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PurposeTo compare axial length (AL) with vitreous cavity length (VCL) in patients with keratoconus and to ascertain whether graft size can be tailored to reduce myopic refractive error in patients with keratoconus undergoing penetrating keratoplasty (PK).Patients and methodsThe AL and VCL were measured prospectively in patients with keratoconus not undergoing PK (Group 1) and in normal phakic, emmetropic individuals (Group 2). A retrospective analysis of these measurements in patients with keratoconus who had undergone PK (Group 3) was also performed. The postoperative spherical equivalent (SE) was then correlated to size of donor buttons.ResultsKeratoconus patients tended to have a longer mean VCL than emmetropic normal individuals. The mean VCL of these patients (Group 1) was 16.49 mm±SD 1.13 compared to the mean VCL of 15.94 mm±SD 0.56 in normals (Group 2, P<0.0001). Patients with keratoconus who had an undersized graft showed reduced myopic refractive error compared to those with same size or oversized grafts.ConclusionVCL measurement is more accurate than AL measurement in deciding upon graft-host size disparity for corneal graft in patients with keratoconus. In patients with increased VCL, undersizing the donor button helps in reducing postoperative myopia. We recommend VCL measurement as part of the routine workup in all keratoconus patients undergoing corneal transplants.

PurposeTo describe a novel use of fibrin glue in managing leaking blebs and leaking wounds following trauma or surgery.MethodsInterventional case series.ResultsWe report eight patients, including three where intra-operative or immediate post-penetrating keratoplasty recalcitrant leaks from the graft–host junction and/or openings created by the needle pass, were noted. All three had thin recipient beds in the sector of leak. This was managed by intra-cameral injection of fibrin glue in the affected quadrant. This stopped the leak and allowed the defect to heal. One patient of Descemets-stripping-endothelial-keratoplasty had leak from the surgical wound, which was also sealed with fibrin glue. Two patients with leaking glaucoma-surgery-related blebs were treated with intra-bleb injection of fibrin glue to stop the leak. One patient with a penetrating corneal injury with a metal wire had a brisk leak upon removal of the wire. This was sealed with fibrin glue. Another patient of chemical burn with spontaneous leaks was managed by glue injection in the perforations. Transient rise of intraocular pressure in one patient with a leaking bleb was the only adverse event recorded.ConclusionThis novel adaptation of the application of fibrin glue can help to deal with persistent intra-operative, post-operative and traumatic aqueous and air leaks.

PurposeTo measure the pressure and volume of air required to create a big bubble (BB) in simulated deep anterior lamellar keratoplasty (DALK) in donor eyes and ascertain the bursting pressure of the BB.Patients and methodsTwenty-two human sclera-corneal discs were used. Air was injected into the corneal stroma to create a BB and the pressure measured by means of a pressure converter attached to the system via a side port. A special clamp was designed to prevent air leak from the periphery of the discs. The pressure at which air emerged in the corneal tissue; the bursting pressure measured after advancing the needle into the bubble cavity and injecting more air; the volume of air required to create a BB and the volume of the BB were ascertained.ResultsType-1 BB were achieved in 19 and type-2 BB in 3 eyes. The maximum pressure reached to create a BB was 96.25+/− 21.61 kpa; the mean type-1 intrabubble pressure was 10.16 +/− 3.65 kpa. The mean bursting pressure of a type-1 BB was 66.65 +/− 18.65 kpa, while that of a type-2 BB was 14.77 +/− 2.44 kpa. The volume of air required to create a type-1 BB was 0.54 ml and the volume of a type-1 BB was consistently 0.1 ml.ConclusionsDua’s layer baring DALK can withstand high intraoperative pressures compared to Descemet’s membrane baring DALK. The study suggests that it could be safe to undertake procedures such as DALK-triple with a type-1 BB but not with a type-2 BB.

PurposeTo design and produce a clamp that enables good handling of donor sclera-corneal disks, allows air to be injected in the corneal stroma and consistently provides pre-Descemets endothelial keratoplasty (PDEK) tissue by creation of a type-1 big bubble (BB) avoiding a type-2BB by shutting the fenestrations in the periphery of the pre-Descemets layer and preventing escape of air.Patients and methodsA clamp with spring loaded handles attached to two rings of 9 mm diameter and 1 mm width with a side port for insertion of a needle for air injection was designed and produced. The clamp was tested in 20 human donor sclera-corneal disks and refined over three prototypes. The final design was used for PDEK in 8 patients.ResultsThe clamp was able to prevent any escape of air or the formation of a type-2BB. By preventing air leak, it provided better control over the volume of air injected allowing for consistent formation of a type-1BB, which could be expanded to its full diameter by advancing the tip of the needle into the bubble cavity. Centration of tissue in the clamp was important. The clamp was used successfully to obtain PDEK tissue from 8 donor sclara-corneal disks for transplant in three eyes with pseudophakic bullous keratopathy and five eyes with pseudophakia and Fuchs’ endothelial dystrophy.ConclusionThe PDEK clamp is a simple and useful instrument that will give surgeons the confidence of consistently obtaining PDEK tissue with ease and without the risk of separating the Descemets membrane in a type-2BB.

PurposeTo investigate the effect of cyclosporine on corneal ultrastructure and on major signs and symptoms of patients with dry eye disease.Patients and methodsIn this prospective cohort study, patients with dry eye disease were treated with a drop of cyclosporine 0.05% twice daily. Clinical evaluation was carried out at baseline and at months 1, 3, and 6. All patients completed the Ocular Surface Disease Index (OSDI) questionnaire, and tear film break-up time (BUT), fluorescein and lissamine green staining, and Schirmer test were carried out. In vivo confocal microscopy was also performed and epithelial cellular density, keratocyte activation, and subbasal plexus morphology were assessed.ResultsA total of 40 patients completed the study. After 6 months, OSDI, BUT, and fluorescein and lissamine green staining showed a clinically significant improvement. During the 6-month follow-up, density of intermediate epithelial cells increased from 1969.5±85.4 cell/mm2to 4881.2±175.7 cell/mm2(P<0.01); average grade of keratocyte activation decreased from 3.6±0.1 to 1.8±0.1 (P<0.001); average grade of number of subbasal nerves decreased from 5.3±0.2 to 2.6±0.2 (P<0.001); average grade of nerve reflectivity decreased from 3.8±0.1 to 2.1±0.2 (P<0.001); and average grade of nerve tortuosity decreased from 3.8± 0.1 to 2.2±0.2 (P<0.001).ConclusionCyclosporine was effective in controlling symptoms and signs of dry eye disease. In vivo confocal microscopy showed an increase in cell density of intermediate epithelium cells, a decrease in hyperreflective keratocytes, and a decrease in density, tortuosity, and reflectivity of nerve fibers.

PurposeTo define optical coherence tomography (OCT) characteristics of type-1, type-2, and mixed big bubbles (BB) seen in deep anterior lamellar keratoplasty.MethodsHuman sclero-corneal discs were obtained from UK (30) and Canada (16) eye banks. Air was injected into corneal stroma until a BB formed. UK samples were fixed in formalin before scanning with Fourier-domain (FD-OCT). One pair of each type of BB was scanned fresh. All BB obtained from Canada were scanned fresh with time-domain (TD-OCT). For each OCT machine used, type-1 BB from which Descemets membrane (DM) was partially peeled, were also scanned. The morphological characteristics of the scans were studied.ResultsFD-OCT of the posterior wall of type-1 (Dua’s layer (DL) with DM) and type-2 BB (DM alone) both revealed a double-contour hyper-reflective curvilinear image with a hypo-reflective zone in between. The anterior line of type-2 BB was thinner than that seen with type-1 BB. In mixed BB, FD-OCT showed two separate curvilinear images. The anterior image was a single hyper-reflective line (DL), whereas the posterior image, representing the posterior wall of type-2 BB (DM) was made of two hyper-reflective lines with a dark space in between. TD-OCT images were similar with less defined component lines, but the entire extent of the BB could be visualised.ConclusionOn OCT examination the DM and DL present distinct features, which can help identify type-1, type-2, and mixed BB. These characteristics will help corneal surgeons interpret intraoperative OCT during lamellar corneal surgery.

PurposeFibrin glue has been used successfully in numerous ophthalmic surgical procedures. Recently, fibrin glue has been used in limbal stem cell transplantation to reduce both operative time and to negate the need for sutures. The aim of this study was to determine the effects of fibrin glue on epithelial cell migration in vitro.MethodsCorneoscleral rims were split to retain the epithelial layer, Bowman’s layer, and anterior stroma. Rims were cut into eight equal-sized pieces and were placed directly on culture plates or affixed with fibrin glue. Rims were maintained in culture for 25 days and epithelial cell growth was monitored. Cells were photographed to measure area or growth and immunofluorescence staining of explants for fibrin was performed.ResultsExplants that were glued demonstrated significantly delayed epithelial cell growth and migration as compared with explants without glue. By day 16, all fibrin glue had dissolved and coincided with onset of cell growth from glued explants. Cell growth commenced between days 3 and 4 for control explants without glue and around days 14–16 for explants with fibrin glue.ConclusionsFibrin glue delays epithelial cell migration by acting as a physical barrier and can potentially interfere with explant-derived limbal epithelial cell migration on to the corneal surface. We propose that glue should be used to attach the conjunctival frill of the limbal explant but care should be taken to ensure that the glue does not wrap around the explant if used to secure the explant as well. Strategic use of glue, to attach the recessed conjunctiva, can be advantageous in delaying conjunctival cell migration and reducing the need for sequential sector conjunctival epitheliectomy.

In India the incidence of breast cancer has increased in the urban population with 1 in every 22 women diagnosed with breast cancer. It is important to know the Her2/neugene status for a better prognostication of these patients.

Purpose To compare the safety and efficacy of a new enhanced viscosity ophthalmic formulation of tobramycin, given twice daily (BID), with the existing four times daily (QID) treatment regimen in patients with acute bacterial conjunctivitis.Methods This was a 12-day, multicenter, observer-masked, randomized, parallel group study. Patients received one drop of tobramycin 0.3% (3 mg/mL) enhanced viscosity ophthalmic solution BID or tobramycin 0.3% (3 mg/mL) ophthalmic solution QID in the affected eyes for 7 days. The primary efficacy variable was the percentage of patients with sustained cure/presumed bacterial eradication based on clinical judgment at the test-of-cure visit (Day 12). Pretherapy bacterial isolates were obtained and tested for susceptibility to tobramycin by determination of minimum inhibitory concentrations (MIC).Results A total of 276 patients were enrolled in the study and 203 of these were culture positive and attended all follow-up examinations. In this group, 98% of those treated with tobramycin enhanced viscosity ophthalmic solution and 99% of those treated with tobramycin 0.3% ophthalmic solution were categorized as having sustained cure/presumed eradication at the test-of-cure visit (p=0.6037). Reported adverse events were not serious, mild to moderate in severity, and generally did not prevent continuation in the study. Several pretreatment pathogens demonstrated tobramycin resistance (MIC > 4 mg/mL). However, therapy with both treatments was effective in the majority of the cases.Conclusions Tobramycin enhanced viscosity ophthalmic solution is well tolerated and has equivalent efficacy to the established treatment regimen with a simplified posology. The formulation provides an alternative therapy for acute bacterial conjunctivitis that should improve patient compliance and satisfaction.

Abstract Background/aims The antimicrobial activity of the tear film exceeds the activity of its known constituents. The authors postulate that this excess activity is the result of antimicrobial peptides called defensins, and they aimed to look for defensins in the human eye. Methods Evidence of defensin production was sought by reverse transcriptase polymerase chain reaction (RT-PCR). Intron spanning primers were designed for β defensins 1 and 2, and α defensins 5 and 6. RT-PCR was performed on cornea, conjunctiva, and lacrimal gland samples, and reaction products were size fractionated and sequenced to confirm their identity. A monoclonal antibody was utilised for the detection of α defensins 1, 2, and 3 in tissue sections and in immunoblots of tears. Results RT-PCR revealed β defensin 1 message in samples of conjunctiva, cornea, and lacrimal gland. β Defensin 2 message was detected in the conjunctiva and cornea but was absent from the lacrimal gland. α Defensin 5 and 6 message was absent in these tissues but α defensins 1, 2, and 3 were detected in normal tears, lacrimal gland, and inflamed conjunctiva by immunochemistry. Conclusion The data suggest the human eye innately produces a spectrum of antimicrobial defensin peptides. Defensins hold therapeutic potential in ocular infections as they have a broad spectrum of antimicrobial activity (bacteria fungi and viruses ) and accelerate epithelial healing.

S-Antigen, a photoreceptor cell protein, is highly efficient in inducing experimental autoimmune uveoretinitis (EAU), a severe inflammation of the uveal tract and retina of the eye. S-Antigen and six synthetic peptides, all of which correspond to known T-cell or B-cell recognition sites, were tested for their abiity to induce oral tolerance to EAU in LEW rats. Feeding three 1-mg doses of native S-Antigen or three doses of one synthetic peptide, designated BSA(343-362) (200 μ per dose), reduced the incidence and severity of EAU induced by immunization with 50 μ of S-Antigen. Another peptide, BSA(270-289), was able to inhibit EAU only when a low dose (10μ) of the uveitogenic S-Antigen was used to induce EAU. Animals which received 200 μ doses of four other immunologically active peptides, BSA(31-51), BSA (143-162), BSA(303-327) and BSA(333-352), were not significantly protected. Furthermore, animals fed BSA(343-362) were significantly less susceptible to EAU induced by adoptive transfer (tEAU) of the uveitogenic R9 T-cell lines. Con A-acti-vated lymphocytes purified from spleens of rats fed peptide BSA(343-362) transferred partial resistance to tEAU induced by adoptive transfer of R9 line cells. The resistance of orally tolerized rats to induction of EAU by adoptive transfer of an activated, pathogenic T-cell line, and the ability of lymphocytes from orally-tolerized animals to transfer resistance to tEAU shows that effector mechanisms can be inhibited by oral feeding as well as the afferent mechanisms reported here and elsewhere. Circulating levels of IgG specific for S-Antigen were not affected by feeding any of the peptides.

A significant amount of information concerning immunologic domains of an antigenic molecule can be obtained by studying its peptides. We describe a method for identifying and characterizing immunologically relevant T-cell and B-cell epitopes in S-antigen, a well-characterized, highly pathogenic retinal autoantigen for the induction of experimental autoimmune uveitis. The method involves the generation of peptide fragments by enzymatic treatment of native S-antigen and by the simultaneous synthesis of large numbers of peptides in small quantities for screening and testing. Peptides demonstrating T- or B-cell activity are then synthesized in large quantity for additional studies. Although useful information was obtained by the use of enzymatically generated peptides, synthetic peptides provided the greatest flexibility and specificity, allowing the precise localization of amino acid sequences of S-antigen required for a particular immunological function such as antibody binding, T-cell proliferative responses, pathogenicity, and the induction of tolerance. These studies have wide applicability to the study of other antigenic molecules and have led to a better understanding of the immune mechanisms involved in the pathogenesis of experimental autoimmune uveitis. This, in turn, provides a basis for the processes that may be occurring in certain forms of human uveitis.Copyright 1993, 1999 Academic Press

Retinal s-antigen (SAg) and interphotoreceptor retinol-binding protein (IRBP) induced experimental autoimmune uveitis (EAU) and experimental autoimmune pinealitis (EAP) are good models for studying the mechanisms involved in autoimmune diseases. Many immunogenetically active epitopes have been identified in these proteins but immunodominance of one or more epitopes in vivo, has not yet been established. In this paper we present and discuss some experiments that led to the discovery of a dominant "tolerogenic" epitope in SAg. We also demonstrate the presence of cross reactive epitopes in the two potent retinal antigens, SAg and IRBP and finally introduce early data on a unique anti S2.4.C5 idiotypic (Id) monoclonal antibody (MAb) which appears to be a site non associated antibody that binds not only to S2.4.C5 but also to SAg.

Experimental autoimmune uveoretinitis (EAU) has been extensively studied as a model for human posterior uveitis, however, few ultrastructural studies of EAU in the rat have been reported. In the present report we document a systematic time-course study of the posterior segment changes in the Lewis rat. The disease varied somewhat in severity in animals sacrificed at identical times after immunisation. In the prodromal stage of the disease, usually around day 14, early pathological changes included mild peripapillary vasculitis and low grade mononuclear and neutrophilic infiltration of the subretinal space with phagocytosis of the rod outer segments. The features of the severe or active diseases were most evident on day 21 and included mixed cellular infiltrate of the vitreous, subretinal serohaemorrhagic exudate, focal retinal detachment and necrosis. Outer retinal destruction was often most severe adjacent areas of retinal vasculitis. Focal monocytic subpigment epithelial microgranulomas, reminiscent of Dalen-Fuch's nodules in humans, were also identified. By day 28 and 49 active inflammation had subsided and large segments of the outer retina were completely destroyed. The retinal pigment epithelium (RPE) also showed signs of activation in the vicinity of focal retinochoroidal mononuclear infiltrates including multilayering, proliferation and increased phagocytosis. Finally, neovascularisation of the RPE by non-fenestrated capillaries derived from the retinal vasculature was evident in the late stages of the disease.

Retinal S-antigen induced experimental autoimmune uvettis (EAU) is a severe, predominantly T-cell mediated inflammatory disease of the uveal tract and retina of the eye. Pretreatment of LEW rats with the monoclonal antibody, MAbS2.4.C5, which defines an epitope in S-antigen, has been shown to effectively inhibit the subsequent induction of EAU with S-antigen. Using synthetic peptides and cyanogen bromide fragments of S-anligen we found the binding site of MAbS2.4.C5 to be located at the carboxy terminus of the molecule corresponding to amino acid positions 375 to 380. Limited Staphylococcus aweus V8 protease digestion yielded several polypeptide fragments including one large 43 kD fragment which retained antibody binding to a variety of both polyclonal and monoclonal antibodies which identify epitopes that span the length of the S-antigen. This treatment, however, completely destroys the MAbS2.4.C5 binding site and dramatically reduces uveitopathogenicity. Limited trypsin and papain digestion, on the other hand, had little effect on pathogenicity or on MAbS2.4.C5 binding to S-antigen or its peptide fragments. These results indicate that the carboxy-terminus of S-antigen plays a predominant role in the pathogenesis of EAU.

Experimental autoimmune uveoretinitis (EAU) is a useful model of human posterior uveitis and as such, permits the analysis of strategies for immuno-intervention. Modulation of the autoimmune response may be attempted at the stages of induction of EAU, during homing of autoreactive lymphocytes to the target organ, the retina, or during the effector stage of the disease. This paper presents a brief overview of current immuno-therapeutic modalities and assesses the usefulness for extrapolation to human disease.

Experimental autoimmune uveoretinitis (EAU) induced by retinal antigens is a CD4+ (Th) lymphocyte mediated disease. Generation of autoreactive CD4+ cells requires the processing and presentation of autoantigen by antigen presenting cells (APC) in combination with MHC Class II antigen. Efficient presentation of antigen to T cells has also been shown to depend on accessory molecules of adhesion such as intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-1 (LFA-1).Aberrant expression of Class II antigens by local tissue cells has been suggested as a possible mechanism in autoimmune processes. Several ocular cells express Class II antigens during inflammation, while other cells such as Muller cells inhibit antigen presentation in vitro. We have also shown that retinal pigment epithelial cells (constitutively) and endothelial cells (after induction) express ICAM-1 and that CD4+ lymphocyte adhesion to these cells is inhibited by antibodies to ICAM-1. Accessory molecules may therefore be important, not only in local presentation of antigen but in recruitment of circulating autoreactive cells to the eye since these cells represent the site of the blood-retinal barrier. Regulation of the local immune response in the eye therefore, may occur at several levels.

Uveitis is a term which encompasses many clinical syndromes which would appear to be discrete entities. Both clinically and experimentally, the separation of uveitis affecting only the anterior segment from that affecting the posterior segment has a sound pathogenetic basis. However, clear distinctions among the various forms of endogenous posterior uveitis are more difficult to maintain in the light of evidence from experimental models of autoimmune uveitis (EAU). EAU can be induced by a variety of retinal antigens and each antigen has been shown to induce somewhat different forms of EAU, depending on such factors as dose of antigen, species and strains of animal model, and the type(s) of adjuvant used. However, within each model a similar spectrum of uveoretinal responses can be induced by each antigen suggesting that the pathogenetic mechanisms are probably similar also. In addition, if these models are analogous to human disease, then each clinical entity within this apparently heterogeneous group of clinical posterior uveitis syndromes may represent one aspect of a general organ-specific uveoretinal response to autoantigens.