Ted W. Reid

Immunohistochemical evidence that human pterygia originate from an invasion of vimentin-expressing altered limbal epithelial basal cells

The goal of this study was to determine the cell origin of human pterygia. In order to determine the origin of these cells, longitudinal cryostat sections through five primary and two recurrent pterygia were studied immunohistochemically by finding limbal basal stem cell staining patterns as defined by monoclonal antibodies AE1 (staining positive) and AE5 (staining negative). In addition, sections were stained with antivimentin antibody. Altered limbal basal cells invading normal cornea along the basement membrane were identified in seven human pterygia with these specific monoclonal antibodies. A group of limbal basal cells (vimentin and AE1 positive) was always present between the dissolved edge of Bowmans layer and vascularized conjunctiva which contained goblet cells. Scattered patches of cells staining positive with both vimentin and AE5 (in addition to their AE1 staining) were also found in conjunctival epithelium growing on corneal basement membrane adjacent to the migrating limbal cells, indicating local infiltration by the altered limbal basal cells. This same pattern was also found in recurrent pterygia. Based on this data we propose that the pathogenesis of pterygia is due to a normal stationary parental limbal epithelial basal cell becoming altered and giving rise to a zone of motile daughter cells, the pterygium cells, which leave the limbal region and migrate as a group centripetally along the corneal basement membrane dissolving Bowmans layer. Since these altered limbal basal cells are found at the microscopic advancing edge over Bowmans layer with no fibroblast mass under them, the pterygium cell apparently precedes the rapid growth of the fibroblasts from the stroma.(ABSTRACT TRUNCATED AT 250 WORDS)

Synergistic effects of substance P with insulin-like growth factor-1 on epithelial migration of the cornea

We find that substance P (SP) and insulin‐like growth factor‐1 (IGF‐1) demonstrate a synergistic effect on the stimulation of rabbit corneal epithelial migration in an organ culture. The addition of either SP or IGF‐1 alone did not affect epithelial migration, while the combination of SP and IGF‐1 stimulated epithelial migration in a dose‐dependent fashion. The synergistic effects of SP and IGF‐1 on corneal epithelial migration were nulled by the addition of a SP antagonist or enkephalinase. Among neurotransmitters (vasoactive intestinal peptide, calcitonin gene‐related peptide, acethylcholine chloride, norepinephrine, serotonin) or tachykinins (neurokinin A, neurokinin B, kassinin, eledoisin, physalaemin), only SP demonstrated a synergistic effect with IGF‐1 on cellular migration. In contrast, the combination of SP and IGF‐1 did not affect the incorporation of 3H‐thymidine into corneal epithelial cells. The attachment of the corneal epithelial cells to fibronectin, collagen type IV, and laminin matrices increased after treatment of the cells with SP and IGF‐1, but SP or IGF‐1 by themselves did not affect the attachment of the cells to these extracellular matrix proteins. An identical synergistic effect on corneal epithelial migration was observed when an NK‐1 receptor agonist was used in place of SP, suggesting the synergistic effect of SP and IGF‐1 might be mediated through the NK‐1 receptor system. These results suggest that the maintenance of the normal integrity of the corneal epithelium might be regulated by both humoral and neural factors.

17 E. coli Alkaline Phosphatase

Publisher Summary This chapter discusses the molecular properties and catalytic properties of E. coli alkaline phosphatase. Purified preparations of alkaline phosphatase from E. coli, judged homogeneous when examined in the analytical ultracentrifuge, contain several isozymes, because several bands which contain enzymic activity are obtained in starch-gel and disc-gel electrophoresis. Although most workers find three brands, four and five equally spaced bands have been found. Single gene mutations apparently affect each isozyme, for the mobility of all the bands are affected without altering the spacing. Levinthal concluded that all of the isozymes were coded for by the same gene. Studies have obtained large single crystals of E. coli alkaline phosphatase. Initial X-ray studies by Hanson show the crystals to be of the space group P 3121, each unit cell containing three dimers. The unit cell dimensions are a = 70.5 A, b = 70.5 A, c = 155.6 A, β = 120°, which is consistent with the globular form predicted by the hydrodynamic frictional ratio.

Dimethyldiselenide and Methylseleninic Acid Generate Superoxide in an In Vitro Chemiluminescence Assay in the Presence of Glutathione: Implications for the Anticarcinogenic Activity of L-Selenomethionine and L-Se-Methylselenocysteine

The reduction of cancer incidence by dietary supplementation with L-selenomethionine, L-Se-methylselenocysteine, and other methylated selenium compounds and metabolites is believed to be due to the metabolic generation of the monomethylated selenium species methylselenol. Dimethyldiselenide and methylseleninic acid were reduced by glutathione in an in vitro chemiluminescent assay in the presence of lucigenin for the detection of superoxide (O2 - ). The methylselenol produced on reduction of dimethyldiselenide and methylseleninic acid was found to be highly catalytic, continuously generating a steady state of O2 - . The O2 - detected by the chemiluminescence generated by methylselenol was fully quenched by superoxide dismutase, causing a complete cessation of chemiluminescence. In contrast, dimethyldisulfide in the presence of glutathione was not catalytic to any measurable extent and did not generate any superoxide. These in vitro results suggest that methylselenol catalysis is possible in vivo, and if metabolism generates sufficient concentrations of methlylselenol from L-selenomethionine or L-Se-methylselenocysteine in vivo, it could change the redox status of cells and oxidatively induce cellular apoptosis.

The science of pterygia

Pterygium is an ocular surface disease of humans attributed to chronic ultraviolet-B exposure. Clinically, the condition involves invasive centripetal growth with associated inflammation and neovascularisation. Previous clinical studies focused primarily on the clinical characteristics and surgical management of pterygia and, because of this, the pathogenesis of pterygia remains incompletely understood. However, considerable progress in this area has been achieved, providing additional insight into this complex disease. This recent evidence implicates antiapoptotic mechanisms, immunological mechanisms, cytokines, growth factors, extracellular matrix modulators, genetic factors, viral infections and other possible causative factors. Limited investigation regarding differences in pathogenesis of primary and recurrent pterygia has been performed. We summarise many of these recent discoveries concerning the pathogenesis of pterygia and describe reported differences between primary and recurrent pterygia.

Organoselenium Coating on Cellulose Inhibits the Formation of Biofilms by Pseudomonas aeruginosa and Staphylococcus aureus

ABSTRACT Among the most difficult bacterial infections encountered in treating patients are wound infections, which may occur in burn victims, patients with traumatic wounds, necrotic lesions in people with diabetes, and patients with surgical wounds. Within a wound, infecting bacteria frequently develop biofilms. Many current wound dressings are impregnated with antimicrobial agents, such as silver or antibiotics. Diffusion of the agent(s) from the dressing may damage or destroy nearby healthy tissue as well as compromise the effectiveness of the dressing. In contrast, the antimicrobial agent selenium can be covalently attached to the surfaces of a dressing, prolonging its effectiveness. We examined the effectiveness of an organoselenium coating on cellulose discs in inhibiting Pseudomonas aeruginosa and Staphylococcus aureus biofilm formation. Colony biofilm assays revealed that cellulose discs coated with organoselenium completely inhibited P. aeruginosa and S. aureus biofilm formation. Scanning electron microscopy of the cellulose discs confirmed these results. Additionally, the coating on the cellulose discs was stable and effective after a week of incubation in phosphate-buffered saline. These results demonstrate that 0.2% selenium in a coating on cellulose discs effectively inhibits bacterial attachment and biofilm formation and that, unlike other antimicrobial agents, longer periods of exposure to an aqueous environment do not compromise the effectiveness of the coating.

Prevention of bacterial colonization of contact lenses with covalently attached selenium and effects on the rabbit cornea.

Purpose: Although silicone hydrogel materials have produced many corneal health benefits to patients wearing contact lenses, bacteria that cause acute red eye or corneal ulcers are still a concern. A coating that inhibits bacterial colonization while not adversely affecting the cornea should improve the safety of contact lens wear. A covalent selenium (Se) coating on contact lenses was evaluated for safety using rabbits and prevention of bacterial colonization of the contact lenses in vitro. Methods: Contact lenses coated with Se were worn on an extended-wear schedule for up to 2 months by 10 New Zealand White rabbits. Corneal health was evaluated with slit-lamp biomicroscopy, pachymetry, electron microscopy, and histology. Lenses worn by the rabbits were analyzed for protein and lipid deposits. In addition, the ability of Se to block bacterial colonization was tested in vitro by incubating lenses in a Pseudomonas aeruginosa broth followed by scanning electron microscopy of the contact lens surface. Results: The covalent Se coating decreased bacterial colonization in vitro while not adversely affecting the corneal health of rabbits in vivo. The Se coating produced no noticeable negative effects as observed with slit-lamp biomicroscopy, pachymetry, electron microscopy, and histology. The Se coating did not affect protein or lipid deposition on the contact lenses. Conclusion: The data from this pilot study suggest that a Se coating on contact lenses might reduce acute red eye and bacterial ulceration because of an inhibition of bacterial colonization. In addition, our safety tests suggest that this positive effect can be produced without an adverse effect on corneal health.

An Organoselenium Compound Inhibits Staphylococcus aureus Biofilms on Hemodialysis Catheters In Vivo

ABSTRACT Colonization of central venous catheters (CVCs) by pathogenic bacteria leads to catheter-related bloodstream infections (CRBSIs). These colonizing bacteria form highly antibiotic-resistant biofilms. Staphylococcus aureus is one of the most frequently isolated pathogens in CRBSIs. Impregnating CVC surfaces with antimicrobial agents has various degrees of effectiveness in reducing the incidence of CRBSIs. We recently showed that organoselenium covalently attached to disks as an antibiofilm agent inhibited the development of S. aureus biofilms. In this study, we investigated the ability of an organoselenium coating on hemodialysis catheters (HDCs) to inhibit S. aureus biofilms in vitro and in vivo. S. aureus failed to develop biofilms on HDCs coated with selenocyanatodiacetic acid (SCAA) in either static or flowthrough continuous-culture systems. The SCAA coating also inhibited the development of S. aureus biofilms on HDCs in vivo for 3 days. The SCAA coating was stable and nontoxic to cell culture or animals. This new method for coating the internal and external surfaces of HDCs with SCAA has the potential to prevent catheter-related infections due to S. aureus.

Successful six-day kidney preservation using trophic factor supplemented media and simple cold storage

This study examined the effect of trophic factor supplementation [TFS; bovine neutrophil peptide‐1 (bactenecin), 1 mg/L; substance P, 2.5 mg/L; nerve growth factor, 20 μg/L; epidermal growth factor, 10 μg/L; insulin‐like growth factor‐1, 10 μg/L] during cold storage with UW lactobionate solution. Dogs transplanted with kidneys stored for 4 days in TFS‐UW had significantly lower peak serum creatinine values (mean 2.9 ± 0.2 mg/dL) and returned to normal values faster (6 days) than kidneys stored for 3 days in unmodified UW solution (4.2 ± 0.3 mg/dL and 14 days, respectively). Kidneys stored for 5 days in TFS‐UW (mean peak creatinine 3.7 ± 0.3) functioned equivalently to kidneys stored for 3 days and better than kidneys stored for 4 days in UW alone. Dogs with kidneys stored for 6 days in TFS‐UW had mean peak creatinines of 5.7 ± 0.4 mg/dL. These returned to normal creatinine values in 14 days, equal to 3‐day stored and significantly better than kidneys stored for 4 days in UW alone (20 days recovery time). This study shows trophic factor deprivation appears to be a critical mechanism of injury in organ preservation with current synthetic storage media, and marks the initial development of a synthetic biologically active preservation solution, the next generation of preservation media.

Doxycycline's effect on ocular angiogenesis: an in vivo analysis.

PURPOSE To determine the in vivo effect of doxycycline on choroidal angiogenesis and pterygium growth by using a choroidal neovascular (CNV) murine model, a directed in vivo angiogenesis assay (DIVAA) and a pterygium murine model. DESIGN Experimental study. PARTICIPANTS Three murine models were investigated with 4 mice minimum per group and 22 maximum per group. METHODS Mice received water with or without doxycycline. For the CNV, the neovascular lesion volume was determined in choroid-retinal pigment epithelial flat mounts using confocal microscopy 7 days after laser induction. For DIVAA, silicone capsules containing 10,000 human pterygium epithelial cells were implanted in the flanks of mice subcutaneously. After 11 days, neovascularization (NV) was quantified using spectrofluorometry after murine tail-vein injection of fluorescein isothiocyanate-labeled dextran. A pterygium epithelial cell model was developed by injecting 10,000 human pterygium epithelial cells in the nasal subconjunctival space in athymic nude mice. Doxycycline was started on day 6 at 50 mg/kg per day; corneal lesions that resulted from the injections were compared at days 6 and 15. MAIN OUTCOME MEASURES The Student t-test was used to evaluate the data for the CNV and DIVAA models and histologic preparations were used to evaluate pterygia lesions. RESULTS There was significantly less NV and lesion volume with doxycycline taken in drinking water versus plain water. With doxycycline treatment, the laser-induced CNV showed a maximal 66% decrease in choroidal blood vessel volume (P< or =0.008) and the DIVAA showed a 30% reduction of blood vessel growth and migration (P<0.004). Histologic preparations demonstrated that pterygium cell lesions regressed when mice were administered doxycycline for 9 days. CONCLUSIONS Doxycycline significantly inhibited angiogenesis in 3 murine models. The most dramatic effect was found in the CNV model followed by the pterygia epithelial cell DIVAA model. The anterior segment pterygium model also showed regression histologically. This suggests that doxycycline may be successful as an adjunctive treatment for CNV and pterygia in humans; clinical trials would be necessary to determine if there is a benefit.