Thrombospondin-2 is up-regulated by TGFß2 and increases fibronectin expression in human trabecular meshwork cells.

Elevated intraocular pressure (IOP) is a major risk factor for the development of primary open-angle glaucoma (POAG). This is from an increased aqueous humour (AH) outflow resistance through the trabecular meshwork (TM). The pathogenic mechanisms leading to the increase in TM outflow resistance are poorly understood but are thought to be from a dysregulation of the TM extracellular matrix (ECM) environment. ECM modification and turnover are crucial in regulating the resistance to aqueous outflow. ECM turnover is influenced by a complex interplay of growth factors such as transforming growth factors (TGFß) family and matrix metalloproteinases (MMPs). Elevated TGFß2 levels result in an increase in ECM deposition such as fibronectin leading to increased resistance. Fibronectin is a major component of TM ECM and plays a key role in its maintenance. Thrombospondins (TSP)-1 and -2 are important regulators of the ECM environment. TSP-1 has been implicated in the pathogenesis of POAG through activation of TGFß2 within the TM. TSP-2 does not contain the catalytic domain to activate latent TGFß, but is able to mediate the activities of MMP 2 and 9, thereby influencing ECM turnover. TSP-2 knock out mice show lower IOP levels compared to their wild type counterparts, suggesting the involvement of TSP-2 in the pathogenesis of POAG but its role in the pathogenesis of POAG remains unclear. The purpose of this study was to investigate the role of TSP-2 in trabecular meshwork ECM regulation and hence the pathogenesis of POAG. TSP-1 and TSP-2 expressions in immortalised glaucomatous TM cells (GTM3) and primary human non-glaucomatous (NTM) and glaucomatous cells (GTM) were determined by immunocytochemistry, immuno-blot analysis and qPCR following treatment with TGFß2 and Dexamethasone. The level of ECM protein fibronectin was determined in TM cells using immuno-blot analysis following treatment with TSP-1 or -2. TM cells secrete TSP-1 and -2 under basal conditions at the protein level and TSP-2 mRNA and protein levels were increased in response to TGFß2 three days post treatment. Exogenous treatment with TSP-2 up-regulated the expression of fibronectin protein in GTM3 cells, primary NTM and GTM cells. TSP-1 did not affect fibronectin protein levels in GTM3 cells. This suggests that the role of TSP-2 might be distinct from that of TSP-1 in the regulation of the TM cell ECM environment. TSP-2 may be involved in the pathogenesis of POAG and contribute to increased IOP levels by increasing the deposition of fibronectin within the ECM in response to TGFß2.

Poly-ε-lysine based hydrogels as synthetic substrates for the expansion of corneal endothelial cells for transplantation.

Dysfunction of the corneal endothelium (CE) resulting from progressive cell loss leads to corneal oedema and significant visual impairment. Current treatments rely upon donor allogeneic tissue to replace the damaged CE. A donor cornea shortage necessitates the development of biomaterials, enabling in vitro expansion of corneal endothelial cells (CECs). This study investigated the use of a synthetic peptide hydrogel using poly-ε-lysine (pεK), cross-linked with octanedioic-acid as a potential substrate for CECs expansion and CE grafts. PεK hydrogel properties were optimised to produce a substrate which was thin, transparent, porous and robust. A human corneal endothelial cell line (HCEC-12) attached and grew on pεK hydrogels as confluent monolayers after 7 days, whereas primary porcine CECs (pCECs) detached from the pεK hydrogel. Pre-adsorption of collagen I, collagen IV and fibronectin to the pεK hydrogel increased pCEC adhesion at 24 h and confluent monolayers formed at 7 days. Minimal cell adhesion was observed with pre-adsorbed laminin, chondroitin sulphate or commercial FNC coating mix (fibronectin, collagen and albumin). Functionalisation of the pεK hydrogel with synthetic cell binding peptide H-Gly-Gly-Arg-Gly-Asp-Gly-Gly-OH (RGD) or α2ß1 integrin recognition sequence H-Asp-Gly-Glu-Ala-OH (DGEA) resulted in enhanced pCEC adhesion with the RGD peptide only. pCECs grown in culture at 5 weeks on RGD pεK hydrogels showed zonula occludins 1 staining for tight junctions and expression of sodium-potassium adenosine triphosphase, suggesting a functional CE. These results demonstrate the pεK hydrogel can be tailored through covalent binding of RGD to provide a surface for CEC attachment and growth. Thus, providing a synthetic substrate with a therapeutic application for the expansion of allogenic CECs and replacement of damaged CE.

Novel Colorimetric and Light Scatter Methods to Identify and Manage Peritoneal Dialysis-Associated Peritonitis at the Point-of-Care.

Introduction: Peritoneal dialysis (PD)-related peritonitis (PDRP) is a common cause of transfer to hemodialysis, patient morbidity, and is a risk factor for mortality. Associated patient anxiety can deter selection of PD for renal replacement therapy. Diagnosis relies on hospital laboratory tests; however, this might be achieved earlier if such information was available at the point-of-care (POC), thereby significantly improving outcomes. The presence of culturable microbes and the concentration of leukocytes in effluent both aid peritonitis diagnosis, as specified in the International Society for Peritoneal Dialysis (ISPD) diagnostic guidelines. Here, we report the development of 2 new methods providing such information in simple POC tests. Methods: One approach uses a tetrazolium-based chemical reporting system, primarily focused on detecting bacterial contamination and associated vancomycin-sensitivity. The second approach uses a novel forward light-scatter device (QuickCheck) to provide an instant quantitative cell count directly from PD patient effluent. Results: The tetrazolium approach detected and correctly distinguished laboratory isolates, taking 10 hours to provide non-quantitative results. We compared the technical performance of the light scatter leukocyte counting approach with spectrophotometry, hemocytometer counting and flow cytometry (Sysmex) using patient effluent samples. QuickCheck had high accuracy (94%) and was the most precise (coefficient of variation <4%), showing minimal bias, overall performing similarly to flow cytometry. Conclusion: These complementary new approaches provide a simple means to obtain information to assist diagnosis at the POC. The first provides antibiotic sensitivity following 10 hours incubation, whereas the second optical approach (QuickCheck), provides instant accurate total leukocyte count.

FluoroPi Device With SmartProbes: A Frugal Point-of-Care System for Fluorescent Detection of Bacteria From a Pre-Clinical Model of Microbial Keratitis.

Purpose: Rapid and accurate diagnosis of microbial keratitis (MK) could greatly improve patient outcomes. Here, we present the development of a rapid, accessible multicolour fluorescence imaging device (FluoroPi) and evaluate its performance in combination with fluorescent optical reporters (SmartProbes) to distinguish bacterial Gram status. Furthermore, we show feasibility by imaging samples obtained by corneal scrape and minimally invasive corneal impression membrane (CIM) from ex vivo porcine corneal MK models. Methods: FluoroPi was built using a Raspberry Pi single-board computer and camera, light-emitting-diodes (LEDs), and filters for white-light and fluorescent imaging, with excitation and detection of bacterial optical SmartProbes: Gram-negative, NBD-PMX (exmax 488 nm); Gram positive, Merocy-Van (exmax 590 nm). We evaluated FluoroPi with bacteria (Pseudomonas aeruginosa and Staphylococcus aureus) isolated from ex vivo porcine corneal models of MK by scrape (needle) and CIM with the SmartProbes. Results: FluoroPi provides <1 µm resolution and was able to readily distinguish bacteria isolated from ex vivo models of MK from tissue debris when combined with SmartProbes, retrieved by both scrape and CIM. Single bacteria could be resolved within the field of view, with limits of detection demonstrated as 103 to 104 CFU/mL. Sample preparation prior to imaging was minimal (wash-free), and imaging and postprocessing with FluoroPi were straightforward, confirming ease of use. Conclusions: FluoroPi coupled with SmartProbes provides effective, low-cost bacterial imaging, delineating Gram-negative and Gram-positive bacteria directly sampled from a preclinical model of MK. Translational Relevance: This study provides a crucial stepping stone toward clinical translation of a rapid, minimally invasive diagnostic approach for MK.

Corneal Endothelial Cell Loss in Glaucoma and Glaucoma Surgery and the Utility of Management with Descemet Membrane Endothelial Keratoplasty (DMEK).

The corneal endothelium has a crucial role in maintaining a clear and healthy cornea. Corneal endothelial cell loss occurs naturally with age; however, a diagnosis of glaucoma and surgical intervention for glaucoma can exacerbate a decline in cell number and impairment in morphology. In glaucoma, the mechanisms for this are not well understood and this accelerated cell loss can result in corneal decompensation. Given the high prevalence of glaucoma worldwide, this review aims to explore the abnormalities observed in the corneal endothelium in differing glaucoma phenotypes and glaucoma therapies (medical or surgical including with new generation microinvasive glaucoma surgeries). Descemet membrane endothelial keratoplasty (DMEK) is increasingly being used to manage corneal endothelial failure for glaucoma patients and we aim to review the recent literature evaluating the use of this technique in this clinical scenario.

Amoebicidal Activity of Poly-Epsilon-Lysine Functionalized Hydrogels.

Purpose: To determine the amoebicidal activity of functionalized poly-epsilon-lysine hydrogels (pɛK+) against Acanthamoeba castellanii. Methods: A. castellanii trophozoites and cysts were grown in the presence of pɛK solution (0-2.17 mM), pɛK or pɛK+ hydrogels, or commercial hydrogel contact lens (CL) for 24 hours or 7 days in PBS or Peptone-Yeast-Glucose (PYG) media (nutrient-deplete or nutrient-replete cultures, respectively). Toxicity was determined using propidium iodide and imaged using fluorescence microscopy. Ex vivo porcine corneas were inoculated with A. castellanii trophozoites ± pɛK, pɛK+ hydrogels or commercial hydrogel CL for 7 days. Corneal infection was assessed by periodic acid-Schiff staining and histologic analysis. Regrowth of A. castellanii from hydrogel lenses and corneal discs at 7 days was assessed using microscopy and enumeration. Results: The toxicity of pɛK+ hydrogels resulted in the death of 98.52% or 83.31% of the trophozoites at 24 hours or 7 days, respectively. The toxicity of pɛK+ hydrogels resulted in the death of 70.59% or 82.32% of the cysts in PBS at 24 hours or 7 days, respectively. Cysts exposed to pɛK+ hydrogels in PYG medium resulted in 75.37% and 87.14% death at 24 hours and 7 days. Ex vivo corneas infected with trophozoites and incubated with pɛK+ hydrogels showed the absence of A. castellanii in the stroma, with no regrowth from corneas or pɛK+ hydrogel, compared with infected-only corneas and those incubated in presence of commercial hydrogel CL. Conclusions: pɛK+ hydrogels demonstrated pronounced amoebicidal and cysticidal activity against A. castellanii. pɛK+ hydrogels have the potential for use as CLs that could minimize the risk of CL-associated Acanthamoeba keratitis.

Antimicrobial Activity of Poly-epsilon-lysine Peptide Hydrogels Against Pseudomonas aeruginosa.

Purpose: To determine the antimicrobial activity of poly-epsilon-lysine (pɛK) functionalization of hydrogels against Pseudomonas aeruginosa. Methods: Antimicrobial activities of pɛK and pɛK+ hydrogels were tested against both keratitis and a laboratory strain of Paeruginosa at a range of inocula sizes, over 4 and 24 hours. The number of viable CFU on pɛK and pɛK+ hydrogels or commercial contact lenses (CL) was investigated. Ex vivo porcine corneas were inoculated with Paeruginosa PAO1 (103 CFU) and incubated with pɛK+ hydrogels or commercial hydrogel CL for 24 hours and the effects of infection determined. Results: PɛK+ hydrogels showed log reductions in viable CFU compared with pɛK hydrogels for all Paeruginosa strains, depending on inocula sizes and incubation time. After 24 hours pɛK+ hydrogels showed >5 and >7.5 log reduction in CFU compared with commercial hydrogel CL at 103 and 106 CFU, respectively. In an ex vivo porcine corneal infection model, pɛK+ hydrogels led to a significant decrease in viable PAO1 CFU and histologic analysis indicated a decreased infiltration of PAO1 into the stroma. Conclusions: PɛK+ hydrogels demonstrated enhanced antimicrobial activity versus nonfunctionalized pɛK hydrogels against clinically relevant Paeruginosa strains. PɛK+ hydrogels have the potential to be used as a bandage CL with innate antimicrobial characteristics to minimize the risk of microbial keratitis.

Information management for high content live cell imaging.

BACKGROUND: High content live cell imaging experiments are able to track the cellular localisation of labelled proteins in multiple live cells over a time course. Experiments using high content live cell imaging will generate multiple large datasets that are often stored in an ad-hoc manner. This hinders identification of previously gathered data that may be relevant to current analyses. Whilst solutions exist for managing image data, they are primarily concerned with storage and retrieval of the images themselves and not the data derived from the images. There is therefore a requirement for an information management solution that facilitates the indexing of experimental metadata and results of high content live cell imaging experiments. RESULTS: We have designed and implemented a data model and information management solution for the data gathered through high content live cell imaging experiments. Many of the experiments to be stored measure the translocation of fluorescently labelled proteins from cytoplasm to nucleus in individual cells. The functionality of this database has been enhanced by the addition of an algorithm that automatically annotates results of these experiments with the timings of translocations and periods of any oscillatory translocations as they are uploaded to the repository. Testing has shown the algorithm to perform well with a variety of previously unseen data. CONCLUSION: Our repository is a fully functional example of how high throughput imaging data may be effectively indexed and managed to address the requirements of end users. By implementing the automated analysis of experimental results, we have provided a clear impetus for individuals to ensure that their data forms part of that which is stored in the repository. Although focused on imaging, the solution provided is sufficiently generic to be applied to other functional proteomics and genomics experiments. The software is available from: fhttp://code.google.com/p/livecellim/

Biomaterials for Regenerative Medicine Approaches for the Anterior Segment of the Eye.

The role of biomaterials in tissue engineering and regenerative medicine strategies to treat vision loss associated with damage to tissues in the anterior segment of the eye has been studied for several years. This has mostly involved replacement and support for the cornea and conjunctiva. These are complex tissues with specific functional requirements for different parts of the tissue. Amniotic membrane (AM) is used in clinical practice to transplant autologous or allogenic cells to the corneal surface. Fibrin gels have also progressed to clinical use under specific conditions. Alternatives to AM such as collagen gels, other natural materials, for example keratin and silks, and synthetic polymers have received considerable attention in laboratory and animal studies. This experience is building a body of evidence to demonstrate the potential of tissue engineering and regenerative medicine in corneal and conjunctival reconstruction and can also lead to other applications in the anterior segment of the eye, for example, the trabecular meshwork. There is a real clinical need for new procedures to overcome vision loss but there are also opportunities for developments in ocular applications to lead to biomaterials innovations for use in other clinical areas.

Automated tracking of gene expression in individual cells and cell compartments.

Many intracellular signal transduction processes involve the reversible translocation from the cytoplasm to the nucleus of transcription factors. The advent of fluorescently tagged protein derivatives has revolutionized cell biology, such that it is now possible to follow the location of such protein molecules in individual cells in real time. However, the quantitative analysis of the location of such proteins in microscopic images is very time consuming. We describe CellTracker, a software tool designed for the automated measurement of the cellular location and intensity of fluorescently tagged proteins. CellTracker runs in the MS Windows environment, is freely available (at http://www.dbkgroup.org/celltracker/), and combines automated cell tracking methods with powerful image-processing algorithms that are optimized for these applications. When tested in an application involving the nuclear transcription factor NF-kappaB, CellTracker is competitive in accuracy with the manual human analysis of such images but is more than 20 times faster, even on a small task where human fatigue is not an issue. This will lead to substantial benefits for time-lapse-based high-content screening.

Controlling drug release from non-aqueous environments: Moderating delivery from ocular silicone oil drug reservoirs to combat proliferative vitreoretinopathy.

In a number of cases of retinal detachment, treatment may require the removal of the vitreous humour within the eye and replacement with silicone oil to aid healing of the retina. The insertion of silicone oil offers the opportunity to also deliver drugs to the inside of the eye; however, drug solubility in silicone oil is poor and release from this hydrophobic drug reservoir is not readily controlled. Here, we have designed a range of statistical graft copolymers that incorporate dimethylsiloxane and ethylene glycol repeat units within the side chains, allowing short chains of oligo(ethylene glycol) to be solubilised within silicone oil and provide hydrogen bond acceptor sites to interact with acid functional drug molecules. Our hypothesis included the potential for such interactions to be able to delay/control drug release and for polymer architecture and composition to play a role in the silicone oil miscibility of the targeted polymers. This strategy has been successfully demonstrated using both ibuprofen and all-trans retinoic acid; drugs with anti-inflammatory and anti-proliferation activity. After the copolymers were shown to be non-toxic to retinal pigment epithelial cells, studies of drug release using radiochemical approaches showed that the presence of 10v/v% of a linear graft copolymer could extend ibuprofen release over three-fold (from 3days to >9days) whilst the release of all-trans retinoic from the silicone oil phase was extended to >72days. These timescales are highly clinically relevant showing the potential to tune drug delivery during the healing process and offer an efficient means to improve patient outcomes.

Pharmacokinetics of Meropenem for Use in Bacterial Keratitis.

PURPOSE: To investigate the toxicity and corneal pharmacokinetics of meropenem as a potential antimicrobial for bacterial keratitis. METHODS: Corneal epithelial cell and keratocyte toxicity was investigated using methyl thiazolyl tetrazolium (MTT) and LIVE/DEAD assays. The penetration of meropenem through the human cornea was measured using an artificial anterior chamber. In one group of corneas, the epithelial and endothelial layers were removed and in a second group these layers were left intact. We applied 50 µL (10 mg/mL) meropenem to the corneal surface and collected samples in the anterior chamber from 45 minutes up to 24 hours. Meropenem concentrations were estimated with a bioassay and HPLC. RESULTS: Meropenem had significantly higher cellular metabolic activity (MTT assay) at both 5 mg/mL and 2.5 mg/mL compared with moxifloxacin (P = 0.029 and P = 0.018, respectively), with 96% cell viability (LIVE/DEAD assay). The measured values for meropenem concentrations in corneal and aqueous samples were significantly higher using a bioassay than with HPLC (P = 0.004). For both intact and denuded corneas, the concentrations in the anterior chamber increased from 0.48 µg/mL (SD 0.89) and 0.89 µg/mL (SD 0.81) to 6.35 µg/mL (SD 0.81) and 13.48 µg/mL (SD 14.82) using HPLC, and from 0.68 µg/mL (SD 1.50) and 1.31 µg/mL (SD 1.55) to 47.03 µg/mL (SD 5.51) and 43.69 µg/mL (SD 27.22) measured with a bioassay. CONCLUSIONS: Meropenem has very low toxicity in vitro. It has good corneal penetration, achieving anterior chamber concentrations above MIC90 for bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, streptococci, coagulase-negative staphylococci, and the Enterobacteriaceae.

NF-kappaB signalling is inhibited by glucocorticoid receptor and STAT6 via distinct mechanisms.

NF-kappaB transcription factors are involved in the cellular response to stress, and are regulated by inhibitor (IkappaB) proteins, which prevent NF-kappaB-mediated transcription by maintaining NF-kappaB in the cytoplasm. Proteins from other pathways are also known to regulate NF-kappaB negatively, notably the glucocorticoid receptor (GR) and IL-4-responsive STAT6. Both pathways were shown to inhibit NF-kappaB-mediated transcription, by expressing either STAT6 or GR and activating the respective pathways. Using fluorescent fusion proteins, we show that GR alters the timing of activated p65 NF-kappaB nuclear occupancy by increasing the export rate of p65 and is independent of whether GR is present as a dimer or monomer. Expression of STAT6 was also shown to alter p65 nuclear occupancy but appeared to affect the import rate and hence the overall maximal level of p65 translocation. Activating STAT6 with IL-4 prior to activating NF-kappaB significantly increased this inhibition. Investigation of IkappaBa showed that activated STAT6 inhibited TNFalpha-mediated IkappaBa phosphorylation and degradation, whereas GR activation did not alter IkappaBalphakinetics. This demonstrates a clear separation of two distinct mechanisms of inhibition by STAT6 and GR upon the NF-kappaB pathway.