Microbiological environmental monitoring in high-risk departments during building activities in a hospital site

Background. This study examines the microbial and fungal contamination associated with the presence of renovation works in a hospital site in Sardinia (Italy). Methods. Microbiological environmental monitoring was carried out before, during and at the conclusion of the works in the Ophthalmology Department in view of a risk assessment procedure. Results. Although the median values of microbial and fungal counts were found raised during the works, protective measures set out by the internal procedures limited the contamination level. Conclusions. This study emphasizes the benefits of environmental surveillance for airborne contamination to help prevent outbreaks of nosocomial mycosis associated with construction work

Consecutive dosing of UVB irradiation induces loss of ABCB5 expression and activation of EMT and fibrosis proteins in limbal epithelial cells similar to pterygium epithelium

Pterygium pathogenesis is often attributed to a population of altered limbal stem cells, which initiate corneal invasion and drive the hyperproliferation and fibrosis associated with the disease. These cells are thought to undergo epithelial to mesenchymal transition (EMT) and to contribute to subepithelial stromal fibrosis. In this study, the presence of the novel limbal stem cell marker ABCB5 in clusters of basal epithelial pterygium cells co-expressing with P63α and P40 is reported. ABCB5-positive pterygium cells also express EMT-associated fibrosis markers including vimentin and α-SMA while their β-catenin expression is reduced. By using a novel in vitro model of two-dose UV-induced EMT activation on limbal epithelial cells, we could observe the dysregulation of EMT-related proteins including an increase of vimentin and α-SMA as well as downregulation of β-catenin in epithelial cells correlating to downregulation of ABCB5. The sequential irradiation of limbal fibroblasts also induced an increase in vimentin and α-SMA. Taken together, these data demonstrate for the first time the expression of ABCB5 in pterygium stem cell activity and EMT-related events while the involvement of limbal stem cells in pterygium pathogenesis is exhibited via sequential irradiation of limbal epithelial cells. The later in vitro approach can be used to further study the involvement of limbal epithelium UV-induced EMT in pterygium pathogenesis and help identify novel treatments against pterygium growth and recurrence

Distinct gene subsets in pterygia formation and recurrence: dissecting complex biological phenomenon using genome wide expression data

<p>Abstract</p> <p>Background</p> <p>Pterygium is a common ocular surface disease characterized by fibrovascular invasion of the cornea and is sight-threatening due to astigmatism, tear film disturbance, or occlusion of the visual axis. However, the mechanisms for formation and post-surgical recurrence of pterygium are not understood, and a valid animal model does not exist. Here, we investigated the possible mechanisms of pterygium pathogenesis and recurrence.</p> <p>Methods</p> <p>First we performed a genome wide expression analysis (human Affymetrix Genechip, >22000 genes) with principal component analysis and clustering techniques, and validated expression of key molecules with PCR. The controls for this study were the un-involved conjunctival tissue of the same eye obtained during the surgical resection of the lesions. Interesting molecules were further investigated with immunohistochemistry, Western blots, and comparison with tear proteins from pterygium patients.</p> <p>Results</p> <p>Principal component analysis in pterygium indicated a signature of matrix-related structural proteins, including fibronectin-1 (both splice-forms), collagen-1A2, keratin-12 and small proline rich protein-1. Immunofluorescence showed strong expression of keratin-6A in all layers, especially the superficial layers, of pterygium epithelium, but absent in the control, with up-regulation and nuclear accumulation of the cell adhesion molecule CD24 in the pterygium epithelium. Western blot shows increased protein expression of beta-microseminoprotein, a protein up-regulated in human cutaneous squamous cell carcinoma. Gene products of 22 up-regulated genes in pterygium have also been found by us in human tears using nano-electrospray-liquid chromatography/mass spectrometry after pterygium surgery. Recurrent disease was associated with up-regulation of sialophorin, a negative regulator of cell adhesion, and <it>never in mitosis a</it>-5, known to be involved in cell motility.</p> <p>Conclusion</p> <p>Aberrant wound healing is therefore a key process in this disease, and strategies in wound remodeling may be appropriate in halting pterygium or its recurrence. For patients demonstrating a profile of 'recurrence', it may be necessary to manage as a poorer prognostic case and perhaps, more adjunctive treatment after resection of the primary lesion.</p

Molecular Effects of Doxycycline Treatment on Pterygium as Revealed by Massive Transcriptome Sequencing

Pterygium is a lesion of the eye surface which involves cell proliferation, migration, angiogenesis, fibrosis, and extracellular matrix remodelling. Surgery is the only approved method to treat this disorder, but high recurrence rates are common. Recently, it has been shown in a mouse model that treatment with doxycycline resulted in reduction of the pterygium lesions. Here we study the mechanism(s) of action by which doxycycline achieves these results, using massive sequencing techniques. Surgically removed pterygia from 10 consecutive patients were set in short term culture and exposed to 0 (control), 50, 200, and 500 µg/ml doxycycline for 24 h, their mRNA was purified, reverse transcribed and sequenced through Illumina’s massive sequencing protocols. Acquired data were subjected to quantile normalization and analyzed using cytoscape plugin software to explore the pathways involved. False discovery rate (FDR) methods were used to identify 332 genes which modified their expression in a dose-dependent manner upon exposure to doxycycline. The more represented cellular pathways included all mitochondrial genes, the endoplasmic reticulum stress response, integrins and extracellular matrix components, and growth factors. A high correlation was obtained when comparing ultrasequencing data with qRT-PCR and ELISA results

Aberrant DNA Methylation of Matrix Remodeling and Cell Adhesion Related Genes in Pterygium

10.1371/journal.pone.0014687PLoS ONE62

Human ganglion cells express the alpha-2 adrenergic receptor: relevance to neuroprotection

Background/aim: Alpha-2α adrenergic receptor (α(2)-AR) agonists are thought to be neuroprotective, preventing retinal ganglion cell death independent of pressure reduction. Previous studies have identified α(2)-ARs in rat retina. The authors aimed to demonstrate the presence and localisation of α(2)-ARs in human and rat retina and on the rat retinal ganglion cell line, RGC-5. Methods: Seven postmortem human and three postmortem rat eyes were paraformaldehyde fixed and frozen. RGC-5 cells were also paraformaldehyde fixed. The expression of α(2A)-ARs was determined by antibody immunofluorescence. Results: α(2A)-AR expression was identified in the human retina, on ganglion cells, and cells in the inner and outer nuclear layers (INL, ONL). Differential α(2A)-AR staining patterns in the INL and ONL suggest a further restriction to as yet unidentified neuronal subclasses. The RGC-5 cell line also expressed α(2A)-ARs in undifferentiated cells and an increased expression upon fully differentiated cells. Conclusion: α(2)-AR agonists in addition to their pressure lowering effects in the eye, may act directly upon retinal neurons, including retinal ganglion cells. The presence of α(2)-ARs on the RGC-5 cell line allows future investigation of these possible direct effects using in vitro glaucoma model systems

Identification and visualization of a distinct microbiome in ocular surface conjunctival tissue

PURPOSE. Knowledge of whether microorganisms reside in protected niches of the conjunctiva is potentially significant in terms of minimizing risks of contact lens inflammation/infection and endophthalmitis. We define if and how microbial communities from limbal and forniceal conjunctival tissue differ from those on the conjunctival surface. METHODS. Human limbal and forniceal conjunctival tissue was obtained from 23 patients undergoing pterygium surgery and analyzed with data from a recent study of conjunctival surface swabs (n = 45). Microbial communities were analyzed by extracting total DNA from tissue samples and surface swabs and sequencing the 16S rRNA gene using the Illumina MiSeq platform. Sequences were quality filtered, clustered into operational taxonomic units (OTUs) at 97% similarity. OTUs associated with blank extraction and sampling negative controls were removed before analysis. Fluorescent in situ hybridization (FISH) was performed on cyrosections of limbal and forniceal conjunctival tissue. RESULTS. There was a significant difference in bacterial community structure between the conjunctival surface and fornix (P = 0.001) and limbus (P = 0.001) tissue. No difference was found in bacterial communities between the limbus and fornix (P = 0.764). Fornix and limbal samples were dominated by OTUs classified to the genus Pseudomonas (relative abundance 79.9%), which were found only in low relative abundances on conjunctival surfaces (6.3%). Application of FISH showed the presence of Pseudomonas in the forniceal tissue sample. CONCLUSIONS. There is a discrete tissue-associated microbiome in freshly-collected human limbal and fornix tissue, which is different from the microbial community structure and composition of the ocular surface microbiome