Cells Of Cornea Research Articles

Age-Related Decrease in Human Corneal γ-Glutamyltranspeptidase Activity

To investigate age-related effects on human corneal γ-glutamyltranspeptidase (GGT) (ectoenzyme important to maintaining corneal hydration and antioxidant potential via glutathione recapture). Age-related differences between total, endothelial, and epithelial GGT activity and endothelial cell density were determined for corneas from 29 donors (mean age, 53 ± 17 years; age range, 13-83 years). GGT activity was determined using a standard colorimetric assay based on the transpeptidation reaction. Corneal GGT localization and expression was determined by immunohistochemistry. Total corneal, endothelial, and epithelial GGT activities in the young (<50 years) donor corneas were 37% (P = 0.02), 44% (P = 0.001), and 36% (P = 0.06) higher, respectively, than in the senior (≥50 years) corneas. The age-related rates of decline for GGT activity were 1.0 unit per year for total cornea, 0.4 to 0.5 unit per year for endothelium, and 0.3 to 0.4 unit per year for epithelium. Notably, endothelial cell density in the young corneas was 14% (P = 0.001) higher than in the senior corneas declining about 100 cells per square millimeter per decade (0.3% per year). GGT activity per 10 endothelial cells decreased at about 0.2 units per year and GGT activity per 10 endothelial cells in the young corneas was 41% higher (P = 0.01) than in the senior corneas. Fewer immunoreactive GGT-positive epithelial cells were detected in senior cornea. The age-related loss of human corneal GGT activity was associated with reductions in endothelial and epithelial GGT activity, being because of reduced number of GGT-positive endothelial and epithelial cells and reduced GGT activity per endothelial cell.

Identification of keratocyte-like cells differentiated from circulating bone marrow-derived cells in the mouse cornea

Bone marrow (BM)-derived stem cells have the potential to differentiate into multiple lineages of tissue resident cells. BM-derived cells have been detected in the mouse cornea, but most of these cells were found to be CD45(+) or CD11b(+) immunocompetent cells. Although some BM-derived cells in the cornea were negative for these cell surface markers, it has remained unclear whether cells of BM origin can differentiate into corneal resident cells. To address this issue, we subjected wild-type mice that had been exposed to a lethal dose of radiation to intravenous injection with BM cells from green fluorescent protein (GFP) transgenic mice. Two months after cell transplantation, fluorescence microscopy revealed the presence of numerous GFP(+) cells throughout the cornea, with intense GFP fluorescence being apparent around the limbal region. Immunohistofluorescence analysis of corneal cross-sections revealed that most of the BM-derived GFP(+) cells expressed CD45 or CD11b, although a few GFP(+) cells were negative for these markers. Immunostaining of individual cells isolated from the corneal stroma, however, showed that a small proportion (~1%) of GFP(+) BM-derived cells expressed the keratocyte-specific proteoglycan keratocan. Our results suggest that BM-derived cells introduced intravenously are able to differentiate into resident cells of the corneal stroma.

Heat Effect Induces Production of Inflammatory Cytokines Through Heat Shock Protein 90 Pathway in Cornea Cells

Purpose: Many studies have been conducted to elucidate the molecular consequences of ultraviolet irradiation, whereas little is known about the effect of infrared radiation on ocular disease. Heat is generated as a consequence of infrared irradiation, in addition to photons, and heat shock is widely considered to be an environmental stressor. Our previous study has demonstrated that heat shock (42 °C) induces apoptosis of Statens Seruminstitut Rabbit Cornea (SIRC) cells. However, the biological effect of mild heat shock (39 °C), which does not induce apoptosis, on SIRC cells has not been studied yet.Methods: The SIRC cells were treated with mild heat shock and the expression of inflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-6 (IL-6), in the mRNA and protein levels were then assayed with reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The levels of heat shock protein 90 (HSP90), phosphorylated AKT, phosphorylated IκBα and nuclear NF-κB were assessed with immunoblot assays; the involvement of these molecules in the signaling pathway were further assessed by using specific inhibitor for HSP90, AKT or NF-κB.Results: Mild heat shock increased the production of inflammatory cytokines, IL-1β and IL-6. Mild heat shock increased expression of HSP90, which increased the phosphorylation of AKT. The activated AKT then increased the phosphorylation of IκBα, resulting in nuclear translocation of NF-κB, which up-regulated the expression of IL-1β and IL-6 in SIRC cells.Conclusions: These findings suggest a critical role for the HSP90-AKT-NF-κB signaling pathway in mild heat shock-induced inflammatory response of cornea cells.

Ocular chemical injuries and their management

Chemical burns represent potentially blinding ocular injuries and constitute a true ocular emergency requiring immediate assessment and initiation of treatment. The majority of victims are young and exposure occurs at home, work place and in association with criminal assaults. Alkali injuries occur more frequently than acid injuries. Chemical injuries of the eye produce extensive damage to the ocular surface epithelium, cornea, anterior segment and limbal stem cells resulting in permanent unilateral or bilateral visual impairment. Emergency management if appropriate may be single most important factor in determining visual outcome. This article reviews the emergency management and newer techniques to improve the prognosis of patients with chemical injuries.

Peptide functionalized nanoparticles for nonviral gene delivery

Much attention has been directed toward improving nonviral gene delivery in order to replace viral vectors and overcome the barriers to clinical gene therapy. Specifically targeting nonviral gene delivery vehicles to the disease tissue can significantly improve their delivery efficiency. Targeting peptides have been developed for growth factor receptors, integrins and tumor vasculature to target cancer, neurotrophin or neurotoxin receptors aiming to give specificity for neurodegenerative disease, airway epithelial cells to target cystic fibrosis and other tissue specific cells and receptors in bone tissue, cornea and dendritic cells. These highly specific, high-affinity peptides were identified using phage display or through rational design, and were evaluated within this review for their potential and ability to improve transfection, highlighting the successful applications of this technology and discussing the features of the design that contribute to this success. It was found that effective peptide targeting of gene delivery relied both on peptide ligand performance and on nonviral vectors that exploit the appropriate intracellular trafficking mechanisms.

Membrane nanotubes in myeloid cells in the adult mouse cornea represent a novel mode of immune cell interaction

Membrane nanotubes (MNTs) are newly discovered cellular extensions that are either blind-ended or can connect widely separated cells. They have predominantly been investigated in cultured isolated cells, however, previously we were the first group to demonstrate the existence of these structures in vivo in intact mammalian tissues. We previously demonstrated the frequency of both cell-cell or bridging MNTs and blind-ended MNTs was greatest between major histocompatibility complex (MHC) class II(+) cells during corneal injury or TLR ligand-mediated inflammation. The present study aimed to further explore the dynamics of MNT formation and their size, presence in another tissue, the dura mater, and response to stress factors and an active local viral infection of the murine cornea. Confocal live cell imaging of myeloid-derived cells in inflamed corneal explants from Cx(3)cr1(GFP) and CD11c(eYFP) transgenic mice revealed that MNTs form de novo at a rate of 15.5 μm/min. This observation contrasts with previous studies that demonstrated that in vitro these structures originate from cell-cell contacts. Conditions that promote formation of MNTs include inflammation in vivo and cell stress due to serum starvation ex vivo. Herpes simplex virus-1 infection did not cause a significant increase in MNT numbers in myeloid cells in the cornea above that observed in injury controls, confirming that corneal epithelium injury alone elicits MNT formation in vivo. These novel observations extend the currently limited understanding of MNTs in live mammalian tissues.

Silencing HLA Class I Expression in Human Corneas to Decrease the Risk of Graft Rejection after Keratoplasty

The high variability of the human histocompatibility leukocyte antigen (HLA) complex, and of the minor histocompatibility antigens significantly contributes to elicit an immune response after allogeneic cornea transplantation, and may lead to the development of acute or chronic rejection. In addition, often inevitable second cornea transplantation is always associated with an increased risk of graft rejection. This project aims to permanently reduce HLA expression on cornea transplants to decrease the risk of graft rejection after keratoplasty. This method represents a new therapeutic concept and may contribute to prevent graft rejection. For this purpose, lentiviral vectors encoding short hairpin RNAs (shRNA) targeting β2-microglobulin were used to induce the downregulation of HLA class I expression. Levels of β2-microglobulin mRNA or cell surface HLA class I expression were determined by Real-Time PCR and flow cytometry, respectively. Cell viability tests using 7-Aminoactinomycin (7-AAD) and Propidium iodide (PI) staining were performed after transduction. Light and fluorescence microscopic analysis were carried out to evaluate the integrity of the corneal endothelium. A mean of 92%±7% of the total cornea cells were transduced. In comparison to the non-engineered cornea, the delivery of the β2-microglobulin-specific shRNA caused a reduction by up to 95% in β2-microglobulin transcript levels as detected at day 5 and day 15 after cornea transduction. Furthermore, the reduction of β2-microglobulin levels induced a decrease in the cell surface expression of HLA class I proteins by up to 90%. Microscopic analysis of the cornea endothelium showed that this cell layer was not affected by the transduction procedure or the silencing effect. The integrity of the HLA class I silenced endothelium and the absence of a positive signal upon 7-AAD/PI staining indicated the absence of off target effects that might impair the endothelial function or viability. This data demonstrates the feasibility of silencing HLA class I expression in the original 3D tissue-structure by RNAi-mediated nucleic acid targeting. As in vivo data in an allogeneic rat model showed a superior survival of RT1-silenced cells, silencing HLA expression has the potential to overcome alloimmunization and tissue rejection caused by HLA mismatches in allogeneic keratoplasty.

Enhanced cornea cell growth on a keratoprosthesis material immobilized with fibronectin or EGF

The surface of keratoprosthesis (KPro) material was modified with functional proteins to enhance corneal cell growth. Surface modification of poly(methyl methacrylate) (PMMA) substrates resulted in greater relative cornea cells growth. The increased growth rate of fibroblast on PMMA with immobilized functional proteins, fibronectin or epidermal growth factor (EGF), was approx. 39% or 19%, respectively. The modified PMMA surface with fibronectin or EGF also increased the human corneal limbal epithelial cell line (HCLE) growth to a maximum of 27% or 73%, respectively. However, PMMA surfaces with non-covalently adsorbed functional proteins did not significantly promote the growth of fibroblast or HCLE. To achieve the maximum growth of cornea cells, the bulk protein concentrations were in the range of 50 and 100 μg/mL for both proteins. This study found the relationship between specific cell growth and functional protein concentrations. Due to its ability to stimulate cell growth with known protein concentration, the newly designed KPro material with selective immobilized proteins may promote integration between KPro and the host tissue. Open image in new window

Expression analysis of MDR1, BCRP and MRP3 transporter proteins in different in vitro and ex vivo cornea models for drug absorption studies

Ocular drug absorption studies are required for the development of new drugs or drug delivery systems for eye treatment. Such preclinical investigations on transcorneal drug absorption are performed ex vivo with the excised corneas of experimental animals or in vitro using corneal cell culture models. The data currently available on the expression of ABC transporter proteins in corneal tissue is limited or contradictory. This study describes, for the first time, the comparison of the expression of ABC transporters, in particular, MDR1, BCRP and MRP3, between human cornea cell culture models and the most commonly used ex vivo models, namely, rabbit and porcine corneas, conducted in the same laboratory. The expression levels and functionality were determined by means of PCR, western blot, immunohistochemistry and bidirectional permeation studies using specific substrates and inhibitors. The results clearly indicate species-dependent expression of the studied efflux transporters. In the rabbit cornea, the expression and activity of MDR1 transporter was confirmed, whereas human cell culture models and porcine corneas did not show MDR1 expression. However, human cornea models possessed MRP3 and BCRP expression, whereas no functional expression was found in rabbit and porcine corneas. Therefore, the translation of transcorneal permeation data from animal experiments to humans should be performed with caution.

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-1β, 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-1β, 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.

Identification and evaluation of a thinning agent compatible with MegaCell DCS, an animal product-free corneal storage medium

PurposeMegaCell DCS, an animal product-free culture medium formulated for storing corneas, is superior to the traditionally used MEM (Eagle’s) with Earles salts, Hepes, and supplemented with foetal calf serum (2 %), glutamine and an antibiotic cocktail (EB MEM). Because this medium does not prevent corneal swelling, and Dextran T500, which is traditionally used for reversing this process before transplant may have adverse effects on corneas, the purpose of the current investigation was to identify an alternative polymer that is compatible with MegaCell DCS.MethodsCorneas maintained in MegaCell DCS or EB MEM were transferred to either EB MEM 5 % Dextran T500 or MegaCell DCS containing 5 % Dextran T500, 4 % polyethylene glycol (PEG) 10,000, PEG 35,000 (2 %, 3 %, 4 %) or Poloxamer 188 (4 %). Endothelial cell losses were determined and corneal hydration levels measured. Stromal cell cultures were generated and immunostained with anti α-SMA antibody. Janus Green was used to compare the viability of endothelial cells of corneas maintained in MegaCell DCS and EB MEM and respectively thinned with PEG 35,000 and Dextran T500.ResultsThe rates of endothelial cell loss from corneas held in MegaCell DCS and thinned in MegaCell DCS containing 5 % Dextran T500, 4 % PEG 10,000 and 4 % Poloxamer 188 for 6 days were similar. When explants of these corneas were cultured myofibroblasts were generated. Although at concentrations of 4 % (w/v) both PEG 10,000 and Poloxamer 188 caused excessive dehydration, the hydration levels of corneas held in MegaCell DCS containing 3 % PEG 35,000 were similar to those of corneas held in EB MEM 5 % Dextran T500. Endothelial cell losses after 6 days were negligible, explants of the corneas generated uniform fibroblastic stromal cell cultures and the extents of Janus Green staining were similar. Over 20 days the inclusion of 5 % Dextran T500 in EB MEM but not 3 % PEG 35,000 in MegaCell DCS, increased the rate of endothelial cell loss.ConclusionPEG 35,000 at a concentration of 3 % w/v does not induce endothelial cell loss and is compatible with MegaCell DCS for thinning corneas prior to transplantation.

Identification of label‐retaining endothelial cells in adult human corneas: a new clue for the existence of endothelial stem cells

AbstractPurpose The lack of self‐renewal capacity of human corneal endothelial cells (EC) in vivo was explained by cell cycle arrest in the G1‐phase due to cell contact inhibition, TGF‐beta signaling, and stress induced premature senescence. Nevertheless, their residual ability to divide in primary culture suggests the existence of progenitor cells, probably located at the endothelial periphery (Whikehart.MolVis2005; He.StemCells2012). Stem cells are slow‐cycling cells characterized by their quiescent state in niches and their ability to retain for a long time markers of S‐Phase like BrDU or EdU. Aim: to search for the presence of label‐retaining EC in human corneasMethods Label retaining EC were searched by 5‐Ethynyl‐2’‐Deoxyuridine (Click‐it EdU) incorporation during long‐term culture: 30 days for organ cultured human corneas (n=10) or 15 days for in vitro primary cultured EC (n=5), both followed by a 30‐day culture without EdU. Flat‐mouted corneas and EC cultures were observed with an inverted fluorescent microscopeResults Label‐retaining EC were observed in the peripheral area of all OC corneas, varying from 1 to 50. Numerous label‐retaining EC were also present in all primary cultures, always attached to Descemet membrane fragmentsConclusion The presence of label‐retaining EC constitutes a new clue for the existence of corneal endothelial stem cells in human. Their apparent scarcity is consistent with the inability of the human corneal endothelium to repair in vivo, but isolation and expansion of these endothelial stem cells or progenitors could allow development of bioengineered endothelium

Expression of Lubricin mRNA and protein in human ocular surface tissues

AbstractPurpose Friction is an inherent potential source of wounding for ocular surface tissues during the blink and bulbus movements and is identified as a major contributor to ocular surface disease of the dry eye type. The expression of lubricin, a boundary lubricant in articulating joints that protects against frictional forces, cell adhesion and protein deposition, was investigated at the ocular surface.Methods Human corneal, conjunctival and control cartilage tissues were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned serially, exposed to antigen retrieval buffers, and stained for lubricin by immunohistochemistry (IHC) with a rabbit polyclonal anti‐human lubricin antibody. Several negative and positive controls were performed. Respective fresh tissues were analysed by reverse transcriptase polymerase chain reaction (RT‐PCR) in order to verify the presence of the mRNA.Results The mRNA for lubricin was found in human corneal and conjunctival cell lines and in complete ocular surface tissues similar to cartilage. The mRNA is translated and expressed into lubricin protein and protein staining was observed along the complete corneal and conjunctival epithelial surface, most strongly expressed in the corneal epithelium . The specificity of staining was verified in several positive and negative controls including use of irrelevant antibodies and preincubation of the anti‐lubricin antibody with a pure peptide, which had served to generate the antibody.Conclusion These findings verify that lubricin is synthesized and expressed by epithelial cells of the human cornea and conjunctiva and conceivably serves protective functions against frictional forces at the human ocular surface similar to the joint.

Surface Chemistry Study of the Interactions of Pharmaceutical Ingredients with Human Meibum Films

To perform a surface chemistry study of the interactions between the benzalkonium chloride (BAC)-preserved eyedrops Travatan, the SofZia-preserved TravatanZ, and the Polyquad-preserved DuoTrav, and tear film (TF) constituents. The interactions of TF compounds with the individual preservatives, BAC, SofZia, and Polyquad, were also examined. Langmuir surface balance measurements were used to examine the interactions between the pharmaceuticals and films of human meibum and rabbit corneal cell lipid extracts. Surface pressure-area isocycles were used to assess the sample's capability to compress and spread during dynamic area changes. The dilatational rheologic properties of human meibum films, pure and in the presence of preservatives, were probed by stress-relaxation studies. Lipid film morphology was monitored by Brewster angle microscopy. The viability of SofZia- and Polyquad-treated Statens Seruminstitut Rabbit Cornea (SIRC) cell cultures was also evaluated. The interactions between BAC-preserved eyedrops and lipids resulted in impaired lipid spread, formation of discontinuous nonuniform surface layers, and increased surface pressure-area hysteresis during compression/expansion. In contrast, TravatanZ, DuoTrav, and the individual preservatives SofZia and Polyquad proved to be safe to the lipid film structure and isothermal reversibility. The stress-relaxation experiments revealed that the viscoelastic properties of meibomian film are impaired by BAC, and remain unaffected by SofZia and Polyquad. SIRC cells' viability and capability to form confluent cellular monolayer were also maintained after exposure to SofZia and Polyquad. Surface chemistry studies present criteria for preclinical in vitro molecular scale characterization of the interactions between eyedrop compounds and TF constituents.

Distribution of Antigen Presenting Cells in the Human Cornea: Correlation of In Vivo Confocal Microscopy and Immunohistochemistry in Different Pathologic Entities

Purpose: The purpose of this study was to determine the quantity and distribution of antigen presenting cells (APC) in various inflammatory and non-inflammatory corneal diseases, comparing in vivo confocal microscopy (IVCM) and immunohistochemistry.Material and methods: Corneae of 41 eyes, composed of group 1 (status post herpes-keratitis), group 2 (keratoconus) and group 3 (graft rejection after keratoplasty) were investigated. IVCM was used preoperatively to assess the distribution and density of dendritic cells in the corneal center versus the paracentral area. Afterwards, all patients underwent penetrating keratoplasty. The host corneas were analyzed by immunohistochemistry for antigen presenting cell distribution, density and characterization by using specific markers for CD207/Langerin, CD209/DC-SIGN and HLA-DR. The IVCM findings were compared with immunohistochemistry results in the corneal epithelium.Results: Cells with branching dendritic morphology were visualized by IVCM mainly in the basal epithelial layer and subepithelial nerve plexus of the central and paracentral cornea. The density of APC in IVCM decreased in all groups towards the central part of the cornea. The highest gradient was observed in group 2, followed by groups 1 and 3. The corneal paracenter showed similiar distribution of APC in group 1 and 2 (76.7 cells/mm2 and 74.4 cells/mm2). The highest density of central APC was observed in group 1 (53.76 cells/mm2), followed by group 3 (27.0 cells/mm2) and group 2 (24.2 cells/mm2). In immunohistochemistry positive stained, APC were distributed similarly to IVCM but with a higher density (p < 0.05).Conclusion: Distribution, density and stage of maturation of corneal epithelial APCs can be evaluated on morphological basis by IVCM. However, the corneal APCs density was about three-fold lower compared to immunohistochemistry findings.

Cathelicidin gene therapy: a new therapeutic option in ulcerative colitis and beyond?

Cathelicidins are natural antimicrobial peptides synthesized and stored in neutrophils, macrophages and also in epithelial cells of gut, skin, cornea and lungs.1, 2, 3, 4 Cathelicidins are present in all mammalian species and exert a potent, broad-spectrum antimicrobial activity against bacteria, viruses, fungi and some parasites by disrupting the membranes of these microorganisms.1, 2, 3 In mice, the cathelicidin cathelin-related antimicrobial peptide (CRAMP) is encoded by the gene Cnlp. In humans, the cathelicidin LL-37 is encoded by the gene camp.1, 2, 3, 5

Mucopolysaccharidosis type VI in a Miniature Poodle-type dog caused by a deletion in the arylsulphatase B gene

AIM: To investigate and characterise an inborn error of metabolism in a dog with skeletal and ocular abnormalities. METHODS: A 2.5-year-old small male Miniature Poodle-like dog was presented with gross joint laxity and bilateral corneal opacities. Clinical examination was augmented by routine haematology, serum chemistry, radiographs, pathology, enzymology and molecular genetic studies. Euthanasia was requested when the dog was 3 years of age because of progressively decreasing quality of life. RESULTS: Radiology revealed generalised epiphyseal dysplasia, malformed vertebral bodies, luxation/subluxation of appendicular and lumbosacral joints with hypoplasia of the odontoid process and hyoid apparatus. These clinical and radiographic findings, together with a positive urinary Berry spot test for mucopolysaccharides, and metachromatic granules in leucocytes, were indicative of a mucopolysaccharidosis (MPS), a lysosomal storage disease. Histological lesions included vacuolation of stromal cells of the cornea, fibroblasts, chondrocytes, macrophages and renal cells. The brain was essentially normal except for moderate secondary Wallerian-type degeneration in motor and sensory tracts of the hind brain. Dermatan sulphate-uria was present and enzymology revealed negligible activity of N-acetylgalactosamine-4-sulphatase, also known as arylsulphatase B, in cultured fibroblasts and liver tissue. A novel homozygous 22 base pair (bp) deletion in exon 1 of this enzyme's gene was identified (c.103_124del), which caused aframe-shift and subsequent premature stop codon. The “Wisdom pure breed-mixed breed” test reported the dog as a cross between a Miniature and Toy Poodle. CONCLUSIONS: The clinicopathological features are similar to those of MPS type VI as previously described in dogs, cats and other species, and this clinical diagnosis was confirmed by enzymology and molecular genetic studies. This is an autosomal recessively inherited lysosomal storage disease. CLINICAL RELEVANCE: The prevalence of MPS VI in Miniature or Toy Poodles in New Zealand and elsewhere is currently unknown. Due to the congenital nature of the disorder, malformed pups may be subject to euthanasia without investigation and the potential genetic problem in the breed may not be fully recognised. The establishment of a molecular genetic test now permits screening for this mutation as a basis to an informed breeding policy.

Retina Expression and Cross-Species Validation of Gene Silencing by PF-655, a Small Interfering RNA Against RTP801 for the Treatment of Ocular Disease

PF-655, a synthetic 19-mer siRNA, targeting the RTP801 gene is currently in clinical trials for the treatment of wet age-related macular degeneration and diabetic macular edema. Preclinical studies have shown a dose-related suppression of RTP801 expression in rat disease models. Investigative studies were conducted with PF-655 to validate the Dutch-Belted rabbit as a biologically relevant species for gene silencing to support nonclinical ocular toxicity and continual dosing studies. Cross-species comparison and DNA sequencing was done to determine the level of homology between PF-655 and rabbit RTP801. Human (HEK 293) and rabbit (SIRC cornea) cell lines were stimulated with CoCl(2) to mimic hypoxic stress (an inducer of RTP801 expression) and treated with PF-655. Taqman-polymerase chain reaction and immunoblot analysis were performed to gauge RTP801 expression in cell culture and rabbit retinas. Sequence analysis showed a 1-base mismatch in the PF-655 targeting site from genomic DNA of Dutch-Belted rabbit and the SIRC cell line, a cornea cell derived from the New Zealand White rabbit. HEK and SIRC CoCl(2)-stressed cells induced RTP801 expression 10-20-fold above control conditions. Treatment with 20 or 100 nM PF-655 showed a decrease in gene expression, 40%-50% relative to appropriate controls. RTP801 mRNA was detectable in primary rabbit retina tissues, with cycle threshold values showing a large linear range for the assay. These results support our investigation into cross-species validation of gene suppression by a therapeutic siRNA designed to a human gene. The SIRC cell line was utilized as a surrogate to test the degree of RTP801 gene silencing induced by PF-655 in vitro. With a 1-base mismatch, the level of silencing in a rabbit ocular cell line was comparable to that of a human cell line. Sequence analysis and expression data confirmed the relevance of the RTP801 target gene in rabbits and the utility of this species as a relevant animal model. Additionally, our work outlines a tractable method that validates relevant larger non-rodent species for ophthalmic drug testing.

Identification of Mono- and Disulfated N-Acetyl-lactosaminyl Oligosaccharide Structures as Epitopes Specifically Recognized by Humanized Monoclonal Antibody HMOCC-1 Raised against Ovarian Cancer

A humanized monoclonal antibody raised against human ovarian cancer RMG-I cells and designated as HMOCC-1 (Suzuki, N., Aoki, D., Tamada, Y., Susumu, N., Orikawa, K., Tsukazaki, K., Sakayori, M., Suzuki, A., Fukuchi, T., Mukai, M., Kojima-Aikawa, K., Ishida, I., and Nozawa, S. (2004) Gynecol. Oncol. 95, 290-298) was characterized for its carbohydrate epitope structure. Specifically, a series of co-transfections was performed using mammalian expression vectors encoding specific glycosyltransferases and sulfotransferases. These experiments identified one sulfotransferase, GAL3ST3, and one glycosyltransferase, B3GNT7, as required for HMOCC-1 antigen formation. They also suggested that the sulfotransferase CHST1 regulates the abundance and intensity of HMOCC-1 antigen. When HEK293T cells were co-transfected with GAL3ST3 and B3GNT7 expression vectors, transfected cells weakly expressed HMOCC-1 antigen. When cells were first co-transfected with GAL3ST3 and B3GNT7 and then with CHST1, the resulting cells strongly expressed HMOCC-1 antigen. However, when cells were transfected with a mixture of GAL3ST3 and CHST1 before or after transfection with B3GNT7, the number of antigen-positive cells decreased relative to the number seen with only GAL3ST3 and B3GNT7, suggesting that CHST1 plays a regulatory role in HMOCC-1 antigen formation. Because these results predicted that HMOCC-1 antigens are SO(3) → 3Galβ1 → 4GlcNAcβ1 → 3(±SO(3) → 6)Galβ1 → 4GlcNAc, we chemically synthesized mono- and disulfated and unsulfated oligosaccharides. Immunoassays using these oligosaccharides as inhibitors showed the strongest activity by disulfated tetrasaccharide, weak but positive activity by monosulfated tetrasaccharide at the terminal galactose, and no activity by nonsulfated tetrasaccharides. These results establish the HMOCC-1 epitope, which should serve as a useful reagent to further characterize ovarian cancer.

A tiered approach combining the short time exposure (STE) test and the bovine corneal opacity and permeability (BCOP) assay for predicting eye irritation potential of chemicals

For the evaluation of eye irritation, one in vitro alternative test may not completely replace the Draize test. Therefore, a tiered approach combining several in vitro assays, including cytotoxicity assays, is proposed in order to estimate the eye irritation potential of a wide range of chemical classes. The Short Time Exposure (STE) test, a relatively newer alternative eye irritation test, involves exposing Statens seruminstitut rabbit cornea (SIRC) cells for 5 min to two concentrations (5% and 0.05%) of test material. In the present study, we examined the predictive capacity of a tiered approach analyzing the results from the STE test and then the results of the bovine corneal opacity and permeability (BCOP) assay for assessing globally harmonized system (GHS) eye irritation rankings of various chemicals. The accuracy of predicting the GHS rankings was slightly improved when the tiered approach combination of STE test and BCOP assay was used compared to when the STE test irritation rank classification was used alone. Moreover, the under prediction rate was substantially improved when this tiered approach was used. From these results, the tiered approach of combining the data analysis of the STE test and BCOP assay might be a promising alternative eye irritation test strategy.