Roles of Epithelial and Mesenchymal TRP Channels in Mediating Inflammatory Fibrosis.

The maintenance of normal vision is dependent on preserving corneal transparency. For this to occur, this tissue must remain avascular and its stromal architecture needs to be retained. Epithelial transparency is maintained provided the uppermost stratified layers of this tissue are composed of terminally differentiated non-keratinizing cells. In addition, it is essential that the underlying stromal connective tissue remains avascular and scar-free. Keratocytes are the source of fibroblasts that are interspersed within the collagenous framework and the extracellular matrix. In addition, there are sensory nerve fibers whose lineage is possibly either neural crest or mesenchymal. Corneal wound healing studies have been undertaken to delineate the underlying pathogenic responses that result in the development of opacification following chemical injury. An alkali burn is one type of injury that can result in severe and long- lasting losses in ocular transparency. During the subsequent wound healing process, numerous different proinflammatory cytokines and proteolytic enzymes undergo upregulation. Such increases in their expression levels induce maladaptive expression of sustained stromal inflammatory fibrosis, neovascularization, and losses in the smooth optical properties of the corneal outer surface. It is becoming apparent that different transient receptor potential channel (TRP) isoforms are important players in mediating these different events underlying the wound healing process since injury upregulates both their expression levels and functional involvement. In this review, we focus on the involvement of TRPV1, TRPA1 and TRPV4 in mediating some of the responses that underlie the control of anterior ocular tissue homeostasis under normal and pathological conditions. They are expressed on both different cell types throughout this tissue and also on corneal sensory nerve endings. Their roles have been extensively studied as sensors and transducers of environmental stimuli resulting from exposure to intrinsic modulators and extrinsic ligands. These triggers include alteration of the ambient temperature and mechanical stress, etc., that can induce pathophysiological responses underlying losses in tissue transparency activated by wound healing in mice losses in tissue transparency. In this article, experimental findings are reviewed about the role of injury-induced TRP channel activation in mediating inflammatory fibrotic responses during wound healing in mice.

An immune response to the avascular lens following wounding of the cornea involves ciliary zonule fibrils.

The lens and central cornea are avascular. It was assumed that the adult lens had no source of immune cells and that the basement membrane capsule surrounding the lens was a barrier to immune cell migration. Yet, microfibril-associated protein-1 (MAGP1)-rich ciliary zonules that originate from the vasculature-rich ciliary body and extend along the surface of the lens capsule, form a potential conduit for immune cells to the lens. In response to cornea debridement wounding, we find increased expression of MAGP1 throughout the central corneal stroma. The immune cells that populate this typically avascular region after wounding closely associate with this MAGP1-rich matrix. These results suggest that MAGP1-rich microfibrils support immune cell migration post-injury. Using this cornea wound model, we investigated whether there is an immune response to the lens following cornea injury involving the lens-associated MAGP1-rich ciliary zonules. Our results provide the first evidence that following corneal wounding immune cells are activated to travel along zonule fibers that extend anteriorly along the equatorial surface of the lens, from where they migrate across the anterior lens capsule. These results demonstrate that lens-associated ciliary zonules are directly involved in the lens immune response and suggest the ciliary body as a source of immune cells to the avascular lens.

Cellular Infiltrate in Rheumatoid Arthritis-associated Paracentral Corneal Ulceration.

PURPOSE: To investigate an immunopathogenesis of central and paracentral corneal ulceration associated with rheumatoid arthritis. METHODS: Sparse infiltrating cells in the ulcer area were identified by immunohistochemistry applied to archived formalin fixed, paraffin embedded tissues that had been recovered from patients undergoing penetrating keratoplasty necessitated by rheumatoid-associated central or paracentral corneal ulceration. RESULTS: Clinically, the ulcers presented as non-infiltrated lesions with a modicum of other ocular inflammation. Sparse T-lymphocytes were consistently identified in the subepithelial areas adjacent to the ulcer, with some neutrophils and macrophages in the stroma. B-lymphocytes were not detected. MHC Class II antigens reactivity was noted on some infiltrating cells and on corneal endothelium of two specimens. CONCLUSIONS: Immunohistochemistry of archival tissue facilitated detection and identification of sparse infiltrate in this infrequent corneal melting. Selective, consistent finding of T-lymphocyte infiltration in the ulcer area supports an immunopathogenesis of this clinical entity.

Novel model of innate immunity in corneal infection.

The cornea functions as the major refractive interface for vision and protects the internal eye from insult. Current understanding of innate immune responses to corneal infection derives from a synthesis of in vitro and in vivo analyses. However, monolayer cell cultures and mouse models do not accurately duplicate all aspects of innate immunity in human patients. Here, we describe a three-dimensional culture system that incorporates human cells and extracellular matrix to more completely simulate the human cornea for studies of infection. Human corneal stromal fibroblasts were mixed with type I collagen in 3-µm pore size transwell inserts, and overlayed with Matrigel to simulate a human corneal stroma and epithelial basement membrane. These were then infected with a cornea-tropic adenovirus, and exposed on their inferior side to leukocytes derived from human peripheral blood. Subsequent analyses were performed with histology, confocal microscopy, ELISA, and fluorescence-activated cell sorting (FACS). CXCL8, a neutrophil chemokine shown previously as the first cytokine induced in infection of human corneal cells, increased upon adenovirus infection of facsimiles in a dose-responsive fashion. Myeloperoxidase-positive cells infiltrated infected corneal facsimiles in a sub-Matrigel location, possibly due to CXCL8 colocalization with heparan sulfate, a Matrigel constituent. Cellular infiltration was significantly inhibited by treatment with chemical inhibitors of p38 MAPK and Src kinase, both constituents of a signaling cascade previously suggested to regulate inflammation after adenovirus infection. FACS analysis determined that both virus and corneal fibroblasts were necessary for the induction of leukocyte migration into the facsimiles. The corneal facsimile, literally a cornea in a test tube, permits mechanistic studies on human tissue in a highly tractable system.

Prevention of herpes simplex virus induced stromal keratitis by a glycoprotein B-specific monoclonal antibody.

The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.

SLURP-1 modulates corneal homeostasis by serving as a soluble scavenger of urokinase-type plasminogen activator.

PURPOSE: Our previous study revealed the immunomodulatory property of the secreted lymphocyte antigen (Ly6)/urokinase-type plasminogen activator receptor (uPAR)-related protein-1 (SLURP1), abundantly expressed in the cornea and associated with the hyperkeratotic disorder Mal de Meleda. Here, we test the hypothesis that SLURP1 modulates the functions of membrane-tethered uPAR by acting as a soluble scavenger of its ligand urokinase-type plasminogen activator (uPA). METHODS: Human corneal limbal epithelial (HCLE) and mouse corneal stromal fibroblast MK/T-1 cells were employed to examine the effect of SLURP1 on cell proliferation and migration. Human corneal limbal epithelial cell clones stably expressing SLURP1 under the control of cytomegalovirus (CMV) promoter were generated using lentiviral vectors. Recombinant 6× His-mouse Slurp1 and maltose-binding protein (MBP)-mouse uPA were expressed in Escherichia coli and partially purified using nickel-ion and amylose columns, respectively. Slurp1 interaction with uPA was detected using ligand blots, ELISA, pull-down assays, and immunofluorescent staining. RESULTS: Stable expression of SLURP1 in HCLE cells was confirmed by immunoblots and immunofluorescent staining. Human corneal limbal epithelial and MK/T-1 cell proliferation and migration rates were suppressed by exogenous SLURP1. Ligand blots, ELISA, and pull-down assays indicated that Slurp1 efficiently interacts with uPA. Immunofluorescent staining demonstrated that exogenous SLURP1 decreased the amount of cell surface-bound uPA in the leading edges of migrating cells. In gap-filling assays, wild-type HCLE cells responded to uPA by increasing their velocity and closing larger area, while the SLURP1-expressing HCLE cells failed to do so. CONCLUSIONS: SLURP1 modulates corneal homeostasis by serving as a soluble scavenger of uPA and regulating the uPA-dependent functions of uPAR.

A novel association between resident tissue macrophages and nerves in the peripheral stroma of the murine cornea.

PURPOSE: To characterize the interactions between resident macrophage populations and nerves in naïve and injured corneas of the mouse eye. METHODS: Corneas from wild-type (WT) C57BL/6J, BALB/cJ, and transgenic Cx3cr1-eGFP mice were subjected to a 1-mm central epithelial debridement injury. The eyes were fixed and immunostained as flat mounts with a range of antibodies to identify macrophages, neurons, and Schwann cells. Interactions between nerves and immune cells were analyzed and quantitated using three-dimensional reconstructions of confocal microscopy images. Naïve eyes acted as controls. RESULTS: A distinctive association between resident immune cells and corneal nerves was noted in the peripheral or perilimbal stromal nerve trunks. These epineurial cells were mostly Cx3cr1(+) Iba-1(+) major histocompatibility complex (MHC) class II(+) F4/80(+) CD11b(+) macrophages. The number of nerve-associated macrophages was greater in WT BALB/c mice than in C57BL/6J mice. There were no qualitative or quantitative differences in the circumferential distribution of nerve-associated macrophages in the cornea. Sterile corneal epithelial debridement led to a dissociation of macrophages from peripheral nerve trunks as early as 2 hours postinjury, with numbers returning to baseline after 72 hours. This dissociation was Cx3cr1 dependent. CONCLUSIONS: This study is the first to highlight a direct physical association between nerves and resident immune cells in the murine cornea. Furthermore, we reveal that this association in normal eyes is responsive to central corneal epithelial injury and is partly mediated by Cx3cr1 signaling. This association may serve as an indicator of malfunctioning neuroimmune communication in disease states such as neurotrophic keratitis and peripheral neuropathy.

Equine deep stromal abscesses (51 cases - 2004-2009)--Part 2: the histopathology and immunohistochemical aspect with attention to the histopathologic diagnosis, vascular response, and infectious agents.

PURPOSE: To investigate histopathologic and immunohistochemical aspects of equine deep stromal abscesses (DSA) with a focus on the histopathologic diagnosis, presumptive etiology, and the immunohistochemical expression of three angiogenesis-related factors: vascular endothelial growth factor-A (VEGF-A), pigment epithelium-derived factor (PEDF), and interleukin-1 receptor antagonist (IL-1ra). SAMPLE POPULATION: Paraffin-embedded biopsy samples from 51 DSA. The biopsies were collected from full-thickness penetrating keratoplasty or split-thickness lamellar keratoplasty surgeries at the University of Florida Veterinary Medical Center in the period from 2004 to 2009. PROCEDURE: The histopathologic and immunohistochemical findings were tested for association between each other. Prevalence calculation and test for association with qualitative data analysis was used for data evaluation. RESULTS: Fungal hyphae were found histologically in 47.1% (n = 24) of the DSA cases. Histopathologically, most fungal DSA showed suppurative keratitis (n = 34; 66.7%) and little to no stromal vascularization infiltrating the abscess (negative association, P = 0.005). All three angiogenesis-related factors were expressed to some degree in DSA tissue. A negative association between VEGF-A and PEDF when compared to the presence of fungal hyphae (P < 0.001, P = 0.023) indicated that cases positive for these two factors will most probably not have fungal hyphae present. CONCLUSION: Abnormally decreased VEGF-A expression is suggested as the reason for the slow vascularization and delayed resolution of fungal DSA, whereas PEDF and IL-ra did not seem to have any influence on the vascularization process. Clinical and histopathologic characteristics of DSA make it possible to suggest an etiology for an equine DSA with an unknown etiology.

IL1beta, IL6 and IL8 gene polymorphisms involvement in recurrent corneal erosion in patients with hereditary stromal corneal dystrophies.

TGFBI gene mutations cause corneal stromal dystrophies of autosomal dominant inheritance. The most frequent complication of stromal dystrophies is recurrent corneal erosion with varying degree of accompanying inflammation. IL-1beta, IL-6 and IL-8 are main cytokines involved in corneal erosion healing. This study aimed to investigate the association between IL1B gene -511C/T, IL6 gene -174G/C and IL8 gene -781C/T polymorphisms and risk of recurrent erosion development in patients with hereditary corneal stromal dystrophies. A trend to decrease of IL1B gene -511TT genotype frequency in group with erosion (3.7%) comparing to control (6.7%) was observed. IL6 gene -174C allele carriers frequency in control group (65.9%) was significantly (P < 0.05) lower comparing to patients with erosion (80.5%). Frequency of IL8 -781TT genotype was significantly (P < 0.05) lower in the group with erosion (10.7%) comparing to patients without erosion (30.8%) and control (25%). IL6 gene -174C allele may be considered as genetic marker of corneal erosion risk in patients with hereditary stromal corneal dystrophies, whereas IL8 -781TT genotype is associated with negative recurrent erosion prognosis in such patients.

Immunolocalization of different collagens in the cornea of human fetal eyes: a developmental approach.

PURPOSE: To study the corneal development in the human fetal eye with particular emphasis on the epithelial basement membrane and Bowman's layer. Thus, immunohistochemical markers supposed to stain this region were employed. MATERIAL AND METHODS: 19 formalin-fixed fetal eyes and a 16-day-old newborn's cornea without any obvious irregularities of the anterior segment were investigated. The age of the fetal eyes ranged from 11 to 38 week of gestation (WoG). The eyes (including the corneal thickness) were measured and, in addition to routine hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) stains, immunohistochemical labeling with antibodies to collagen IV, V, IX, and XVII was performed. RESULTS: Analysis of the H&E stains revealed that measurements of corneal thickness correlated well with corneal development as a basic indicator for maturation. In a more detailed immunohistochemical analysis, collagen IV was expressed in the epithelial basement membrane (BM) of the cornea, conjunctiva, and Descemet's membrane in fetal eyes up to the age of 23 WoG. In fetal eyes older than 23 WoG, staining was confined to the limbal area only. With the antibody against collagen V, the corneal stroma and the BM were intensely stained. Bowman's layer (first detected at 17 WoG by light microscopy) was not labeled. Anti-collagen IX labeled predominantly the conjunctival and corneal epithelium. With anti-collagen XVII, the BM of the cornea and conjunctiva was stained in all fetal eyes, whereas intracellular expression in the epithelium increased with age. CONCLUSION: Our results indicate maturation-associated variations of collagen expression in the human cornea. Measurements of the corneal thickness may serve as an additional parameter to narrow down the developmental age with possible implications for pediatric pathology and forensic issues.

Murine corneal stroma cells inhibit LPS-induced dendritic cell maturation partially through TGF-ß2 secretion in vitro.

PURPOSE: The peripheral cornea contains mature and immature resident dendritic cells (DCs) while the central cornea is exclusively equipped with immature DCs. There must be some factors that cause immature DCs. This study investigated whether corneal stroma cells (CSCs) inhibit DC maturation by secreting cytokines. METHODS: The messenger ribonucleic acid (mRNA) and protein level of transforming growth factor beta 2 (TGF-ß(2)) was analyzed using reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Immature DCs were induced to mature in the presence of lipopolysaccharide (LPS) and with concentrations of CSC culture supernatant (containing and not containing neutralizing TGF-ß(2) antibodies). Then, the DC phenotypic and functional maturation were analyzed. RESULTS: CSCs exhibited positive expressions of TGF-ß(2) mRNA and secreted high concentrations of TGF-ß(2) protein. In the presence of LPS, DCs, which were treated with a CSC culture supernatant, displayed reduced expressions of cluster of differentiation 80 (CD80), CD86, and major histocompatibility complex II (MHC II) in a dose-dependent manner. Moreover, treated DCs showed lower T-cell stimulation capacity and a higher endocytosis function. However, these phenotypic and functional modifications were partially reversed after the application of neutralizing TGF-ß(2) antibodies. CONCLUSIONS: This study demonstrates that CSCs can partially inhibit LPS-induced DC maturation through TGF-ß(2) secretion in vitro.

Expression of toll-like receptor 4 contributes to corneal inflammation in experimental dry eye disease.

Purpose. To investigate the corneal expression of toll-like receptor (TLR) 4 and determine its contribution to the immunopathogenesis of dry eye disease (DED). Methods. Seven to 8-week-old female C57BL/6 mice were housed in a controlled environment chamber and administered scopolamine to induce experimental DED. Mice received intravenous TLR4 inhibitor (Eritoran) to block systemic TLR4-mediated activity. The expression of TLR4 by the corneal epithelium and stroma was evaluated using real-time polymerase chain reaction and flow cytometry. Corneal fluorescein staining (CFS) was performed to evaluate clinical disease severity. The corneal expression of proinflammatory cytokines (IL-1ß, IL-6, TNF, and CCL2), corneal infiltration of CD11b(+) antigen-presenting cells, and lymph node frequency of mature MHC-II(hi) CD11b(+) cells were assessed. Results. The epithelial cells of normal corneas expressed TLR4 intracellularly; however, DED significantly increased the cell surface expression of TLR4. Similarly, flow cytometric analysis of stromal cells revealed a significant increase in the expression of TLR4 proteins by DED-induced corneas as compared with normal corneas. DED increased the mRNA expression of TLR4 in corneal stromal cells, but not epithelial cells. TLR4 inhibition decreased the severity of CFS and significantly reduced the mRNA expression of IL-1ß, IL-6, and TNF. Furthermore, TLR4 inhibition significantly reduced the corneal infiltration of CD11b(+) cells and the lymph node frequency of MHC-II(hi) CD11b(+) cells. Conclusions. These results suggest that DED increases the corneal expression of TLR4 and that TLR4 participates in the inflammatory response to ocular surface desiccating stress.

Substance P in the corneal stroma regulates the severity of herpetic stromal keratitis lesions.

PURPOSE: To determine whether substance P (SP) in herpes simplex virus-1 (HSV-1) infected cornea regulates the severity of herpetic stromal keratitis (HSK) lesions in a mouse model. METHODS: C57BL/6 mice were infected ocularly with HSV-1 (RE). The corneas with HSK lesions, on Day 15 postinfection, were grouped on the basis of the corneal opacity as mild (≤2) or severe (>2). The amount of SP was determined in the corneas with mild or severe HSK lesions by enzyme immunosorbent assay (EIA) and confocal microscopy. Subconjunctival inoculation of spantide I, SP receptor antagonist, was carried out during the clinical phase of HSK. ELISA and flow cytometry were used to determine the level of cytokines, chemokines, and influx of immune cell types in the corneal lesions. RESULTS: The authors determined a significantly higher level of SP in the corneas with severe HSK lesions in comparison with mild lesions. The corneas with a higher level of SP also exhibited higher amounts of proinflammatory cytokines (IL-6, IFN-γ) and chemokines (CCL3, CXCL2) when compared with the corneas with a lower level of SP. SP receptor NK1R expression was determined in CD45- and CD45+ cells in infected cornea. SP present in the corneal stroma of the eyes with severe HSK lesions colocalized with ß-III tubulin(+) and IA(b+) cell types. Subconjunctival inoculation of spantide I during the clinical phase of HSK resulted in significant reduction in the corneal opacity and angiogenesis. CONCLUSIONS: Collectively, these results demonstrate the relative contribution of substance P in regulating the clinical severity of HSK lesions in a mouse model.

Investigation of the differential potentials of TLR agonists to elicit uveitis in mice.

TLRs are critical for host defense and innate immunity. Emerging evidence also supports a role for TLRs in many chronic inflammatory diseases, including inflammatory eye disease, known as uveitis. The activation of TLR4 by endotoxin induces a standard model of murine uveitis. How activation of additional TLRs influences the onset and/or severity of anterior uveitis has not been examined. We sought to elucidate the potential of TLRs (TLR1/2, TLR2/6, TLR3, TLR4, TLR5, TLR7/8, and TLR9) to trigger uveitis in mice. Directly stimulated iris/ciliary body explants demonstrated a marked increase in production of inflammatory cytokines TNF-α, IL-6, IP-10/CXCL10, MCP-1, and KC with relatively little production of IFN-γ, IL-10, IL-12p40, IL-12p70, IL-17, IL-1ß, IL-4, or RANTES. The cytokine-response profiles were comparable amongst the TLR agonists, albeit some differences were noted, such as greater IP-10 production following TLR3 activation. Intra-ocular injection of TLR agonists increased leukocyte interactions with the endothelium of the iris vasculature and resulted in chemotaxis into the iris tissue. Assessment of leukocytic responses by ivt videomicroscopy and histology revealed quantitative differences amongst responses to the TLR agonists with respect to the timing and numbers of rolling, adhering, iris-infiltrating, and aqueous humor-infiltrating cells, along with cytokine levels in vivo. Our data demonstrate the eye's responsiveness to a diverse array of microbial products, which activates TLRs, and reveal differences in relative cellular response among the various TLR agonists in vivo.

Long-term follow-up of a supradescemetic keratoprosthesis in rabbits: an immunofluorescence study.

OBJECTIVE: To evaluate the long-term clinical and immunohistological outcome of two different non-penetrating keratoprosthesis (KPro) implanted in non-injured rabbit corneas. MATERIALS AND METHODS: Three rabbits underwent implantation of a pHEMA-MMA(34) synthetic cornea in the supradescemetic space, and PMMA synthetic corneas in the supradescemetic space and within the central stroma. Animals were followed for at least 24 months before euthanasia. Periodic evaluation was performed with slit-lamp examination and photography. At the end of the follow-up, histological examination including hematoxylin eosin staining and immunocharacterization against collagen IV, alpha smooth muscle actin (α-SMA) and macrophages was performed. RESULTS: The pHEMA-MMA(34) implant was not extruded, and remained transparent until the end of follow-up. This material did not induce any cell infiltration, corneal scarring or tissue remodeling in the surrounding stroma as shown by immunofluorescence. In contrast, synthetic corneas made of PMMA-induced myofibroblast differentiation, stromal remodeling and macrophage infiltration. This reaction was even more significant in the rabbit with the PMMA implant within the corneal stroma. CONCLUSION: pHEMA-MMA(34) was clinically biocompatible, and did not induce any inflammatory reaction or scarring when implanted in the supradescemetic space. This material showed more promising biocompatibility results than for PMMA, whether implanted within the central cornea stroma or in the supradescemetic space.

The antigenicity of ex vivo cultivated human corneal limbal epithelial and stromal cells: temporal changes in vitro.

PURPOSE: To investigate the effect of ex vivo culture on the expression of human leukocyte antigen (HLA) in human corneal limbal epithelial cells (LECs), and to evaluate the expression of HLA and costimulatory molecules on human corneal limbal stromal cells (LSCs). METHODS: The expression of HLA-ABC and HLA-DR were evaluated using flow cytometry on cultured human LECs and LSCs after serial passage and compared with that on freshly isolated cells. Additionally, the expression of costimulatory molecules CD80, CD86, CD40, and immunoregulatory molecule HLA-G were investigated on LSCs with or without an addition of interferon (IFN) γ (250 U/mL, 4 days). RESULTS: HLA-ABC and HLA-DR were expressed in 99.12 ± 1.02% and 25.86 ± 4.34% of freshly isolated LECs, respectively. After culture, the HLA-DR-expressing cells significantly decreased with serial passage, whereas the percentage of HLA-ABC-expressing cells did not change. More than 90% of LSCs expressed HLA-ABC and HLA-G, whereas the expression of HLA-DR, CD80, CD86, and CD40 was negligible. This expression profile did not change after serial passage. However, the treatment with IFN-γ induced a significant increase in HLA-DR (0.17% vs. 17.68%) and CD40 (0.19% vs. 13.19%) expression on LSCs. CONCLUSIONS: The immunogenicity of LECs can be lowered after ex vivo cultivation, whereas LSCs can be immunogenic in the presence of IFN-γ.

Immunotactoid microtubular corneal deposits in bilateral paraprotein crystalline keratopathy.

PURPOSE: To report an ultrastructurally distinct form of paraprotein crystalline keratopathy. METHODS: Three corneas submitted from a single patient including one native cornea from each eye and a failed corneal graft from the right eye. Light microscopy, immunohistochemistry, immunofluorescence, and transmission electron microscopic examination were performed. RESULTS: The transmission electron microscopy showed "immunotactoid" extracellular microtubular deposits measuring >30 nm in diameter with a central lucent core. CONCLUSIONS: Immunotactoid keratopathy is a distinct type of paraprotein crystalline keratopathy associated with a monocolonal immunoglobulin G kappa light chain (IgGk) protein. Larger case series are needed to determine the clinical significance of this entity.

Amniotic membrane transplantation induces apoptosis in T lymphocytes in murine corneas with experimental herpetic stromal keratitis.

PURPOSE: To investigate the effect of human amniotic membrane transplantation (AMT) on T-cell immune response in murine corneas with herpetic stromal keratitis (HSK). METHODS: Herpes simplex virus (HSV)-1-infected BALB/c mice with necrotizing HSK were treated with AMT. CD3(+) cell apoptosis was determined in treated corneas and in vitro by flow cytometric analysis using the annexin V/7-AAD system. The effect of interleukin (IL)-2, cyclosporine, rapamycin, or Fas on T-cell survival was measured. Activation phenotype was measured by (3)H-thymidine uptake and flow cytometry (CD25, CD69, major histocompatibility complex class II). Cytokine/chemokine secretion from amniotic membrane (AM)-treated corneas or draining lymph node cells was measured. The immune-modulating capacity of long-term AMT treatment and adoptive transfer of AM-treated splenocytes was tested. RESULTS: After AMT, HSK and corneal inflammatory cell infiltration improved, and T-lymphocyte apoptosis occurred. T-cell apoptosis was also induced in vitro, independently of rIL-2, cyclosporine, rapamycin, or Fas. AMT-treated corneas and cultured lymphocytes had reduced IL-2, IL-10, IL-12, CRG-2, and CCL-2 content. Long-term AMT treatment decreased the proliferative response and type 1 helper T-cell cytokine level in draining lymph node cells. The improvement in HSK did not persist. Delayed-type hypersensitivity or HSV-1-specific cytotoxicity was not altered CONCLUSIONS: The results suggest that murine HSK improves after AMT through reduced local T-helper cell immune responses by inducing apoptosis in T lymphocytes, independently of passive apoptosis or activation-induced cell death. AM also reduces local T-helper cytokine and chemokine levels but does not result in immune deviation. Immunologic memory against HSV-1 is not affected by AMT, and long-term protection or tolerance is not induced.

The use of phospholipase A(2) to prepare acellular porcine corneal stroma as a tissue engineering scaffold.

This study was to develop a method using phospholipase A(2) (PLA(2)) to prepare acellular porcine corneal stroma (APCS) for tissue engineering. The APCS was prepared from native porcine cornea (NPC) that was treated with 200 U/ml PLA(2) and 0.5% sodium deoxycholate (SD). The removal of DNA content, representing decellularization efficiency, reached to 91%, while all hydroxyproline and 80% of glycosaminoglycan were retained in the APCS when compared with NPC. The residual PLA(2) and SD were 0.35+/-0.04 U/mg dry weight and 4.3+/-0.8 ng/mg dry weight respectively. The extracts of APCS had no inhibitory effects on proliferation of corneal epithelial and endothelial cells as well as keratocytes. There was no sign of infiltration of neutrophilic leukocytes or leukomonocytes at 2 weeks after subcutaneous implantation of APCS. The prepared APCS displayed similar light transmittance to NPC. There were no significant differences in the areal modulus and curvature variation between APCS and NPC. Rabbit lamellar keratoplasty showed that the grafts of APCS were epithelialized completely in 8+/-2 days, and their transparency was restored in 84+/-11 days when the light transmittance of APCS-transplanted corneas displayed no significant difference compared with native corneas. Corneal neovascularization, corneal deformation, and graft degradation were not observed within 12 months.

Gamma interferon and interleukin-1 receptor 1 regulate neutrophil recruitment to the corneal stroma in a murine model of Onchocerca volvulus keratitis.

Toll-like receptor 2 (TLR2) is an essential mediator of corneal inflammation induced by the filarial nematode Onchocerca volvulus, which harbors endosymbiotic Wolbachia bacteria. TLR2 is also required for dendritic cell activation, gamma interferon (IFN-gamma) production, and neutrophil recruitment to the cornea. To examine the role of IFN-gamma in O. volvulus keratitis, C57BL/6 and IFN-gamma(-/-) mice were immunized subcutaneously, and a soluble antigen extract from O. volvulus adult worms (OvAg) was injected into the corneal stroma of each animal. We found that, in the absence of IFN-gamma, neutrophil recruitment to the cornea was significantly impaired, whereas there was no effect on eosinophil infiltration. Since the cornea contains resident macrophages and fibroblasts and our previous studies showed that CXC chemokines mediate neutrophil recruitment, we examined the role of recombinant IFN-gamma (rIFN-gamma) on each cell type. We found no effect of rIFN-gamma on CXC chemokine production by macrophages or corneal fibroblasts, either alone or with filarial extracts; in contrast, rIFN-gamma was found to enhance OvAg-induced tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), IL-1alpha, and IL-1beta in macrophages. Furthermore, we found that rTNF-alpha, rIL-1alpha, or rIL-1beta induced CXC chemokine production by corneal fibroblasts but not by macrophages. To determine the relative contributions of endogenous cytokines, we injected OvAg into the corneal stroma of C57BL/6, IL-1 receptor 1(-/-) (IL-1R1(-/-)), and TNF-alphaR1/2(-/-) mice and examined neutrophil recruitment. We found that neutrophil infiltration was impaired in IL-1R1(-/-) mice but not in TNF-alphaR1/2(-/-) mice. IFN-gamma therefore appears to regulate neutrophil recruitment to the corneal stroma by enhancing TLR2 expression and OvAg-induced IL-1alpha and IL-1beta production by macrophages in the cornea, which then induce IL-1R1-dependent production of CXC chemokine by resident corneal fibroblasts.