Evaluation of methods for cultivating limbal mesenchymal stromal cells

Background: Mesenchymal stromal cells (MSC) with similar properties to bone marrow derived mesenchymal stromal cells (BM-MSC) have recently been grown from the limbus of the human cornea. We presently contribute to this novel area of research by evaluating methods for culturing human limbal MSC (L-MSC). Methods: Four basic strategies are compared: serum-supplemented medium (10% foetal bovine serum; FBS), standard serum-free medium supplemented with B-27, epidermal growth factor, and fibroblast growth factor 2, or one of two commercial serum-free media including Defined Keratinocyte Serum Free Medium (Invitrogen), and MesenCult-XF (Stem Cell Technologies). The phenotype of resulting cultures was examined using photography, flow cytometry (for CD34, CD45, CD73, CD90, CD105, CD141, CD271), immunocytochemistry (α-sma), differentiation assays (osteogenesis, adipogenesis, chrondrogenesis), and co-culture experiments with human limbal epithelial (HLE) cells. Results: While all techniques supported to varying degrees establishment of cultures, sustained growth and serial propagation was only achieved in 10% FBS medium or MesenCult-XF medium. Cultures established in 10% FBS medium were 70-80% CD34-/CD45-/CD90+/CD73+/CD105+, approximately 25% α-sma+, and displayed multi-potency. Cultures established in MesenCult-XF were >95% CD34-/CD45-/CD90+/CD73+/CD105+, 40% CD141+, rarely expressed α-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of MesenCult-XF-grown L-MSC. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker ∆Np63, along with the corneal differentiation marker cytokeratin 3. Conclusions: We conclude that MesenCult-XF® is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells.

1,1,1-Trichloro-2,2-bis(4-ethoxyphenyl)ethane

In the title compound, C18H19Cl3O2, which is the 4-ethoxyphenyl analogue of the insecticidally active 4-methoxyphenyl compound methoxychlor, the dihedral angle between the two benzene rings is 60.38(13)deg. An intramolecular aromatic C-H...Cl interaction is present.

Isopropyl 2,2-bis(4-bromophenyl)-2-hydroxyacetate

In the structure of the title compound C17H16Br2O3, which is a restricted commercial acaricide (common name bromopropylate), has two independent and conformationally similar molecules in the asymmetric unit [dihedral angle between the planes of the two phenyl rings in each, 68.7(4) and 77.4(5)deg]. The C-atoms of the isopropyl group of one of the molecules are disordered over two sites with occupancies of 0.638 and 0.362. Minor non-merohedral twinning was also present in the crystal. Intermolecular hydrogen-bonding interactions involving the hydroxy groups and carboxyl O-atom acceptors give separate centrosymmetric homodimers through cyclic hydrogen-bonding motifs [graph set R2/2(10)].

Relationship among CFH and ARMS2 genotypes, macular pigment optical density, and neuroretinal function in persons without age-related macular degeneration

Purpose: To determine whether there is a difference in neuroretinal function and in macular pigment optical density between persons with high- and low-risk gene variants for age-related macular degeneration (AMD) and no ophthalmoscopic signs of AMD, and to compare the results on neuroretinal function to patients with manifest early AMD. Methods and Participants: Neuroretinal function was assessed with the multifocal electroretinogram (mfERG) for 32 participants (22 healthy persons with no AMD and 10 early AMD patients). The 22 healthy participants with no AMD had high- or low-risk genotypes for either CFH (rs380390) and/or ARMS2 (rs10490924). Trough-to-peak response densities and peak-implicit times were analyzed in 5 concentric rings. Macular pigment optical densitometry was assessed by customized heterochromatic flicker photometry. Results: Trough-to-peak response densities for concentric rings 1 to 3 were, on average, significantly greater in participants with high-risk genotypes than in participants with low-risk genotypes and in persons with early AMD after correction for age and smoking (p<0.05). The group peak- implicit times for ring 1 were, on average, delayed in the patients with early AMD compared with the participants with high- or low-risk genotypes, although these differences were not significant. There was no significant correlation between genotypes and macular pigment optical density. Conclusion: Increased neuroretinal activity in persons who carry high-risk AMD genotypes may be due to genetically determined subclinical inflammatory and/or histological changes in the retina. Neuroretinal function in healthy persons genetically susceptible to AMD may be a useful additional early biomarker (in combination with genetics) before there is clinical manifestation.

Using optical coherence tomography to assess corneoscleral morphology after soft contact lens wear

Purpose. To evaluate the use of optical coherence tomography (OCT) to assess the effect of different soft contact lenses on corneoscleral morphology. Methods. Ten subjects had anterior segment OCT B-scans taken in the morning and again after six hours of soft contact lens wear. For each subject, three different contact lenses were used in the right eye on non-consecutive days, including a hydrogel sphere, a silicone hydrogel sphere and a silicone hydrogel toric. After image registration and layer segmentation, analyses were performed of the first hyper-reflective layer (HRL), the epithelial basement membrane (EBL) and the epithelial thickness (HRL to EBL). A root mean square difference (RMSD) of the layer profiles and the thickness change between the morning and afternoon measurements, was used to assess the effect of the contact lens on the corneoscleral morphology. Results. The soft contact lenses had a statistically significant effect on the morphology of the anterior segment layers (p <0.001). The average amounts of change for the three lenses (average RMSD values) for the corneal region were lower (3.93±1.95 µm for the HRL and 4.02±2.14 µm for the EBL) than those measured in the limbal/scleral region (11.24±6.21 µm for the HRL and 12.61±6.42 µm for the EBL). Similarly, averaged across the three lenses, the RMSD in epithelial thickness was lower in the cornea (2.84±0.84 µm) than the limbal/scleral (5.47±1.71 µm) region. Post-hoc analysis showed that ocular surface changes were significantly smaller with the silicone hydrogel sphere lens than both the silicone hydrogel toric (p<0.005) and hydrogel sphere (p<0.02) for the combined HRL and EBL data. Conclusions. In this preliminary study, we have shown that soft contact lenses can produce small but significant changes in the morphology of the limbal/scleral region and that OCT technology is useful in assessing these changes. The clinical significance of these changes is yet to be determined.

Identification of GABA receptors in chick cornea

Purpose: The cornea has an important role in vision, is highly innervated and many neurotransmitter receptors are present, e.g., muscarine, melatonin, and dopamine receptors. γ-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the retina and central nervous system, but it is unknown whether GABA receptors are present in cornea. The aim of this study was to determine if GABA receptors are located in chick cornea. Methods: Corneal tissues were collected from 25, 12-day-old chicks. Real time PCR, western blot, and immunohistochemistry were used to determine whether alpha1 GABAA, GABAB, and rho1 GABAC receptors were expressed and located in chick cornea. Results: Corneal tissue was positive for alpha1 GABAA and rho1 GABAC receptor mRNA (PCR) and protein (western blot) expression but was negative for GABAB receptor mRNA and protein. Alpha1 GABAA and rho1 GABAC receptor protein labeling was observed in the corneal epithelium using immunohistochemistry. Conclusions: These investigations clearly show that chick cornea possesses alpha1 GABAA, and rho1 GABAC receptors, but not GABAB receptors. The purpose of the alpha1 GABAA and rho1 GABAC receptors in cornea is a fascinating unexplored question.

4-(4-Nitrobenzyl)pyridinium 3-carboxy-4-hydroxybenzenesulfonate

In the title salt, C12H11N2O2+·C7H5O6S-, the dihedral angle between the benzene and pyridine rings in the 4-(4-nitrobenzyl)pyridinium cation is 82.7 (2)°. Within the anion there is an intramolecular hydroxy-O-HO(carboxylic acid) bond. In the crystal, the cation forms a single N+-HOsulfonate hydrogen bond with the anion. These cation-anion pairs interact through duplex anion carboxylic acid O-HOsulfonate hydrogen bonds, giving a centrosymmetric cyclic association [graph set R22(16)]. The crystals studied were non-merohedrally twinned.

Effect of age on components of peripheral ocular aberrations

Purpose: To investigate the effect of age on the contributions of the anterior cornea and internal components to ocular aberrations in the peripheral visual field. Methods: Ocular aberrations were measured in 10 young emmetropes and 7 older emmetropes using a modified commercial Hartmann-Shack aberrometer across 42° x 32° of central visual field. Anterior corneal aberrations were estimated from anterior corneal topography using theoretical ray-tracing. Internal aberrations were calculated by subtracting anterior corneal aberrations from ocular aberrations. Results: Anterior corneal aberrations of young subjects were reasonably compensated by the internal aberrations, except for astigmatism for which the internal contribution was small out to the 21° field limit. The internal coma and spherical aberration of the older subjects were considerably smaller in magnitude than those of the young subjects such that the compensation for anterior corneal aberrations was poorer. This can be explained by age-related changes in the lens shape and refractive index distribution. Conclusion: oss of balance between anterior cornea and internal components of higher order aberrations with increasing age, found previously for on-axis vision, applies also to the peripheral visual field.

Evaluation of Eph receptor and ephrin expression within the human cornea and limbus

Eph receptor tyrosine kinases and their ligands, the ephrins, regulate the development and maintenance of multiple organs but little is known about their potential role within the cornea. The purpose of this study was to perform a thorough investigation of Eph/ephrin expression within the human cornea including the limbal stem cell niche. Initially, immunohistochemistry was performed on human donor eyes to determine the spatial distribution of Eph receptors and ephrins in the cornea and limbus. Patterns of Eph/ephrin gene expression in (1) immortalised human corneal endothelial (B4G12) or corneal epithelial (HCE-T) cell lines, and (2) primary cultures of epithelial or stromal cells established from the corneal limbus of cadaveric eye tissue were then assessed by reverse transcription (RT) PCR. Limbal epithelial or stromal cells from primary cultures were also assessed for evidence of Eph/ephrin-reactivity by immunofluorescence. Immunoreactivity for ephrinA1 and EphB4 was detected in the corneal endothelium of donor eyes. EphB4 was also consistently detected in the limbal and corneal epithelium and in cells located in the stroma of the peripheral cornea. Expression of multiple Eph/ephrin genes was detected in immortalised corneal epithelial and endothelial cell lines. Evidence of Eph/ephrin gene expression was also demonstrated in primary cultures of human limbal stromal (EphB4, B6; ephrinA5) and epithelial cells (EphA1, A2; ephrinA5, B2) using both RT-PCR and immunofluorescence. The expression of Eph receptors and ephrins within the human cornea and limbus is much wider than previously appreciated and suggests multiple potential roles for these molecules in the maintenance of normal corneal architecture.

Risk factors for moderate and severe microbial keratitis in daily wear contact lens users

Objective: To establish risk factors for moderate and severe microbial keratitis among daily contact lens (CL) wearers in Australia. Design: A prospective, 12-month, population-based, case-control study. Participants: New cases of moderate and severe microbial keratitis in daily wear CL users presenting in Australia over a 12-month period were identified through surveillance of all ophthalmic practitioners. Case detection was augmented by record audits at major ophthalmic centers. Controls were users of daily wear CLs in the community identified using a national telephone survey. Testing: Cases and controls were interviewed by telephone to determine subject demographics and CL wear history. Multiple binary logistic regression was used to determine independent risk factors and univariate population attributable risk percentage (PAR%) was estimated for each risk factor.; Main Outcome Measures: Independent risk factors, relative risk (with 95% confidence intervals [CIs]), and PAR%. Results: There were 90 eligible moderate and severe cases related to daily wear of CLs reported during the study period. We identified 1090 community controls using daily wear CLs. Independent risk factors for moderate and severe keratitis while adjusting for age, gender, and lens material type included poor storage case hygiene 6.4× (95% CI, 1.9-21.8; PAR, 49%), infrequent storage case replacement 5.4× (95% CI, 1.5-18.9; PAR, 27%), solution type 7.2× (95% CI, 2.3-22.5; PAR, 35%), occasional overnight lens use (<1 night per week) 6.5× (95% CI, 1.3-31.7; PAR, 23%), high socioeconomic status 4.1× (95% CI, 1.2-14.4; PAR, 31%), and smoking 3.7× (95% CI, 1.1-12.8; PAR, 31%). Conclusions: Moderate and severe microbial keratitis associated with daily use of CLs was independently associated with factors likely to cause contamination of CL storage cases (frequency of storage case replacement, hygiene, and solution type). Other factors included occasional overnight use of CLs, smoking, and socioeconomic class. Disease load may be considerably reduced by attention to modifiable risk factors related to CL storage case practice.

The relationship between CFH and ARMS2 genotypes and neuroretinal function in persons without age-related macular degeneration

Purpose: To determine whether neuroretinal function differs in healthy persons with and without common risk gene variants for age- related macular degeneration (AMD) and no ophthalmoscopic signs of AMD, and to compare those findings in persons with manifest early AMD. Methods and Participants: Neuroretinal function was assessed with the multifocal electroretinogram (mfERG) (VERIS, Redwood City, CA,) in 32 participants (22 healthy persons with no clinical signs of AMD and 10 early AMD patients). The 22 healthy participants with no AMD were risk genotypes for either the CFH (rs380390) and/or ARMS2 (rs10490920). We used a slow flash mfERG paradigm (3 inserted frames) and a 103 hexagon stimulus array. Recordings were made with DTL electrodes; fixation and eye movements were monitored online. Trough N1 to peak P1 (N1P1) response densities and P1-implicit times (IT) were analysed in 5 concentric rings. Results: N1P1 response densities (mean ± SD) for concentric rings 1-3 were on average significantly higher in at-risk genotypes (ring 1: 17.97 nV/deg2 ± 1.9, ring 2: 11.7 nV/deg2 ±1.3, ring 3: 8.7 nV/deg2 ± 0.7) compared to those without risk (ring 1: 13.7 nV/deg2 ± 1.9, ring 2: 9.2 nV/deg2 ±0.8, ring 3: 7.3 nV/deg2 ± 1.1) and compared to persons with early AMD (ring 1: 15.3 nV/deg2 ± 4.8, ring 2: 9.1 nV/deg2 ±2.3, ring 3 nV/deg2: 7.3± 1.3) (p<0.5). The group implicit times, P1-ITs for ring 1 were on average delayed in the early AMD patients (36.4 ms ± 1.0) compared to healthy participants with (35.1 ms ± 1.1) or without risk genotypes (34.8 ms ±1.3), although these differences were not significant. Conclusion: Neuroretinal function in persons with normal fundi can be differentiated into subgroups based on their genetics. Increased neuroretinal activity in persons who carry AMD risk genotypes may be due to genetically determined subclinical inflammatory and/or histological changes in the retina. Assessment of neuroretinal function in healthy persons genetically susceptible to AMD may be a useful early biomarker before there is clinical manifestation of AMD.

Effect of the sterilization method on the properties of Bombyx mori silk fibroin films

We have compared the effects of different sterilization techniques on the properties of Bombyx mori silk fibroin thin films with the view to subsequent use for corneal tissue engineering. The transparency, tensile properties, corneal epithelial cell attachment and degradation of the films were used to evaluate the suitability of certain sterilization techniques including gamma-irradiation (in air or nitrogen), steam treatment and immersion in aqueous ethanol. The investigations showed that gamma-irradiation, performed either in air or in a nitrogen atmosphere, did not significantly alter the properties of films. The films sterilized by gamma-irradiation or by immersion in ethanol had a transparency greater than 98% and tensile properties comparable to human cornea and amniotic membrane, the materials of choice in the reconstruction of ocular surface. Although steam-sterilization produced stronger, stiffer films, they were less transparent, and cell attachment was affected by the variable topography of these films. It was concluded that gamma-irradiation should be considered to be the most suitable method for the sterilization of silk fibroin films, however, the treatment with ethanol is also an acceptable method.

2-(4-Aminobenzenesulfonamido)-4,6-dimetylpyrimidin-1-ium 2-carboxy-4,6-dinitrophenolate

In the structure of the of the phenolate salt of the sulfa drug sulfamethazine with 3,5-dinitrosalicylic acid, C12H15N4O2S+ C7H3N2O7-, the dihedral angle between the pyrimidine and phenyl rings of the cation is 59.70(17)\%. Cation--anion hydrogen-bonding interactions involving pyrimidine N+-H...O(carboxyl) and amine N-H...O(carboxyl) pairs give a cyclic R2/2(8) motif while secondary N-H...O hydrogen bonds between the aniline group and both sulfone and nitro O-atom acceptors give a two-dimensional structure extending along (001).

Conserved and variable features of gag structure and arrangement in immature retrovirus particles

The assembly of retroviruses is driven by oligomerization of the Gag polyprotein. We have used cryo-electron tomography together with subtomogram averaging to describe the three-dimensional structure of in vitro-assembled Gag particles from human immunodeficiency virus, Mason-Pfizer monkey virus, and Rous sarcoma virus. These represent three different retroviral genera: the lentiviruses, betaretroviruses and alpharetroviruses. Comparison of the three structures reveals the features of the supramolecular organization of Gag that are conserved between genera and therefore reflect general principles of Gag-Gag interactions and the features that are specific to certain genera. All three Gag proteins assemble to form approximately spherical hexameric lattices with irregular defects. In all three genera, the N-terminal domain of CA is arranged in hexameric rings around large holes. Where the rings meet, 2-fold densities, assigned to the C-terminal domain of CA, extend between adjacent rings, and link together at the 6-fold symmetry axis with a density, which extends toward the center of the particle into the nucleic acid layer. Although this general arrangement is conserved, differences can be seen throughout the CA and spacer peptide regions. These differences can be related to sequence differences among the genera. We conclude that the arrangement of the structural domains of CA is well conserved across genera, whereas the relationship between CA, the spacer peptide region, and the nucleic acid is more specific to each genus.

Morphometry and abnormalities of the feet of Kaimanawa feral horses in New Zealand

Objective: The present study investigated the foot health of the Kaimanawa feral horse population and tested the hypotheses that horses would have a large range of foot morphology and that the incidence of foot abnormality would be significantly high. Procedures: Abnormality was defined as a variation from what the two veterinarian assessors considered as optimal morphology and which was considered to impact negatively on the structure and/or function of the foot. Fifteen morphometric variables were measured on four calibrated photographic views of all four feet of 20 adult Kaimanawa feral horses. Four morphometric variables were measured from the lateromedial radiographs of the left forefoot of each horse. In addition, the study identified the incidence of gross abnormality observed on the photographs and radiographs of all 80 feet. Results: There was a large variation between horses in the morphometric dimensions, indicating an inconsistent foot type. Mean hoof variables were outside the normal range recommended by veterinarians and hoof care providers; 35% of all feet had a long toe conformation and 15% had a mediolateral imbalance. Abnormalities included lateral (85% of horses) and dorsal (90% of horses) wall flares, presence of laminar rings (80% of horses) and bull-nose tip of the distal phalanx (75% of horses). Both hypotheses were therefore accepted. Conclusions: The Kaimanawa feral horse population demonstrated a broad range of foot abnormalities and we propose that one reason for the questionable foot health and conformation is lack of abrasive wearing by the environment. In comparison with other feral horse populations in Australia and America there may be less pressure on the natural selection of the foot of the Kaimanawa horses by the forgiving environment of the Kaimanawa Ranges. Contrary to popular belief, the feral horse foot type should not be used as an ideal model for the domestic horse foot.