity was evaluated with gelatin zymography. Results: Levels of IL-1β and IL-6 in tear fluid were significantly higher in conjunctivochalasis (p = 0.0062 and p = 0.0134) than in the control group. Pro-MMP-9 levels were significantly elevated in blepharitis (p = 0.013), in allergic eye disease, in dry eye and in conjunctivochalasis (all p < 0.001), in comparison to controls. Conclusions: Pro-MMP-9 levels in tears are elevated in all of the studied pathologies especially in ocular allergy and conjunctivochalasis. However, IL-1β and IL-6 were only found to be overexpressed in conjunctivochalasis. These findings illustrate the selective implication of different molecules in each disorder.

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]-α), 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-α, 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-α (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-α, ICAM-1, and VCAM-1 in the tears of patients with keratoconus. These increased levels are higher in cases with severe keratoconus.

Plasma rich in growth factors as a therapeutic agent for persistent corneal epithelial defects.

Objective: To evaluate the efficacy of topically applied autologous plasma rich in growth factors (PRGF) as a treatment for persistent epithelial defects (PEDs) of the cornea. Methods: A series of prospective noncomparative cases. Participants: Twenty eyes from 18 patients with PED with various underlying etiopathologies: neurogenic, iatrogenic, associated with burning or secondary to severe dry eye. Patients were treated with a PRGF eyedrop solution. Serial photographs of the cornea were taken until epithelialization was complete. We had previously characterized the levels of a panel of growth factors (platelet-derived growth factor, epithelial growth factor, vascular endothelial growth factor, hepatocyte growth factor, fibroblast growth factor, and nerve growth factor) in the PRGF of 11 of these patients. The following variables were additionally recorded: (1) duration of PED before treatment, (2) previous treatments, (3) time for complete epithelialization, and (4) treatments required concomitantly with PRGF. Results: Epithelial defects healed in 17 of 20 cases (85%), with a mean therapeutic time of 10.9 weeks (range 2-39 weeks). Mean progression time before treatment was 26.7 weeks (range 2-104 weeks). Growth factor concentrations were platelet-derived growth factor 12645.9 ± 1690.0 pg/mL, epithelial growth factor 468.9 ± 97.6 pg/mL, vascular endothelial growth factor 204.5 ± 119.4 pg/mL, hepatocyte growth factor 149.5 ± 173.5 pg/mL, fibroblast growth factor 82.6 ± 95.9 pg/mL, and nerve growth factor 37.7 ± 18.6 pg/mL. Conclusion: PRGF, when applied as eyedrops, is a highly effective therapeutic agent for the treatment of a broad etiopathological spectrum of corneal PEDs.

In Vitro Effects of Three Blood Derivatives on Human Corneal Epithelial Cells

PURPOSE We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. METHODS The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. RESULTS We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. CONCLUSIONS PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.

Tear proteome and protein network analyses reveal a novel pentamarker panel for tear film characterization in dry eye and meibomian gland dysfunction.

Dry eye and meibomian gland dysfunction are common ocular surface disorders. Discrimination of both conditions often may be difficult given the overlapping of signs and symptoms, and the lack of correlation with clinical parameters. A total of 144 individuals were included in this study. To search for proteome differences, tear proteins were collected by Merocel sponge and analyzed using 2D-PAGE. Comparative tear protein profile analysis indicated changes in the expression levels of fifteen proteins. Subsequent to MALDI-TOF/TOF protein identification, network analysis revealed expression/interaction connections with other proteins, thereby identifying additional putative markers. A screening validation assay demonstrated the discriminative power of six candidate biomarkers. A further validation study using multiplexed-like ELISA assays in tear samples collected with both sponge and capillary confirmed the high discriminatory power of five biomarkers: S100A6, annexin A1 (ANXA1), annexin A11 (ANXA11), cystatin-S (CST4), and phospholipase A2-activating protein (PLAA) with an area under ROC curve (AUC)≥ 97.9% (sensitivity ≥ 94.3%; specificity ≥ 97.6%) when comparing dry eye and control individuals. This panel also discriminated between dry eye, meibomian gland dysfunction and control individuals, with a global correct assignment (CA) of 73.2% between all groups. Correct assignment was not found to be significantly dependent on the tear collection method.

Efficacy of plasma rich in growth factors for the treatment of dry eye.

Purpose: To evaluate the efficacy of plasma rich in growth factors (PRGF) for the treatment of moderate/severe dry eye. Methods: PRGF treatment was administered to 16 patients who had moderate/severe dry eye diagnosed and who had not responded previously to other standard treatments. We quantified several growth factors present in the PRGF of each patient and obtained quantitative registers of the symptoms (modified score dry eye questionnaire), both before and after PRGF treatment. We also performed impression cytology to determine the degree of squamous metaplasia before and after PRGF treatment. Results: PRGF treatment was associated with a statistically significant improvement in score dry eye questionnaire values (P < 0.001). Results from impression cytology corroborated this improvement, but the reduction in the degree of squamous metaplasia was not statistically significant. In 75% of patients treated with PRGF, no further treatments were required, whereas in the remaining 25% other ocular treatments could be reduced. Conclusions: PRGF led to symptom improvement in patients with moderate/severe dry eye. Surprisingly, the symptoms recorded in the dry eye questionnaire do not always agree with the degree of squamous metaplasia measured by impression cytology.

Changes in tear protein profile in patients with conjunctivochalasis.

Purpose: To compare the protein profiles of tears from normal volunteers and patients with conjunctivochalasis (CCH), with a view to identifying proteins whose expression is altered in this pathology. Methods: Tears from 8 normal subjects and 6 patients with CCH were analyzed by 2-dimensional electrophoresis. Total protein from tears was separated in the first dimension by isoelectric focusing, and the second dimension was carried out using 8%-16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel images were analyzed using Progenesis SameSpot software. Those spots of interest were manually cut out from the gels, and the corresponding proteins were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF). Expression levels of proteins that had been found to be significantly altered were further verified by Western blot. Results: Approximately 250 spot proteins were detected in the whole tear proteome. Twenty-four spots were significantly upregulated in CCH compared with that in controls. Eleven protein spots were identified, which included proteins belonging to the S100 family (A8, A9, A4; 2.44, 1.71, and 2.82 fold upregulation, respectively), guanosine triphosphate-binding protein 2 (1.95 fold), l-lactate dehydrogenase A-like 6B (2.32 fold), fatty acid-binding protein (2.01 fold), keratin type I cytoskeletal 10 (1.81 fold), glutathione S-transferase P (2.27 fold), peroxiredoxin-1, peroxiredoxin-5 (1.79- and 1.92 fold, respectively), and cullin-4B+ glyceraldehyde 3-phosphate dehydrogenase (1.96 fold). Conclusions: We have identified a group of proteins, which is upregulated in CCH tears. Although some of them, such as S100A4, S100A8, and peroxiredoxin-5, are markers of inflammation and oxidative processes, monitoring their levels in CCH might be useful for assessing the severity and progression of the disease.

Corneal dioptric power after penetrating keratoplasty

The dioptric power of the cornea (spherical equivalent) was studied in 60 eyes operated on for penetrating keratoplasty. In order to determine the possible influence of (1) the underlying pathology, (2) the presence of neovascularisation, or (3) the size of the graft the sample was divided into four groups, with the following results: group A (keratoconus, same-sized graft) = 42.25 D; group B (keratoconus, oversized graft) = 45.16 D; group C (keratopathy with minimal or no vascularisation) = 45.34 D; group D (keratopathy with significant vascularisation) = 45.36 D. The results showed that donor-receptor disparity is the main factor determining the outcome of the postoperative corneal spherical power. There was no demonstrable influence from underlying pathology or the presence of vessels in the receptor cornea.

Decreased corneal sensitivity and tear production in fibromyalgia.

PURPOSE To investigate corneal sensitivity to selective mechanical, chemical, heat, and cold stimulation in patients with fibromyalgia (FM). METHODS Twenty patients with FM (18 women, 2 men; 51.9 +/- 2.3 years old) and 18 control subjects (16 women, 2 men; 51.7 +/- 2.4 years) participated voluntarily in the study. Subjective symptoms of ocular dryness were explored and a Schirmer I test was performed. The response to selective stimulation of the central cornea with the Belmonte gas esthesiometer was measured. RESULTS The majority (18/20) of patients with FM reported dry eye symptoms, with the ocular dryness score significantly higher in affected subjects than in healthy ones (2.3 +/- 0.1 vs. 0.05 +/- 0.02; P < 0.001). The Schirmer test results were significantly lower in patients with FM than in those in the control group (10.5 +/- 2.2 and 30.6 +/- 1.6 mm, respectively; P < 0.001). Mean corneal threshold sensitivity values to chemical stimulation (31.16% +/- 2.04% CO(2) FM; 15.72% +/- 0.67% CO(2) control), heat (1.87 +/- 0.11 degrees C FM; 0.99 +/- 0.05 degrees C control), and cold (-2.53 +/- 0.11 degrees C FM; -0.76 +/- 0.05 degrees C control) were increased in patients with FM, whereas threshold responses to mechanical stimulation did not vary significantly (123.0 +/- 8.0 mL/min FM; 107.8 +/- 4.4 mL/min control). CONCLUSIONS The reduced corneal sensitivity of patients with fibromyalgia is attributable to a moderate decrease in corneal polymodal and cold nociceptor sensitivity, which may be the consequence or the cause of the chronic reduction in tear secretion also observed in these patients.