The MyD88+ phenotype is an adverse prognostic factor in epithelial ovarian cancer

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.

Corneal changes following short-term miniscleral contact lens wear

Purpose To examine the influence of short-term miniscleral contact lens wear on corneal shape, thickness and anterior surface aberrations. Methods Scheimpflug imaging was captured before, immediately following and 3 hours after a short period (3 hours) of miniscleral contact lens wear for 10 young (mean 27 ± 5 years), healthy participants. Natural diurnal variations were considered by measuring baseline diurnal changes obtained on a separate control day without contact lens wear. Results Small but significant anterior corneal flattening was observed immediately following lens removal (overall mean 0.02 ± 0.03 mm, p < 0.001) which returned to baseline levels three hours after lens removal. During the three hour recovery period significant corneal thinning (-13.4 ± 10.5 μm) and posterior surface flattening (0.03 ± 0.02 mm) were also observed (both p < 0.01). The magnitude of posterior corneal flattening during recovery correlated with the amount of corneal thinning (r = 0.69, p = 0.03). Central corneal clearance (maximum tear reservoir depth) was not associated with corneal swelling following lens removal (r = -0.24, p > 0.05). An increase in lower-order corneal astigmatism Z(2,2) was also observed following lens wear (mean -0.144 ± 0.075 μm, p = 0.02). Conclusions Flattening of the anterior corneal surface was observed immediately following lens wear, while ‘rebound’ thinning and flattening of the posterior surface was evident following the recovery period. Modern miniscleral contact lenses that vault the cornea may slightly influence corneal shape and power but do not induce clinically significant corneal oedema during short-term wear.

GABAB receptors expressed in human aortic endothelial cells mediate intracellular calcium concentration regulation and endothelial nitric oxide synthase translocation

GABAB receptors regulate the intracellular Ca2+ concentration ([Ca2+]i) in a number of cells (e.g., retina, airway epithelium and smooth muscle), but whether they are expressed in vascular endothelial cells and similarly regulate the [Ca2+]i is not known. The purpose of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in cultured human aortic endothelial cells (HAECs), and to explore if altering receptor activation modified [Ca2+]i and endothelial nitric oxide synthase (eNOS) translocation. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABAB1 and GABAB2 in cultured HAECs. The effects of GABAB receptors on [Ca2+]i in cultured HAECs were demonstrated using fluo-3. The influence of GABAB receptors on eNOS translocation was assessed by immunocytochemistry. Both GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs, and the GABAB1 and GABAB2 proteins were colocated in the cell membrane and cytoplasm. One hundred μM baclofen caused a transient increase of [Ca2+]i and eNOS translocation in cultured HAECs, and the effects were attenuated by pretreatment with the selective GABAB receptor antagonists CGP46381 and CGP55845. GABAB receptors are expressed in HAECs and regulate the [Ca2+]i and eNOS translocation. Cultures of HAECs may be a useful in vitro model for the study of GABAB receptors and vascular biology.

GABAB1 and GABAB2 receptor subunits co-expressed in cultured human RPE cells regulate intracellular Ca2+ via Gi/o-protein and phospholipase C pathways

GABAB receptors associate with Gi/o-proteins that regulate voltage-gated Ca(2+) channels and thus the intracellular Ca(2+) concentration ([Ca(2+)]i), there is also reported cross-regulation of phospholipase C. These associations have been studied extensively in the brain and also shown to occur in non-neural cells (e.g. human airway smooth muscle). More recently GABAB receptors have been observed in chick retinal pigment epithelium (RPE). The aims were to investigate whether the GABAB receptor subunits, GABAB1 and GABAB2, are co-expressed in cultured human RPE cells, and then determine if the GABAB receptor similarly regulates the [Ca(2+)]i of RPE cells and if phospholipase C is involved. Human RPE cells were cultured from 5 donor eye cups. Evidence for GABAB1 and GABAB2 mRNAs and proteins in the RPE cell cultures were investigated using real time PCR, western blots and immunofluorescence. The effects of the GABAB receptor agonist baclofen, antagonist CGP46381, a Gi/o-protein inhibitor pertussis toxin, and the phospholipase C inhibitor U73122 on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo-3. Both GABAB1 and GABAB2 mRNA and protein were identified in cell cultures of human RPE; antibody staining was co-localized to the cell membrane and cytoplasm. One-hundred μM baclofen caused a transient increase in the [Ca(2+)]i of RPE cells regardless of whether Ca(2+) was added to the buffer. Baclofen induced increases in the [Ca(2+)]i were attenuated by pre-treatment with CGP46381, pertussis toxin, and U73122. GABAB1 and GABAB2 are co-expressed in cell cultures of human RPE. GABAB receptors in RPE regulate the [Ca(2+)]i via a Gi/o-protein and phospholipase C pathway.

Fully automated, semiautomated, and manual morphometric analysis of corneal subbasal nerve plexus in individuals with and without diabetes

Purpose The aim of the study was to determine the association, agreement, and detection capability of manual, semiautomated, and fully automated methods of corneal nerve fiber length (CNFL) quantification of the human corneal subbasal nerve plexus (SNP). Methods Thirty-three participants with diabetes and 17 healthy controls underwent laser scanning corneal confocal microscopy. Eight central images of the SNP were selected for each participant and analyzed using manual (CCMetrics), semiautomated (NeuronJ), and fully automated (ACCMetrics) software to quantify the CNFL. Results For the entire cohort, mean CNFL values quantified by CCMetrics, NeuronJ, and ACCMetrics were 17.4 ± 4.3 mm/mm2, 16.0 ± 3.9 mm/mm2, and 16.5 ± 3.6 mm/mm2, respectively (P < 0.01). CNFL quantified using CCMetrics was significantly higher than those obtained by NeuronJ and ACCMetrics (P < 0.05). The 3 methods were highly correlated (correlation coefficients 0.87–0.98, P < 0.01). The intraclass correlation coefficients were 0.87 for ACCMetrics versus NeuronJ and 0.86 for ACCMetrics versus CCMetrics. Bland–Altman plots showed good agreement between the manual, semiautomated, and fully automated analyses of CNFL. A small underestimation of CNFL was observed using ACCMetrics with increasing the amount of nerve tissue. All 3 methods were able to detect CNFL depletion in diabetic participants (P < 0.05) and in those with peripheral neuropathy as defined by the Toronto criteria, compared with healthy controls (P < 0.05). Conclusions Automated quantification of CNFL provides comparable neuropathy detection ability to manual and semiautomated methods. Because of its speed, objectivity, and consistency, fully automated analysis of CNFL might be advantageous in studies of diabetic neuropathy.

Refractive indices with Haag-Streit Lenstar biometer

Purpose: To estimate refractive indices used with the Lenstar biometer. Methods: Axial lengths of model eyes were determined using an IOLMaster biometer and a Lenstar; comparing these lengths gave an overall eye index for the Lenstar. Using the Lenstar Graphical User interface, we determined that boundaries between media could be manipulated so that there were opposite changes in optical pathlength on either side of the boundary and specified changes in distances determined the ratios of media indices. These ratios were combined with the overall eye index to estimate indices. Results: The IOLMaster and Lenstar produced axial length estimates to within ±0.01 mm. Estimations of group refractive indices were 1.340, 1.341, 1.415 and 1.354 for cornea, aqueous, lens and overall eye, respectively. The aqueous and lens indices, but not those for the cornea, are similar to schematic eye indices and reasonable lens indices. Conclusion: The Lenstar appears to use different refractive indices for different ocular media.

Is retinal shape different in Asians and Caucasians? Estimation from peripheral refraction and peripheral eye length methods

Purpose:Race appears to be associated with myopiogenesis, with East Asians showing high myopia prevalence. Considering structural variations in the eye, it is possible that retinal shapes are different between races. The purpose of this study was to quantify and compare retinal shapes between racial groups using peripheral refraction (PR) and peripheral eye lengths (PEL). Methods:A Shin-Nippon SRW5000 autorefractor and a Haag-Streit Lenstar LS900 biometer measured PR and PEL, respectively, along horizontal (H) and vertical (V) fields out to ±35° in 5° steps in 29 Caucasian (CA), 16 South Asian (SA) and 23 East Asian (EA) young adults (spherical equivalent range +0.75D to –5.00D in all groups). Retinal vertex curvature Rv and asphericity Q were determined from two methods: a) PR (Dunne): The Gullstrand-Emsley eye was modified according to participant’s intraocular lengths and anterior cornea curvature. Ray-tracing was performed at each angle through the stop, altering cornea asphericity until peripheral astigmatism matched experimental measurements. Retinal curvature and hence retinal co-ordinate intersection with the chief ray were altered until sagittal refraction matched its measurement. b) PEL: Ray-tracing was performed at each angle through the anterior corneal centre of curvature of the Gullstrand-Emsley eye. Ignoring lens refraction, retinal co-ordinates relative to the fovea were determined from PEL and trigonometry. From sets of retinal co-ordinates, conic retinal shapes were fitted in terms of Rv and Q. Repeated-measures ANOVA were conducted on Rv and Q, and post hoc t-tests with Bonferroni correction were used to compare races. Results:In all racial groups both methods showed greater Rv for the horizontal than for the vertical meridian and greater Rv for myopes than emmetropes. Rv was greater in EA than in CA (P=0.02), with Rv for SA being intermediate and not significantly different from CA and EA. The PEL method provided larger Rv than the PR method: PEL: EA vs CA 87±13 vs 83±11 m-1 (H), 79±13 vs 72±14 m-1 (V); PR: EA vs CA 79±10 vs 67±10 m-1 (H), 71±17 vs 66±12 m-1 (V). Q did not vary significantly with race. Conclusions:Estimates of Rv, but not of Q, varied significantly with race. The greater Rv found in EA than in CA and the comparatively high prevalence rate of myopia in many Asian countries may be related.

Anatomical specializations for enhanced olfactory sensitivity in kiwi, Apteryx mantelli

The ability to function in a nocturnal and ground-dwelling niche requires a unique set of sensory specializations. The New Zealand kiwi has shifted away from vision, instead relying on auditory and tactile stimuli to function in its environment and locate prey. Behavioral evidence suggests that kiwi also rely on their sense of smell, using olfactory cues in foraging and possibly also in communication and social interactions. Anatomical studies appear to support these observations: the olfactory bulbs and tubercles have been suggested to be large in the kiwi relative to other birds, although the extent of this enlargement is poorly understood. In this study, we examine the size of the olfactory bulbs in kiwi and compare them with 55 other bird species, including emus, ostriches, rheas, tinamous, and 2 extinct species of moa (Dinornithiformes). We also examine the cytoarchitecture of the olfactory bulbs and olfactory epithelium to determine if any neural specializations beyond size are present that would increase olfactory acuity. Kiwi were a clear outlier in our analysis, with olfactory bulbs that are proportionately larger than those of any other bird in this study. Emus, close relatives of the kiwi, also had a relative enlargement of the olfactory bulbs, possibly supporting a phylogenetic link to well-developed olfaction. The olfactory bulbs in kiwi are almost in direct contact with the olfactory epithelium, which is indeed well developed and complex, with olfactory receptor cells occupying a large percentage of the epithelium. The anatomy of the kiwi olfactory system supports an enhancement for olfactory sensitivities, which is undoubtedly associated with their unique nocturnal niche.

Evidence for an Auditory Fovea in the New Zealand Kiwi (Apteryx mantelli).

Kiwi are rare and strictly protected birds of iconic status in New Zealand. Yet, perhaps due to their unusual, nocturnal lifestyle, surprisingly little is known about their behaviour or physiology. In the present study, we exploited known correlations between morphology and physiology in the avian inner ear and brainstem to predict the frequency range of best hearing in the North Island brown kiwi. The mechanosensitive hair bundles of the sensory hair cells in the basilar papilla showed the typical change from tall bundles with few stereovilli to short bundles with many stereovilli along the apical-to-basal tonotopic axis. In contrast to most birds, however, the change was considerably less in the basal half of the epithelium. Dendritic lengths in the brainstem nucleus laminaris also showed the typical change along the tonotopic axis. However, as in the basilar papilla, the change was much less pronounced in the presumed high-frequency regions. Together, these morphological data suggest a fovea-like overrepresentation of a narrow high-frequency band in kiwi. Based on known correlations of hair-cell microanatomy and physiological responses in other birds, a specific prediction for the frequency representation along the basilar papilla of the kiwi was derived. The predicted overrepresentation of approximately 4-6 kHz matches potentially salient frequency bands of kiwi vocalisations and may thus be an adaptation to a nocturnal lifestyle in which auditory communication plays a dominant role.

Eye rubbing-induced changes in intraocular pressure and corneal thickness measured at five locations, in subjects with ocular allergy

AIM To assess the effects of eye rubbing on corneal thickness (CT) and intraocular pressure (IOP) measurements obtained 0-30min after habitual eye rubbing in symptomatic patients. METHODS Measurements of IOP and CT were obtained at five locations (central, temporal, superior, nasal and inferior) before, and every 5min for 30min interval after 30s of eye rubbing, for 25 randomly selected eyes of 14 subjects with ocular allergy and 11 age-matched normals. Differences in measurements were calculated in each group [Baseline measurements minus measurements recorded at each time interval after eye rubbing (for IOP), and for each corneal location (for CT)] and comparison were then made between groups (allergic versus control) for differences in any observed effects. RESULTS Within groups, baseline mean IOPs in the allergic patient-group (14.2±3.0 mm Hg) and in the control group (13.1±1.9 mm Hg) were similar at all times, after eye rubbing (P >0.05, for all). The maximum reduction in IOP was 0.8 mm Hg in the control subjects and the maximum increase was also 0.8 mm Hg in the allergic subjects. Between groups (allergic versus control), the changes in IOP remained under 1 mm Hg at all times (P=0.2) after 30min of eye rubbing. Between 0 and 30min of CT measurements after eye rubbing, the mean central CT (CCT), inferior CT (ICT), superior CT (SCT), temporal CT (TCT) and nasal CT (NCT) did not vary significantly from baseline values in the control and allergic-subject groups (P>0.05, for both). Between both groups, changes in CT were similar at all locations (P>0.05) except for the TC which was minimally thinner by about 4.4 µm (P=0.001) in the allergic subjects than in the control subjects, 30min following 30s of eye rubbing. CONCLUSION IOP measured in allergic subjects after 30s of habitual eye rubbing was comparable with that obtained in normal subjects at all times between 0 and 30min. Although, CT in the allergic subjects were similar to those of the control subjects at all times, it varied between +10 and -7.5 µm following eye rubbing, with the temporal cornea showing consistent reductions in thickness in the subjects with allergy. However, this reduction was minimal and was considered to not be clinically relevant.

Evaluation of intra- and extra-epithelial secretory IgA in chlamydial infections

IgA is an important mucosal antibody that can neutralize mucosal pathogens by either preventing attachment to epithelia (immune exclusion) or alternatively inhibit intraepithelial replication following transcytosis by the polymeric immunoglobulin receptor (pIgR). Chlamydia trachomatis is a major human pathogen that initially targets the endocervical or urethral epithelium in women and men, respectively. As both tissues contain abundant SIgA we assessed the protection afforded by IgA targeting different chlamydial antigens expressed during the extra and intraepithelial stages of infection. We developed an in vitro model utilizing polarizing cells expressing the murine pIgR together with antigen-specific mouse IgA, and an in vivo model utilizing pIgR-/- mice. SIgA targeting the extraepithelial chlamydial antigen, the major outer membrane protein (MOMP), significantly reduced infection in vitro by 24 % and in vivo by 44 %. Conversely, pIgR-mediated delivery of IgA targeting the intraepithelial inclusion membrane protein A (IncA) bound to the inclusion but did not reduce infection in vitro or in vivo. Similarly, intraepithelial IgA targeting the secreted protease Chlamydia protease-like activity factor (CPAF) also failed to reduce infection. Together, these data suggest the importance of pIgR-mediated delivery of IgA targeting extra but not intraepithelial chlamydial antigens for protection against a genital tract infection.

Treatment of silk fibroin with poly(ethylene glycol) for the enhancement of corneal epithelial cell growth

A silk protein, fibroin, was isolated from the cocoons of the domesticated silkworm (Bombyx mori) and cast into membranes to serve as freestanding templates for tissue-engineered corneal cell constructs to be used in ocular surface reconstruction. In this study, we sought to enhance the attachment and proliferation of corneal epithelial cells by increasing the permeability of the fibroin membranes and the topographic roughness of their surface. By mixing the fibroin solution with poly(ethylene glycol) (PEG) of molecular weight 300 Da, membranes were produced with increased permeability and with topographic patterns generated on their surface. In order to enhance their mechanical stability, some PEG-treated membranes were also crosslinked with genipin. The resulting membranes were thoroughly characterized and compared to the non-treated membranes. The PEG-treated membranes were similar in tensile strength to the non-treated ones, but their elastic modulus was higher and elongation lower, indicating enhanced rigidity. The crosslinking with genipin did not induce a significant improvement in mechanical properties. In cultures of a human-derived corneal epithelial cell line (HCE-T), the PEG treatment of the substratum did not improve the attachment of cells and it enhanced only slightly the cell proliferation in the longer term. Likewise, primary cultures of human limbal epithelial cells grew equally well on both non-treated and PEG-treated membranes, and the stratification of cultures was consistently improved in the presence of an underlying culture of irradiated 3T3 feeder cells, irrespectively of PEG-treatment. Nevertheless, the cultures grown on the PEG-treated membranes in the presence of feeder cells did display a higher nuclear-to-cytoplasmic ratio suggesting a more proliferative phenotype. We concluded that while the treatment with PEG had a significant effect on some structural properties of the B. mori silk fibroin (BMSF) membranes, there were minimal gains in the performance of these materials as a substratum for corneal epithelial cell growth. The reduced mechanical stability of freestanding PEG-treated membranes makes them a less viable choice than the non-treated membranes.

Effect of ocular hypotony secondary to cyclodialysis cleft on corneal topography

PURPOSE: To report the changes in corneal topography in 2 cases of ocular hypotony induced by cyclodialysis cleft after blunt trauma, which were successfully treated by argon laser photocoagulation. METHODS: For both patients, a full ophthalmic clinical examination and corneal topography were performed before and after argon laser cleft closure. RESULTS: In the first case, the corneal topography showed 3.81-D astigmatism at 96 degrees, which was reduced to 1.1 D at 124 degrees 1 week after treatment and 0.66 D at 122 degrees at 3 weeks after treatment. In the second case, the corneal astigmatism was 3.91 D at 104 degrees, which decreased to 1.44 D at 104 degrees and 0.35 D at 118 degrees at 1 week and 4 months after treatment, respectively. CONCLUSIONS: In both cases, the with-the-rule astigmatism reduced significantly after successful closure of the cleft and an increase in intraocular pressure.

A Bruch’s membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a similar thickness as Bruch’s membrane (3 μm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell). Cultures established on either material developed a cobblestoned morphology with partial pigmentation within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na+/K+-ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned medium collected from above and below both membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrate that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model.