Keratoconus: the possible involvement of inflammatory cytokines in its pathogenesis. An experimental study and review of the literature.

OBJECTIVE: Keratoconus (KC) is generally described as a non-inflammatory disease, characterized by thinning in the central region of the cornea with consequent tissue degradation producing impaired visual acuity. MATERIALS AND METHODS: In our experimental study, we analyzed the presence and implications of several inflammatory cytokines in the corneal tissues of patients suffering from keratoconus by immunohistochemical analysis. RESULTS: The analysis showed increased levels of inflammatory factors in the pathological tissues compared to controls, confirming that KC cannot be considered an entirely non-inflammatory pathology and that its etiopathogenesis includes several chronic inflammatory events. CONCLUSIONS: In the light of these results, the classification of KC as an inflammatory pathology or as a pathology related to inflammation might be useful in directing future research aimed at developing effective anti-inflammatory therapies to pharmacologically target the inflammatory mediators which contribute to the development and progression of the disease.

Pathogenesis and complications of chronic eye rubbing in ocular allergy.

PURPOSE OF REVIEW: To review the updated literature regarding eye rubbing complications and its association with ocular allergy disorders. RECENT FINDINGS: Atopy and ocular allergy disorders, mainly vernal keratoconjunctivitis (VKC), are strongly associated with rubbing-related complications, most probably via itching and watery eye sensations that trigger the habit of chronic eye rubbing. Vigorous and prolonged rubbing may lead to establishment of corneal remodeling and ectatic disorders, such as keratoconus. Keratoconus development in rubbed eyes can be caused by mechanical mechanisms of corneal thinning and its loss of rigidity, by elevated temperature of the epithelium during rubbing, by increased intraocular pressure (IOP) because of distending forces, and by inflammatory molecules that may serve as a causal mediator between eye rubbing and keratoconus. Other eye rubbing complications include acute hydrops and perforation, IOP spikes, iris prolapse and iridoschisis rupture of lens capsule and IOL dislocation, and even posterior segment disorders, such as glaucomatous optic neuropathy, retinal detachment and extrusion of implanted silicone oil in the eye. SUMMARY: Chronic eye rubbing in allergic eye diseases can lead to progression of keratoconus, and to other rare anterior and posterior segment complications. Strategies eliminating eye rubbing and its consequences are vital, mainly among at-risk populations, such as young children and individuals with allergic ocular disorders or corneal transplants.

Innate Immunity Biomarkers for Early Detection of Keratoconus.

Purpose: To compare toll-like receptors 2 (TLR2) and 4 (TLR4) expression in cells of corneal and conjunctival epithelium between unilateral KC patients and control subjects. Methods: Prospective cross-sectional case-control study, including 50 unilateral KC patients and 20 control subjects. TLR2 and TLR4 expression was measured with flow cytometry. Results: Mean expression of TLR2 and TLR4, in corneal and conjunctival cells, was significantly higher in KC than in subclinical and control groups (all p < 0.0001). TLR2 expression in corneal epithelial cells can predict with the highest sensitivity and specificity the probability of KC existence compared to a control (area under the curve 0.995, 95% CI: 0.987-1.000; p < 0.0001). Corneal TLR2 expression also has a predictive capacity to discriminate between subclinical KC and controls (area under the curve 0.989, 95% CI: 0.975-1.000; p < 0.0001). Conclusions: TLR2 and TLR4 expression in corneal and conjunctival epithelial cells may constitute predictive biomarkers for the early detection of KC.

Vernal Keratoconjunctivitis.

Vernal keratoconjunctivitis, a chronic bilateral seasonal allergic inflammatory disease of the eye, is an important cause of visual debilitation and impairment of quality of life in children and young adults in certain parts of the world such as the Mediterranean areas, Central and West Africa, the Middle East, Japan, the Indian subcontinent, and South America. It usually has a self-limiting course; however, in a few cases, the disease is recurrent and leads to long-term visual disabling complications such as keratoconus and limbal stem cell deficiency. The main pathogenic mechanism is immunoglobulin E mediated; however, there may be non-immunoglobulin E and certain nonspecific hypersensitivity mechanisms. The predominant cell types involved are CD4 T cells and eosinophils. The management of vernal keratoconjunctivitis is challenging. Although an acute episode can be well managed with the help of currently available topical agents, the major challenge lies in preventing recurrences and their consequences. Steroids are highly effective in controlling both an acute episode and chronic disease; however, the long-term complications of steroid use often prevent their continued use. Immunomodulators such as tacrolimus and cyclosporine may be used as steroid-sparing agents; however, the dosing and duration of use still need to be clearly defined. Surgery is required for the management of complications such as shield ulcer and corneal ectasia or opacity; however, the disease process and management are largely well defined, and genetic predisposition factors responsible for chronicity and an effective albeit safe treatment modality for the chronic form of the disease need further research.

Allergen-specific exposure associated with high immunoglobulin E and eye rubbing predisposes to progression of keratoconus.

We report two male children with persistent allergic eye disease (AED) and keratoconus (KC). Both presented with symptoms of vernal keratoconjunctivitis and decreased vision. In view of unrelenting AED, serum immunoglobulin E (IgE) quantification and identification of specific allergens were advised. Increased serum IgE levels were observed in both cases. Sunflower and sheep wool were identified as specific allergens for the first and second patient respectively by skin patch test. There was complete resolution of symptoms of AED in both patients following avoidance of causative allergens. However, the progression of KC in both eyes of the first patient and one eye of the second patient was observed. They were advised collagen cross-linking. Elevated serum IgE indicates the presence of systemic allergy. Avoiding implicated allergens help alleviate ocular symptoms. Using serum IgE and identifying specific allergen can guide in the treatment of AED and thus prevent progressive KC due to eye rubbing and resulting inflammation.

An Immunohistochemical Study of Inflammatory Cell Changes and Matrix Remodeling With and Without Acute Hydrops in Keratoconus.

PURPOSE: To determine the inflammatory cell and matrix changes in advanced keratoconus, including acute hydrops, using immunohistochemical analysis. METHODS: The corneal tissue from eight subjects with keratoconus undergoing corneal transplantation (three keratoconic buttons, five buttons post acute hydrops­one of them with extensive neovascularization following hydrops) was compared with tissue from two normal corneoscleral rims (n = 10). The corneas were sectioned and analyzed with specific markers for macrophages, lymphocytes, dendritic cells, and scar associated matrix molecules laminin, fibronectin, tenascin-C, and type III collagen. RESULTS: Populations of cells using markers for macrophages, leucocytes and antigen presenting cells were found to be associated with the epithelium and stroma of keratoconic tissue. Populations of these cells appeared decreased in hydrops-associated keratoconus except for a large increase in leucocytes in the stroma and endothelium associated with neovascularization. Extracellular matrix deposition was found to be uniquely demonstrated in localized areas of the stroma, corresponding to the site of hydrops involvement. CONCLUSIONS: Immunohistochemical analysis revealed a chronic, inflammatory process with recruitment of immunoinflammatory cells and deposition of scar tissue in keratoconus. The inflammatory markers were somewhat attenuated in hydrops-associated keratoconus corneas and thus inflammation was not considered to be a major factor in the development of acute corneal hydrops.

Analysis of superoxide dismutase 1, dual-specificity phosphatase 1, and transforming growth factor, beta 1 genes expression in keratoconic and non-keratoconic corneas.

PURPOSE: To quantitatively assess the superoxide dismutase 1 (SOD1), transforming growth factor, beta 1 (TGF-ß1), and dual-specificity phosphatase 1 (DUSP1) messenger ribonucleic acid (mRNA) expression levels as the main intracellular reactive oxygen species neutralizers, wound healing mediators, and immunomodulators (respectively) in keratoconic (KCN) and non-KCN corneas. METHODS: Total RNA was extracted from normal and keratoconic cultured corneal stromal fibroblasts. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to measure the relative expression levels of mRNAs of the SOD1, TGF-ß1, and DUSP1 genes. RESULTS: The mRNA expression of TGF-ß1 and DUSP1 was augmented in the KCN corneas (three- and fivefold, respectively; both p<0.05). The KCN and non-KCN samples showed no difference in comparative SOD1 mRNA levels. CONCLUSIONS: This study demonstrated a higher level of DUSP1 and TGF-ß1 expression as known molecules in the inflammatory process. These results may provide new insight into the complex molecular pathways underlying KCN for investigating other inflammatory molecules.

Subnormal cytokine profile in the tear fluid of keratoconus patients.

Keratoconus, historically viewed as a non-inflammatory disease, is an ectatic corneal disorder associated with progressive thinning of the corneal stroma. Recently, a few inflammatory mediators have been reported to be elevated in the tear fluid of keratoconus patients. Consequently, we investigated a wide range of inflammation regulating cytokines in the tears and sera of keratoconus and control subjects. Interleukin (IL)-1ß, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, interferon (IFN)-γ, chemokine C-C motif ligand 5 (CCL5) and tumor necrosis factor (TNF)-α were tested in tear samples and sera of keratoconus and control individuals by multiplex immuno-bead assays. Selected cytokines were further tested by standard ELISA on pooled tear samples. All cytokines in the sera were generally low, with no significant changes between keratoconus and control subjects. However, in tear fluids, clear differences were detected between the two groups. These differences include increased IL-6, and decreased IL-12, TNF-α, IFN-γ, IL-4, IL-13 and CCL5 in keratoconus compared to control tear fluids. The decreases in IL-12, TNF-α and CCL5 were statistically significant, while the IL-13 decrease was statistically significant in the severe keratoconus group only. IL-17 could not be detected by multiplex immuno-bead assay, but showed an increase in keratoconus by conventional ELISA on a limited number of pooled tear samples. Our findings confirm increased IL-6, but dispute earlier reports of increased TNF-α, and suggest a cytokine imbalance in keratoconus disrupting corneal homeostasis. Moreover, an increase in IL-17 suggests tissue degenerative processes at work, contributing to the thinning and weakening of the corneal connective tissue in keratoconus.

Corneal abnormalities in the NC/Nga mouse: an atopic dermatitis model.

PURPOSE: To study corneal abnormalities in the NC/Nga mouse, which is an animal model of atopic dermatitis. METHODS: To study histopathologic changes of the eyelid, conjunctiva, and cornea, we extracted the eyeballs together with upper and lower eyelids and fixed them for examination by light and electron microscopy or snap-froze them for immunohistochemistry. Transferase mediated-dUTP digoxigenin nick-end labeling staining was performed to detect apoptotic cells. In order to assess eye scratching behavior and the effect of tacroliums hydrate ointment, we made video recordings. RESULTS: Mice kept in a conventional room suffered from various grades of blepharoconjunctivitis and scratched their eyes furiously. Tacrolimus hydrate ointment reduced their eye-scratching behavior. Histopathologic study of the eyelid and conjunctiva showed that this blepharoconjunctivitis was caused by allergic inflammation. Mice with severe blepharoconjunctivitis showed thinning of the corneal epithelium, an irregular interface between the epithelium and stroma, subepithelial deposition of materials, and neovascularization of the stroma. Their corneas were cone shaped. Many transferase mediated-dUTP digoxigenin nick-end labeling-positive cells were recognized among superficial epithelial cells and keratocytes. CONCLUSIONS: NC/Nga mice are a useful animal model of atopic blepharoconjunctivitis. Various corneal disorders in these mice may depend on their eye-scratching behavior.

Histopathological and immunohistochemical studies of lenticules after epikeratoplasty for keratoconus.

AIMS: To examine histopathological and immunohistochemical changes in lenticules and host of corneal buttons from patients who previously underwent epikeratoplasty for keratoconus. METHODS: 12 penetrating keratoplasty specimens from patients with keratoconus who had previously undergone epikeratoplasty, eight keratoconus, and seven normal corneas were examined. Immunostaining for Sp1, alpha1-proteinase inhibitor (alpha1-PI), and alpha2-macroglobulin (alpha2M) were performed. RESULTS: In nine of the 12 lenticules, the keratoconus-like disruptions were found in Bowman's layer. Peripheral and posterior keratocyte repopulation of the lenticules was observed in all cases. Keratocyte repopulation in the anterior and mid-stromal regions of the lenticules appeared related to the time since epikeratoplasty. Sp1 nuclear staining of the basal and wing epithelial cells was more intense in lenticules and keratoconus corneas than in normal corneas. Lenticular, host, and keratoconus keratocytes showed positive Sp1 staining, whereas staining was absent in normal corneas. Compared to normal corneas, alpha1-PI and alpha2M immunostaining was lower in the lenticules, host, and keratoconus specimens. CONCLUSIONS: The epithelial cells and keratocytes repopulated in the lenticules retain keratoconus-like biochemical abnormalities such as upregulation of Sp1 and downregulation of alpha1-PI and alpha2M. The authors speculate that both keratocytes and the corneal epithelium may participate in the development of keratoconus.

Expression of HLA-G in human cornea, an immune-privileged tissue.

Human leukocyte antigen (HLA)-G retains the capacity to modulate immune responses, favoring the establishment of tolerance in solid-tissue allotransplants. To better understand the mechanisms that promote corneal allograft survival, we investigated whether HLA-G was an immunoregulatory factor involved in corneal immunology. We therefore sought HLA-G expression in corneal tissues. Corneal transplantation consists in replacing the center of a diseased cornea with normal corneal tissue. Two corneal parts are not used in such surgery: diseased central corneal tissue and peripheral normal cornea. For this study, we used healthy corneas obtained from deceased donors and diseased corneas obtained from patients with pseudophakic bullous keratopathy or keratoconus who had undergone corneal transplantation. Immunohistochemical analysis carried out on the cryopreserved corneas showed a positive immunohistochemical staining with anti-HLA-G, anti-HLA-A, -B, and -C, and anti-HLA class I monoclonal antibodies. Staining was obtained for keratocytes, epithelial cells, and endothelial cells from both healthy and pathologic human corneas, revealing the presence of HLA class I proteins, including HLA-G. HLA-G transcripts were detected in normal cornea by reverse transcriptase-polymerase chain reaction with a classical pattern of alternative splicing. The detection of HLA-G protein in adult corneas leads to the conclusion that this protein may contribute to the maintenance of the privileged immune status of cornea.

[Keratocyte apoptosis in keratoconus]. / Apoptoz keratotsitov pri keratokonuse.

Analysis of 37 corneal disks removed in perforating keratoplasty for stages II-IV keratocone and of 15 corneal disks (control) from subjects dead from mechanical injuries at the age of 17-35 years was carried out. The expression of CD95 receptors on keratocyte membranes was evaluated by indirect immunofluorescent label using rabbit antibodies. Keratocyte apoptosis was evaluated by indirect immunofluorescent labeling of nuclear DNA nucleotides. Estimation of the volumic share of keratocytes expressing CD95 on cell membrane showed it to be almost 8-fold higher and apoptosis index almost 5-fold higher in the keratocone corneal disks than in the control. The results indicate the presence of a Fas-mediated mechanism of keratocyte apoptosis and its manifold predominance in the cornea in keratocone. These results are clinically significant for the choice of pathogenetic therapy of patients with keratocone.

[Chronic endothelial cell loss of the graft after penetrating keratoplasty: influence of endothelial cell migration from graft to host]. / Chronischer Endothelzellverlust des Transplantats nach perforierender Keratoplastik: Einfluss der Migration von Endothelzellen vom Transplantat zur Wirtshornhaut.

BACKGROUND: Chronic endothelial cell loss of the graft is common after penetrating keratoplasty. Some kind of subclinical immunological reaction that is not visible at the slitlamp has been suspected as main cause for this phenomenon. Furthermore, migration of graft endothelial cells towards the host cornea has been discussed to add to this loss in special cases. In this study, 3 homogenous patient groups with similar risk of immunological reactions were examined. Main difference between these groups was the potential of graft endothelial cells to migrate towards the host cornea. PATIENTS AND METHODS: Patients with keratoconus without cataract surgery (group I with little migration potential, n = 273), patients with Fuchs endothelial dystrophy without/with cataract surgery (groups IIa/IIb with moderate migration potential, n = 89/n = 165) and patients with bullous keratopathy after cataract surgery (group III with potentially large migration tendency, n = 188) were included in the study. All patients had a first keratoplasty. Patients with glaucoma or further intraocular procedures after keratoplasty were excluded from the study. Clear graft survival and ratio of grafts without immune reactions were estimated according to Kaplan and Meier. Endothelial analysis concerned only patients without immune reactions and with at least 3 postoperative endothelial cell density values of the graft center (76 patients in I, 18 patients in IIa, 41 patients in IIb and 23 patients in III). RESULTS: Mean relative loss of endothelial cells per year was 14.0 +/- 19.0 % in group I, 17.0 +/- 19.1 % in group IIa, 20.8 +/- 18.2 % in group IIb and 29.4 +/- 17.6 % in group III (ANOVA, p < 0.01). Five years postoperatively in group I 99 %, in group IIa 98 %, in group IIb 93 % and in group III 69 % of the grafts were centrally clear (log rank test, p < 0.001). In the same period in group I 88 %, in group IIa 86 %, in group IIb 83 % and in group III 81 % of the grafts were free of immune reactions (log rank test, p < 0.05). Reasons for irreversible graft failure were immune reactions (0 in group I; 0 in group IIa; 1 in group IIb; 9 in group III, ), surface disorders (1 in group I; 0 in IIa; 1 in group IIb; 3 in group III) and endothelial failure (0 in group I; 1 in group IIa; 5 in group IIb; 6 in group III) (chi square test, p < 0.01). CONCLUSIONS: In patients with bullous keratopathy endothelial cell loss via migration seems to add significantly to the general chronic loss which is suspected to be immunological. Peripheral migration of endothelial cells, therefore, most probably contributes to limited prognosis of patients with bullous keratopathy in terms of clear graft survival. In consequence, corneal grafts for bullous keratopathy should be as large as immunologically tolerable, and endothelial cell density should be as high as possible in order to counteract this special endothelial loss factor.

[Characterization of immunological parameters of the lacrimal fluid in patients with various types of the course of keratoconus]. / Kharakteristika immunologicheskikh pokazatelei sleznoi zhidkosti u bol'nykh s razlichnymi tipami techeniia keratokonusa.

Local immunity is analyzed in 81 patients (146 eyes) aged 16-49 years with three clinical patterns of keratocone. During long remission, IgG level was the maximum. In progressive disease, sIgA and transferrin levels were increased. The most pronounced shifts were detected in the patients with acute keratocone: increased concentrations of IgM, C3 and C4 complement components, alpha 1-antitrypsin, orosomucoid, lysozyme activity, and immune complexes. These data prompt the development of pathogenetically based approaches to the treatment of patients with various clinical immunological types of keratocone.

[Loss of an eye due to hyper-IgE syndrome after corneal transplantation]. / Een verloren oog door het hyper-IgE-syndroom na een hoornvliestransplantatie.

A 37-year-old patient with bilateral keratoconus underwent a perforating cornea transplantation after acute onset of pain and hydrops of the right cornea. One day after operation endophthalmitis developed, caused by a viridans streptococcus. Hyper-IgE syndrome was suspected because of the patient's crude facial features. His medical history brought up additional symptoms of this disease. IgE levels were extremely elevated (7320 kU/l), the eosinophil count was slightly raised (0.25 x 10(9)/l). The patient was treated with several local antibiotics but his vision was only light perception at the time of discharge from the hospital. This case illustrates how an usually successful operation may have a disastrous outcome in case of late diagnosis of the hyper-IgE syndrome. The hyper-IgE syndrome can be recognized by the characteristic facial features in combination with the often extensive (juvenile) medical history with infections, and by elevated serum IgE levels. As patients with the hyper-IgE syndrome are extremely susceptible to develop infections, prophylactic antibiotic therapy is indicated in surgical procedures.

[State of local immunity in keratoconus]. / Sostoianie mestnogo immuniteta pri keratokonuse.

Local immunity was studied in 81 patients (146 eyes) aged 11-49 with keratoconus at different stages of the disease running different course. The concentrations of IgG, IgM, secretory IgA, lysozyme, immune complexes, C3 component of the complement, transferrin, and alpha 1-antitrypsin are increased in tears of patients with keratoconus, reflecting the activation of local immunity in this condition.

[Results of immunogenetic typing of lymphocytes of patients with keratoconus]. / Rezul'taty immunogeneticheskogo tipirovaniia limfotsitov bol'nykh keratokonusom.

Histotyping of HLA I antigens in 100 patients with keratoconus. Europeoids of the Chelyabinsk district, and of 701 controls (donors listed in the regional register) showed an increased incidence of HLA A28, B12, and B15, a decreased incidence of HLA B8, B13, and complete absence of HLA A11. Estimation of HLA haplotypes in keratoconus showed an increased incidence of haplotypes A1-B5 (relative risk RR = 5.9), A2-B15 (RR = 3.34), A2-B27 (RR = 2.12), and A9-B21 (RR = 4.13) and decreased incidence of A1-B8 (RR = 0.31). The incidence of incomplete phenotypes was 27%. The most incident of incomplete phenotypes was A3-B35. Reliably linked HLA haplotypes A3-B7, A2-B27, A9-B21, and A9-B35 were typical of men with keratoconus, whereas A2-B21 haplotype was characteristic of the female patients.

[Associations of HLA class I haplotype antigens with various patterns of keratoconus]. / Assotsiatsiia antigenov, gapltipov HLA I klassa s razlichnymi tipami techeniia keratokonusa.

Analysis of deviations in the distribution of HLA antigens and haplotypes characteristic of each of the three patterns of keratocone revealed a genetic predisposition to the development of a certain pattern of the illness. HLA-B27 antigen in the phenotype and increased incidence of haplotypes A2-B27 and A3-B35 were typical of patients with the progressive course of keratocone. In patients with a favorable course of the illness the incidence of HLA-A2 and B15 was the highest. Patients with a history of acute keratocone were characterized by an increased incidence of HLA-A1, A19, B7, and complete absence of HLA-A2. Our findings will help predict the course of keratocone and its relapses presenting as acute keratocone and plan the treatment strategy with due consideration for the immunogenetic features.

[The immunity of patients with keratoconus]. / Osobennosti immuniteta u bolnykh keratokonusom.

Cellular and humoral immunity parameters were studied in patients with keratoconus at different stages, aged 14 to 49 years. The results showed the immune homeostasis to be impaired in patients with local involvement of the cornea. At a total system's level the patients developed shifts in the relative content of T-lymphocytes and their subpopulations, dysimmunoglubulinemia with increased levels of immunoglobulins of the primary immune response in the blood serum, high levels of the C3 and C4 components with a reduction of the total hemolytic activity of the complement, increased levels of NBT-positive and phagocytic cells, and a decrease of the activity of serum lysozyme.