Topical flagellin protects the injured corneas from Pseudomonas aeruginosa infection

Among bacterial pathogens, Pseudomonas (P.) aeruginosa infection is the most sight threatening. The corneal innate immune responses are key mediators of the host’s defense to P. aeruginosa. Using a mouse model of Pseudomonas keratitis, we evaluated the protective effects of topical application of flagellin, a ligand for Toll-Like receptor 5 (TLR5), on the development of Pseudomonas keratitis and elucidated the underlying mechanisms. Topical application of purified flagellin 6 and 24 h prior to P. aeruginosa inoculation on injured mouse corneas significantly attenuated clinical symptoms of P. aeruginosa keratitis, decreased bacterial burden, and suppressed infection induced inflammation in the B6 mouse cornea. Topical application of flagellin on wounded cornea induced PMN infiltration and markedly upregulated cathelicidin-related antimicrobial peptide (CRAMP) expression. In PMN depleted mice, flagellin promoted bacterial clearance in the cornea compared to that of the PBS treated mice, but was unable to prevent corneal perforation and systemic bacterial dissemination and sepses. Deletion of CRAMP increased corneal susceptibility to P. aeruginosa and abolished flagellin-induced protection in B6 mice. Our findings illustrate the profound protective effect of flagellin on the cornea innate defense, a response that can be exploited for prophylactic purposes to prevent contact lens associated Pseudomonas keratitis.     

The role of cytokines and pathogen recognition molecules in fungal keratitis - Insights from human disease and animal models

Fungal infections of the cornea are an important cause of blindness and visual impairment worldwide, with contact lens wear being the main risk factor in the USA and other industrialized countries, and traumatic injury being the main risk factor in developing countries. In this review, we highlight recent advances in the understanding of the host response to Aspergillus and Fusarium species in infected human corneal tissue and in mouse models of fungal keratitis.     

Trichophyton Spp. fungal keratitis in 22 years old female contact lenses wearer

Fungal keratitis represents one of the most difficult forms of microbial keratitis to diagnose and treat successfully. It is difficult to obtain correct diagnosis and topical antifungal preparations. Fungi can cause severe stromal necrosis and enter the anterior chamber by penetrating an intact Descemet membrane. The most common pathogens are filamentous fungi (Aspergillus and Fusarium spp.) and Candida albicans. The incidence of Trichophyton spp. keratitis is 5%. A 22 years old female contact lenses wearer after keratitis developed corneal melting syndrome, spontaneous perforation of the cornea and complicated cataract of the left eye. Conjunctival swab was sterile as well as first sample of corneal tissue and sample from the anterior chamber. Urgent therapeutic perforating keratoplasty (PK), was performed together with extracapsular cataract extraction and the implantation of the intraocular lens in the posterior chamber. The patient was treated with ciprofloxacin and diflucan (systemic therapy); with dexamethason and atropin (subconjunctivaly) and chlorhexidine, brolene, levofloxacin, polimyxin B, and dexamethason/neomycin (topically). Microbiology evaluation was performed once again following excisional biopsy of the intracameral portion of the lesion. The presence of Trichophyton spp. was finally confirmed. Itraconazole and garamycin were included in the systemic therapy. Corneal graft was clear for 17 days but decompensated 28 days after the PK. After two weeks microorganisms invaded the vitreous and caused endophthalmitis. Despite urgent pars plana vitrectomy patient developed endophthalmitis, lost light sensation and developed phthysis. Evisceration and the implantation of silicon prosthesis was done. Perforating keratoplasty is a method of choice in treating severe infectious keratitis unresponsive to conservative treatment but without the eradication of microorganisms it cannot restore the vision or save the eye. Trichophyton spp. may cause a severe disease of the anterior and posterior part of the eye which may finish with the lost of vision/eye. Prompt diagnosis and treatment of Trichophyton spp. keratitis are essential for a good visual outcome.     

Keratitis

Corneal inflammation or keratitis is a significant cause of ocular morbidity around the world. Fortunately, the majority of the cases are successfully managed with medical therapy, but the failure of therapy does occur, leading to devastating consequences of either losing the vision or the eye. This review attempts to provide current information on most, though not all, aspects of keratitis.Corneal inflammation may be ulcerative or nonulcerative and may arise because of infectious or noninfectious causes. The nonulcerative corneal inflammation may be confined to the epithelial layer or to the stroma of the cornea or may affect both. For clarity, this section has been divided into nonulcerative superficial keratitis and nonulcerative stromal keratitis. While the former usually includes hypersensitivity responses to microbial toxins and unknown agents, the latter can be either infectious or noninfectious.In the pathogenesis of ulcerative keratitis, microorganisms such as bacteria, fungi, parasites (Acanthamoeba), or viruses play an important role. Approximately, 12.2% of all corneal transplantations are done for active infectious keratitis. Available world literature pertaining to the incidence of microbial keratitis has been provided special place in this review. On the other hand, noninfectious ulcerative keratitis can be related to a variety of systemic or local causes, predominantly of autoimmune origin.     

Present Status of Corneal Contact Lenses in Medical Practice

Common indications for and contraindications to corneal contact lens wear are outlined. The processes of adaptation of the cornea to a correctly fitted contact lens are described. Biomicroscopic examination of the cornea revealed that 29 of 250 eyes in the authors'' series showed small abrasions at some time during the fitting period. The dangers of infection of a simple corneal abrasion by such pathogens as Pseudomonas aeruginosa are stressed. Prophylactic treatment of abrasions with a topical antibiotic preparation containing polymyxin is recommended. Serious criticism is levelled at the practice of estimating the fit of a contact lens by the fluorescein pattern only, and repeated keratometry readings combined with biomicroscopy are considered to be essential. It is proposed that the evaluation, fitting and after-care of the contact lens patient should be the responsibility of an ophthalmologist.     

Dipeptidyl peptidase IV (DPPIV) activity in the tear fluid as an indicator of the severity of corneal injury: a histochemical and biochemical study

Comparative histochemical and biochemical studies on the catalytically active protease Dipeptidyl peptidase IV (DPPIV), have been performed in the rabbit cornea and the tear fluid using a sensitive fluorogenic substrate, Gly-Pro-7-amino-4-Trifluoromethyl Coumarine (AFC). In both normal and experimentally injured corneas, DPPIV activity was detected histochemically and in the tear fluid biochemically. In contrast to the normal cornea where DPPIV activity was absent and in the tear fluid where it was low, during continuous wearing of contact lenses or repeated irradiation of the cornea with UVB rays, slight DPPIV activity appeared first in the superficial layers of the corneal epithelium, while later increased activity was present in the whole epithelium. This paralleled elevated DPPIV activity in the tear fluid. Moreover, during continuous contact lens wear, the increased DPPIV activity in the tear fluid was, in many cases, coincidental with the presence of capillaries in the limbal part of the corneal stroma. After severe alkali burns when corneal ulcers appeared, collagen fragments were active for DPPIV, which was associated with high DPPIV activity in the tear fluid. In conclusion, Gly-Pro-AFC was found to be useful for comparative histochemical and biochemical studies on DPPIV activity in the experimentally injured rabbit eye. Using the method of the tear film collection by a short touch of substrate punches to the respective site of the cornea or conjunctiva we can show that in experimental injuries (wearing of contact lenses, irradiation of the cornea with UVB rays), the damaged corneal cells were the main source for DPPIV activity in the tear fluid. It is suggested that the activity of DPPIV measured in the tear fluid might serve as an indicator of early corneal disorders, e.g. corneal vascularization related to contact lens wear.     

Toll-like receptor 2 siRNA suppresses corneal inflammation and attenuates Aspergillus fumigatus keratitis in rats

Toll-like receptors (TLRs) are key components of innate immunity that detect microbial infection and trigger host defense responses. However, they are capable of initiating both protective and damaging immune responses, as exaggerated expression of inflammatory components can have devastating effects on the host. We previously reported that TLR2 in corneal epithelium has an important role in the pathogenesis of fungal keratitis, however, how the corneal inflammation is modulated remains to be elucidated. This study aims to investigate the effect of targeting TLR2 on Aspergillus fumigatus keratitis in rats. The control or TLR2 small interfering RNA (siRNA) was applied sub-conjunctively and topically to the cornea. TLR2 immunostaining was performed to determine the feasibility of TLR2 siRNA delivery. Production of inflammatory cytokines and chemokines were determined by real-time quantitative PCR. Polymorphonuclear leukocyte (PMN) infiltration was assessed by myeloperoxidase activity. It was found that rat corneas treated with TLR2 siRNA showed a significant reduction of TLR2 expression in corneal epithelium. TLR2 siRNA treatment improved the outcome of keratitis, which was characterized by decreased corneal opacity, less corneal perforation, suppressed PMN infiltration, reduced production of inflammatory cytokines and chemokines, and less fungal burden. In conclusion, TLR2 siRNA treatment attenuated A. fumigatus keratitis by suppressing corneal inflammation and preventing fungal invasion, suggesting a novel avenue to control fungal infection and avert damage caused by excessive inflammation.     

Bacterial biofilm on contact lenses and lens storage cases in wearers with microbial keratitis

Bacterial biofilm formation on contact lenses (CLs), and CL storage cases may be a risk factor for CL-associated corneal infection and may explain the persistence of organisms in CL storage cases. This study evaluated biofilm formation on, and microbial contamination of, CLs and CL storage cases from patients with microbial keratitis. Contact lenses and CL storage cases from 20 wearers with microbial keratitis were sampled microbiologically and visualized using scanning electron microscopy (SEM). Culture results from the cornea were also noted. Bacterial biofilm was present more frequently ( P < 0·05) on CL storage case surfaces (17/20) compared with CL surfaces (11/20) and biofilm density was significantly greater on case surfaces ( P < 0·05). There was no association between poor compliance and microbial contamination of the CL storage case, nor between poor compliance and biofilm formation or density on the CL or CL storage case. Biofilm formation occurred equally frequently with hydrogen peroxide and chlorine release care systems. Microbial keratitis in CL wearers is frequently associated with bacterial biofilm in the CL storage case. Despite the use of current CL disinfection systems, the CL storage case is a favourable environment for proliferation of certain organisms. Biofilm on CLs may prolong the retention time of organisms at the ocular surface and increase their potential pathogenicity.     

Maintaining transparency: a review of the developmental physiology and pathophysiology of two avascular tissues

The lens and cornea are transparent and usually avascular. Controlling nutrient supply while maintaining transparency is a physiological challenge for both tissues. During sleep and with contact lens wear the endothelial layer of the cornea may become hypoxic, compromising its ability to maintain corneal transparency. The mechanism responsible for establishing the avascular nature of the corneal stroma is unknown. In several pathological conditions, the stroma can be invaded by abnormal, leaky vessels, leading to opacification. Several molecules that are likely to help maintain the avascular nature of the corneal stroma have been identified, although their relative contributions remain to be demonstrated. The mammalian lens is surrounded by capillaries early in life. After the fetal vasculature regresses, the lens resides in a hypoxic environment. Hypoxia is likely to be required to maintain lens transparency. The vitreous body may help to maintain the low oxygen level around the lens. The hypothesis is presented that many aspects of the aging of the lens, including increased hardening, loss of accommodation (presbyopia), and opacification of the lens nucleus, are caused by exposure to oxygen. Testing this hypothesis may lead to prevention for nuclear cataract and insight into the mechanisms of lens aging. Although they are both transparent, corneal pathology is associated with an insufficient supply of oxygen, while lens pathology may involve excessive exposure to oxygen.     

Viral Capsid Is a Pathogen-Associated Molecular Pattern in Adenovirus Keratitis

Human adenovirus (HAdV) infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9) signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9^−/− mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD). These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins.     

Raman Microspectroscopy Analysis in the Treatment of Acanthamoeba Keratitis

Acanthamoeba keratitis is a rare but serious corneal disease, often observed in contact lens wearers. Clinical treatment of infected patients frequently involves the use of polyhexamethylene biguanide (PHMB), a polymer used as a disinfectant and antiseptic, which is toxic also for the epithelial cells of the cornea. Prompt and effective diagnostic tools are hence highly desiderable for both starting early therapy and timely suspension of the treatment. In this work we use Raman microspectroscopy to analyse in vitro a single Acanthamoeba cell in cystic phase. In particular, we investigate the effect of PHMB at the single-cell level, providing useful information on both the underlying biochemical mechanism and the time frame for Acanthamoeba eradication in ocular infections. Furthermore, we demonstrate that Raman spectroscopy, in conjunction with standard multivariate analysis methods, allows discriminating between live and dead Acanthamoebas, which is fundamental to optimizing patients’ treatment.     

Geometric optical optimization of the corneal lens of Notonecta glauca

The optimal shape of the corneal lens of the water bug backswimmer (Notonecta glauca) and the optimal shape and position of the thin transition layer between the distal and proximal units of its cornea are theoretically determined. Using a geometric optical method, first the shape of a geometric interface between the lens units is determined, which eliminates the longitudinal spherical aberration. This interface is investigated for differently formed thick lenses when the medium in contact with the entrance surface of the lens is water or air. The optimal transition layer for the amphibious backswimmer is that, the boundaries of which are the theoretical interfaces for water and air, and the refractive index varies continuously in it. The optimal shape of the corneal lens is determined, with the disadvantageous lenses, with respect to the possible minimal spherical aberration and amount of reflected light from the transition layer, being rejected. The optimal position of the transition layer in the cornea can be obtained from the minimization of the amount of diffracted light on the marginal connection of the layers. The optimal corneal lens for backswimmer has ellipsoid boundary surfaces; the optimal transition layer in it is thin bell-shaped, at the marginal connection of which there is no dimple, the maximum of the layer is on the margin of the cornea. The shape of the theoretically optimal corneal lens, the shape and position of the theoretically optimal transition layer agree well with those of Notonecta glauca. The question posed, the geometric optical method used and the results presented are of general importance, and not only with respect to vision in the bug Notonecta, but also in the fossil trilobites, or in the wave guide theories which have been employed in similar modelling problems, in design of system of lenses without spherical aberration, for example.     

A Dual Role for Corneal Dendritic Cells in Herpes Simplex Keratitis: Local Suppression of Corneal Damage and Promotion of Systemic Viral Dissemination

The cornea is the shield to the foreign world and thus, a primary site for peripheral infections. However, transparency and vision are incompatible with inflammation and scarring that may result from infections. Thus, the cornea is required to perform a delicate balance between fighting infections and preserving vision. To date, little is known about the specific role of antigen-presenting cells in viral keratitis. In this study, utilizing an established murine model of primary acute herpes simplex virus (HSV)-1 keratitis, we demonstrate that primary HSV keratitis results in increased conventional dendritic cells (cDCs) and macrophages within 24 hours after infection. Local depletion of cDCs in CD11c-DTR mice by subconjuntival diphtheria toxin injections, led to increased viral proliferation, and influx of inflammatory cells, resulting in increased scarring and clinical keratitis. In addition, while HSV infection resulted in significant corneal nerve destruction, local depletion of cDCs resulted in a much more severe loss of corneal nerves. Further, local cDC depletion resulted in decreased corneal nerve infection, and subsequently decreased and delayed systemic viral transmission in the trigeminal ganglion and draining lymph node, resulting in decreased mortality of mice. In contrast, sham depletion or depletion of macrophages through local injection of clodronate liposomes had neither a significant impact on the cornea, nor an effect on systemic viral transmission. In conclusion, we demonstrate that corneal cDCs may play a primary role in local corneal defense during viral keratitis and preserve vision, at the cost of inducing systemic viral dissemination, leading to increased mortality.     

Ocular lesions associated with attachment of the copepod Ommatokoita elongata (Lernaeopodidae: Siphonostomatoida) to corneas of Pacific sleeper sharks Somniosus pacificus captured off Alaska in Prince William Sound

Twenty eyes from 10 Pacific sleeper sharks Somniosus pacificus, infected with the copepod Ommatokoita elongata, were collected in Prince William Sound, Alaska, and the eyes of an additional 18 S. pacificus captured in the same area were inspected for copepods. Prevalence of infection by adult female O. elongata was 97% (n = 28); mean intensity of infection was 1.89 (+/-1SD = 0.32) adult female copepods per infected shark and 1.0 (+/- 1SD = 0.0) adult female copepods per infected eye. Five of the 20 collected eyes were infected by O. elongata chalimi, and 9 of 20 eyes had 1 to several remnants of bullae embedded in the cornea. Bullae were each associated with a corneal opacity, and anchoring plugs of chalimi were associated with pinpoint lesions in the cornea or conjunctiva. All eyes exhibited marked edema and erosion of the bulbar conjunctiva, and this torus-shaped lesion corresponded to each O. elongata adult female's presumed feeding and abrasion radius. Histological examinations revealed lesions in the anterior segment of eyes to be generally similar, but graded, in severity, and in all eyes they involved the conjunctiva, cornea, filtration angle, and iris. Epithelial lesions were characterized by corneal ulceration, dysplasia, hyperplasia, and heterophilic keratitis, and by ulcerative conjunctivitis accompanied by epithelial hyperplasia with rete peg formation. Disorganization of fibers, necrosis, mineralization, minimal heterophilic influx, and perilimbic neovascularization were associated with bullae in the corneal stroma. Within the limbus there was diffuse histiocytic and lymphocytic inflammation and marked lymphofollicular hyperplasia. Heterophilic and mononuclear anterior uveitis affecting the filtration angle and anterior surface of the iris was also observed in most eyes. One eye had a partial transcorneal prolapse of a ruptured lens, with degenerative changes in the ruptured lens and severe keratitis associated with the anchoring devices of an adult copepod and several chalimi. Fourteen eyes exhibited 1 to several, randomly distributed, small, round to irregular, corneal opacities or pits that were not associated with copepods, and it is likely that these opacities represented lesions associated with adult female or larval anchoring devices from past infections. The avascular cornea represents a niche that is somewhat shielded from host immune reactions, and this, and the fact that the general body surface of sleeper sharks is covered by tall and sharp placoid scales, may partially explain the corneal attachment of O. elongata adult females. It was concluded that O. elongata infections can lead to severe vision impairment in Pacific sleeper sharks but that these infections do not significantly debilitate hosts because they probably do not need to rely on acute vision for their survival.     

Maintaining corneal integrity how the "window" stays clear

The anterior surface of the eye is composed of the cornea, conjunctiva, and the zone between the two called the limbus. The cornea must maintain optical clarity to retain good vision. However, the ocular surface is vulnerable to trauma, microbial infection, and exposure to environmental toxins. This places the cornea, especially, at risk for disruptions of the epithelial barrier and subsequent immunopathological events. Cell-cell and cell-matrix attachment junctions incorporating adhesion molecules ensure that the epithelial barrier remains intact. Protein components of the basement membrane, including laminins, are vital to the adhesion of corneal epithelial cells to the underlying stroma and function to enhance the strength of the bond between epithelium and connective tissue. Epithelial cells also play an early and crucial role in the initiation of ocular surface responses should a potentially antigenic molecule enter into deeper corneal tissues. For example, epithelial cells may produce and release cytokines such as interleukin-1 (IL-1). The delicate balance between the matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are central to mechanisms regulating dissolution of the extracellular matrix that may be a consequence of infection or wound healing. Adhesion molecules, cytokines and chemokines, and MMPs and TIMPs thus participate in the corneal response to immunologic challenge or wounding. They may also be involved in corneal pathologies associated with genetic diseases, diabetes, and vitamin A deficiency. In addition these molecules are components of cellular pathways underlying the clinical complications often observed with contact lens wear and refractive surgeries used to improve visual acuity.     

An essential role for antibody in neutrophil and eosinophil recruitment to the cornea: B cell-deficient (microMT) mice fail to develop Th2-dependent, helminth-mediated keratitis.

Invasion of the corneal stroma by neutrophils and eosinophils and subsequent degranulation disrupts corneal clarity and can result in permanent loss of vision. In the current study, we used a model of helminth-induced inflammation to demonstrate a novel role for Ab in mediating recruitment of these inflammatory cells to the central cornea. C57BL/6 and B cell-deficient (microMT) mice were immunized s. c. and injected intrastromally with Ags from the parasitic helminth Onchocerca volvulus (which causes river blindness). C57BL/6 mice developed pronounced corneal opacification, which was associated with an Ag-specific IL-5 response and peripheral eosinophilia, temporal recruitment of neutrophils and eosinophils from the limbal vessels to the peripheral cornea and subsequent migration to the central cornea. In contrast, the corneas of microMT mice failed to develop keratitis after intrastromal injection of parasite Ags unless Ags were injected with immune sera. Eosinophils were recruited from the limbal vessels to the peripheral cornea in microMT mice, but failed to migrate to the central cornea, whereas neutrophil recruitment was impaired at both stages. With the exception of IL-5, T cell responses and peripheral eosinophils were not significantly different between C57BL/6 and microMT mice. Taken together, these findings not only demonstrate that Ab is required for the development of keratitis, but also show that recruitment of neutrophils to the cornea is Ab-dependent, whereas eosinophil migration is only partially dependent upon Ab interactions.     

Corneal thickness in congenital glaucoma

Central corneal thickness is very important measurement in glaucoma treatment because it influences the eye pressure measurements. A thinner cornea gives us artifactually lower intraocular pressure and a thicker cornea gives higher intraocular pressure reading, so it has to be corrected in both cases. The aim of this study is to compare central corneal thickness between congenital glaucoma patients and normal subjects. Prospective study included 27 patients with congenital glaucoma and 35 patients in control group. First group was subdivided in two subgroups: A--8 earlier operated patients, B--19 patients treated with topic therapy. Patients had no other corneal disorders, history of trauma, corneal surgery and they were not contact lens wearers. Measurements were performed by specular microscope Tomey EM 3000 on central corneas. This study showed that patients with congenital glaucoma have lower central corneal thickness than normal subjects. Also, the study showed that antiglaucomatous operation doesn't influence central corneal thickness. Central corneal thickness need to be a routine part of examination measurements because of need to correct intraocular pressure according to it, but also the thinner corneas values can suggest congenital glaucoma diagnosis beside the other parameters.     

14例茄病镰刀菌所致角膜溃疡临床分析

目的探讨真菌性角膜溃疡的病原学特点、临床表现及抗真菌综合治疗的方法。方法对2004年10月-2006年10月送检的疑似感染的角膜标本进行镜检、培养及菌种鉴定。对其中14例病历资料完整的茄病镰刀菌所致角膜溃疡患者进行临床分析。结果在33例送检标本中分离出茄病镰刀菌24株（72．7％）。上述14例患者中,有3例角膜穿孔合并眼内炎行眼球内容物除去术,3例行结膜瓣或羊膜移植,1例角膜移植,7例经非手术治疗保留较好视力。结论茄病镰刀菌是我国北方真菌性角膜炎的主要致病菌,可导致视力严重受损且治愈困难。早期诊断配合以抗真菌为主的综合治疗可阻止病情进展,明显改善视力。     

Ubiquitous lens alpha-, beta-, and gamma-crystallins accumulate in anuran cornea as corneal crystallins

Corneal epithelium is known to have high levels of some metabolic enzymes such as aldehyde dehydrogenase in mammals, gelsolin in zebrafish, and alpha-enolase in several species. Analogous to lens crystallins, these enzymes and proteins are referred to as corneal crystallins, although their precise function is not established in any species. Although it is known that after lentectomy, the outer cornea undergoes transdifferentiation to regenerate a lens only in anuran amphibians, major proteins expressed in an anuran cornea have not been identified. This study therefore aimed to identify the major corneal proteins in the Indian toad (Bufo melanostictus) and the Indian frog (Rana tigrina). Soluble proteins of toad and frog corneas were resolved on two-dimensional gels and identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight and electrospray ionization quadrupole time-of-flight. We report that anuran cornea is made up of the full complement of ubiquitous lens alpha-, beta-, and gamma-crystallins, mainly localized in the corneal epithelium. In addition, some taxon-specific lens crystallins and novel proteins, such as alpha- or beta-enolase/tau-crystallin, were also identified. Our data present a unique case of the anuran cornea where the same crystallins are used in the lens and in the cornea, thus supporting the earlier idea that crystallins are essential for the visual functions of the cornea as they perform for the lens. High levels of lens alpha-, beta-, and gamma-crystallins have not been reported in the cornea of any species studied so far and may offer a possible explanation for their inability to regenerate a lens after lentectomy. Our data that anuran cornea has an abundant quantity of almost all the lens crystallins are consistent with its ability to form a lens, and this connection is worthy of further studies.     

Triose phosphate isomerase, a novel enzyme-crystallin, and tau-crystallin in crocodile cornea. High accumulation of both proteins during late embryonic development

Several enzymes are known to accumulate in the cornea in unusually high concentrations. Based on the analogy with lens crystallins, these enzymes are called corneal crystallins, which are diverse and species-specific. Examining crystallins in lens and cornea in multiple species provides great insight into their evolution. We report data on major proteins present in the crocodile cornea, an evolutionarily distant taxon. We demonstrate that tau-crystallin/alpha-enolase and triose phosphate isomerase (TIM) are among the major proteins expressed in the crocodile cornea as resolved by 2D gel electrophoresis and identified by MALDI-TOF. These proteins might be classified as putative corneal crystallins. tau-Crystallin, known to be present in turtle and crocodile lens, has earlier been identified in chicken and bovine cornea, whereas TIM has not been identified in the cornea of any species. Immunostaining showed that tau-crystallin and TIM are concentrated largely in the corneal epithelium. Using western blot, immunofluorescence and enzymatic activity, we demonstrate that high accumulation of tau-crystallin and TIM starts in the late embryonic development (after the 24th stage of embryonic development) with maximum expression in a two-week posthatched animal. The crocodile corneal extract exhibits significant alpha-enolase and TIM activities, which increases in the corneal extract with development. Our results establishing the presence of tau-crystallin in crocodile, in conjunction with similar reports for other species, suggest that it is a widely prevalent corneal crystallin. Identification of TIM in the crocodile cornea reported here adds to the growing list of corneal crystallins.