Genomics of the origin and evolution of Citrus.

The genus Citrus, comprising some of the most widely cultivated fruit crops worldwide, includes an uncertain number of species. Here we describe ten natural citrus species, using genomic, phylogenetic and biogeographic analyses of 60 accessions representing diverse citrus germ plasms, and propose that citrus diversified during the late Miocene epoch through a rapid southeast Asian radiation that correlates with a marked weakening of the monsoons. A second radiation enabled by migration across the Wallace line gave rise to the Australian limes in the early Pliocene epoch. Further identification and analyses of hybrids and admixed genomes provides insights into the genealogy of major commercial cultivars of citrus. Among mandarins and sweet orange, we find an extensive network of relatedness that illuminates the domestication of these groups. Widespread pummelo admixture among these mandarins and its correlation with fruit size and acidity suggests a plausible role of pummelo introgression in the selection of palatable mandarins. This work provides a new evolutionary framework for the genus Citrus.

Characterization and functional performance of a commercial human conjunctival epithelial cell line.

The conjunctiva is a complex tissue that covers the eye beginning at the corneal limbus and extending over the inner surfaces of the eyelids. Due to its important functions in maintaining the health of the ocular surface, adequate in vitro models of conjunctival structure and function are essential to understand its roll in different pathologies. Because there is scarcity of human conjunctival tissue that can be used in research, cell lines are often the only option for initial studies. An immortalized human conjunctival epithelial cell (IM-HConEpiC) line is now commercially available; however, it is not very well characterized. In this study, we have developed a new protocol to culture these cells without the use of collagen-coated culture surfaces, but with a defined cell culture medium. We characterized IM-HConEpiCs cultured under these conditions and corroborated that the cells maintained a conjunctival epithelial phenotype, including acidic and neutral mucins, junctional proteins E-cadherin and zonula occludens 1, and expression of CK8 and CK19, among others. In addition, we analyzed the response to oxidative stress and inflammatory stimuli and found that IM-HConEpiCs respond as expected for conjunctival epithelial tissue. For instance, cells exposed to oxidative stress increased the production of reactive oxygen species, and that increase was blocked in the presence of an antioxidant agent. In addition, after stimulation with TNF-α, IM-HConEpiCs significantly increased the production of IL-1ß, IL-6, IL-8, and IP-10. Therefore, with this study we conclude that IM-HConEpiCs can be a useful tool in functional studies to determine the response of the conjunctiva to pathological conditions and/or to test new therapeutic strategies.

Improved in vitro corneal delivery of a thrombospondin-1-derived peptide using a liposomal formulation.

Peptide delivery to and through ocular sites is a growing field of research interest. However, several barriers restrict the permeation and bioavailability of these molecules to target tissues. The main pharmacological barriers of topical administration are the tear film, rapid drainage of the tear film, and poor corneal permeation. If the administered molecule is a peptide, instability and enzymatic degradation can be significant. Novel approaches such as the design and development of nanocarriers to overcome these drawbacks have been investigated with promising results. Therefore, in continuation of our previous study using a liposome-based (LP) formulation as topical drug delivery system, the aim of this work was to efficiently encapsulate a thrombospondin-1-derived peptide, KRFK, in this formulation and to assess peptide permeability through different ocular surface epithelia. LPs were prepared by the solvent evaporation technique and the labeled peptide FITC-KRFK was incorporated in the aqueous core. Different sonication times were used to optimize encapsulation efficiency. The selected formulation was further analyzed in terms of size, pH, osmolarity, and corneal epithelial cytotoxicity. The permeabilities of the LP-encapsulated and free labeled KRFK peptides were assessed with in vitro models of conjunctival and corneal epithelia. Our results provide a proof of concept that the LP formulation efficiently encapsulates the KRFK peptide and improves corneal permeation. Data reported in this study strongly support that this formulation could be a more effective therapeutic approach than free peptide instillation and warrant further analysis using experimental in vivo models.

Topical Application of TGF-ß-Activating Peptide, KRFK, Prevents Inflammatory Manifestations in the TSP-1-Deficient Mouse Model of Chronic Ocular Inflammation.

Chronic inflammation of the ocular surface poses a risk of vision impairment. The understanding of the molecular mechanisms that are involved in the inflammatory response is critical to identify novel molecular targets. Recently, thrombospondin-1 (TSP-1) has emerged as a key player in ocular surface homeostasis that efficiently activates the TGF-ß2 isoform that is predominantly expressed in the ocular mucosa. Here, the potential of the peptide derived from TSP-1 (KRFK), that can activate TGF-ß, is proposed as a potentially applicable therapeutic for chronic ocular surface inflammatory disorders. Our in vitro results confirm that the chosen peptide activates TGF-ß, reducing the expression of co-stimulatory molecules on dendritic cells, driving them towards a tolerogenic phenotype. For the in vivo studies, the TSP-1-/- mouse is used as a pre-clinical model of chronic ocular inflammation. We observe that the topical application of KRFK alters the peripheral balance of effectors by reducing the proportion of pathogenic Th1 and Th17 cells while increasing Treg cell proportion in cervical lymph nodes. In line with these findings, the development of chronic ocular surface inflammation is significantly prevented in KRFK-treated TSP-1-/- mice, as assessed by clinical parameters and inflammatory cytokine expression in conjunctival and lacrimal gland tissues. Together, our results identify the KRFK peptide as a novel therapeutic option to prevent the development of chronic inflammatory manifestations of the ocular surface.

Novel anti-inflammatory liposomal formulation for the pre-ocular tear film: In vitro and ex vivo functionality studies in corneal epithelial cells.

In ocular surface inflammatory diseases, such as dry eye disease, long-term symptom relief requires targeting the inflammation itself rather than treating only the surface-associated dryness with artificial tears. Therefore, we included an anti-inflammatory agent in an unpreserved liposome-based (LP) formulation used as artificial tears. Our aim was to characterize and study its in vitro and ex vivo cell uptake and functionality. Human corneal epithelial (HCE) cells were used to study MPA-LP-induced effects after 60 min of exposure, using blank LP and non-LP MPA formulations as controls. A fluorescent labeled LP formulation was used to determine uptake by HCE cells and localization in ex vivo porcine corneas. The LP formulation complied with the required physicochemical properties and had no cytotoxicity on HCE cells after 60 min of exposure. HCE cells showed LP-associated fluorescence at 24, 48, and 72 h after 60 min of exposure, and the LP-associated fluorescence was uniformly distributed throughout the porcine corneal epithelium immediately after 5 min of exposure. MPA-LP increased protein expression and nuclear translocation of progesterone receptor in comparison with controls as determined by Western blotting and immunofluorescence. Moreover, MPA-LP significantly reduced the cell proliferation rate and IL-6 and IL-8 production 48 h after the exposure period, as determined by the alamarBlue assay and ELISA, respectively. None of these effects were evident in blank LP-exposed cells and non-LP MPA formulation reduced only IL-6 production. Our results suggest that the LP-based formulation, used to replenish the lipids of the tear film, can be loaded with anti-inflammatory agents that can be delivered into the cells and activate specific drug receptors. These agents can reduce inflammatory cytokine production and may be effective in the treatment of inflammatory processes associated with ocular surface diseases.

Thrombospondin-1 induces differential response in human corneal and conjunctival epithelial cells lines under in vitro inflammatory and apoptotic conditions.

Recently, thrombospondin-1 (TSP-1) has been reported to be critical for maintaining a healthy ocular surface. The purpose of the study was to characterize the expression of TSP-1 and of its receptors CD36 and CD47 in corneal and conjunctival epithelial cells and determine the effect of exogenous TSP-1 treatment on these cells, following the induction of inflammation- and apoptosis-related changes. The expression of TSP-1, CD36 and CD47 by corneal and conjunctival cell lines was firstly characterized by ELISA, immunofluorescence analysis, Western blotting and reverse transcription polymerase chain reaction (RT-PCR). Benzalkonium chloride (BAC) exposure for 5 or 15 min was used as pro-inflammatory and pro-apoptotic stimulus for corneal or conjunctival epithelial cells, respectively. To analyze inflammation and apoptosis-related changes, IL-6 and TGF-ß2 secretion determined by ELISA was used as inflammatory markers, while activated caspase-3/7 levels and cell viability, determined by CellEvent™ Caspase-3/7 Green Detection Reagent and XTT cytotoxicity assay, respectively, were used as apoptotic markers. Changes in CD36 and CD47 mRNA expression were quantified by real time RT-PCR. Corneal epithelial cells secreted and expressed higher protein levels of TSP-1 than conjunctival epithelial cells, although TSP-1 mRNA expression levels were similar and had lower CD36 and CD47, both at protein and mRNA levels. Both cell lines responded to exogenous TSP-1 treatment increasing CD36 at protein and mRNA levels. Blocking experiments revealed a predominance of TSP-1/CD47 rather than TSP-1/CD36 interactions to up-regulate CD36 levels in conjunctival epithelial cells, but not in corneal epithelial cells. BAC exposure increased IL-6 secretion and caspase-3/7 levels and decreased cell viability in both, corneal and conjunctival epithelial cells. Moreover, BAC exposure increased latent TGF-ß2 levels in conjunctival epithelial cells. Interestingly, CD36 mRNA expression was down-regulated after BAC exposure in both cell lines. Exogenous TSP-1 treatment reduced TGF-ß2 up-regulated levels by BAC exposure in conjunctival epithelial cells and less pronounced reduced IL-6 in BAC-exposed corneal epithelial cells. The effect on CD36 and CD47 regulation was less pronounced or even opposite depending on the inflammation- and apoptosis-related markers tested. Our results show evidence of the capacity of corneal and conjunctival epithelial cells to respond to TSP-1 via CD36 or CD47. Experimental simulation of inflammation- and apoptosis-related conditions changed the effects differentially elicited by TSP-1 on corneal and conjunctival epithelial cells, suggesting an unexpected and relevant contribution of TSP-1 on ocular surface homeostasis regulation.

IL-6 as a corneal wound healing mediator in an in vitro scratch assay.

Corneal healing process under inflammatory conditions is not fully understood. We aimed at determining the effect of an inflammatory (presence of IL-6) or anti-inflammatory (presence of IL-10) environment and a mixture of both in the expression of IL-6 signaling pathway mediators, and on corneal wound healing in an in vitro scratch assay. For that purpose, human corneal epithelial cells were cultured until confluence. The effect of IL-6 (10 ng/ml), IL-10 (20 ng/ml) or IL-6 + IL-10 exposure on the expression of IL-6R, gp130, and STAT3 was determined by Western blotting and quantitative PCR, at different time points. The monolayer was mechanically wounded using a sterile 10 µl pipette tip. Wound healing rate in the presence or absence of these cytokines was measured immediately after cytokine exposure and after 4, 8, and 24 h. The effect of mitomycin C on wound healing rate, in control and IL-6-stimulated cells, was also evaluated. Detection of proliferative cells was performed with an EdU imaging kit. For the visualization of migrating cells, cold methanol-fixed cells were incubated with an α-actinin antibody. For the statistical analysis a two-factor design of experiment method was applied. Levene test was used to contrast equality of variances. If variances were equal, ANOVA was performed to test the equality of means. If variances were not equal, a Mood's median test was performed. We observed that IL-6 and IL-10 stimulation, and their combination, increased gp130 production at different time points. STAT3 production was increased in IL-6-stimulated cells, at 72 h. An increase in pSTAT3 production was found in IL-6- and IL-10-stimulated cells, that was sustained in time in IL-6 + IL-10 co-stimulated cultures. Scraped areas had an initial width of 570.57 ± 75.82 µm. In IL-6-exposed cells wound healing closure was faster than in control cells or IL-10-exposed cells. After 8 h, wound width in IL-10-exposed cells, was also significantly smaller than that of control cells. Cells exposed to IL-6 + IL-10 had the slowest wound healing rate, similar to control cells. Wounds were closed after 24 h regardless the experimental condition. Mitomycin C exposure increased the wound closure rate in every experimental condition. No significant differences in the percentage of proliferative cells at the edge of the scratch and in distant areas of the monolayer were found. At the edge of the scratch, some actin filaments of non-proliferative cells were directed through the cell-free area, independently of the stimulating condition. In conclusion, the presence of IL-10 and, most importantly, of IL-6, increased the wound healing rate in an in vitro corneal wound healing model. The combination of both cytokines did not have a synergistic action in wound healing. In our model, wound closure was the result of the combination of cell proliferation and cell migration.

CD44 and RHAMM hyaluronan receptors in human ocular surface inflammation.

BACKGROUND: CD44 and RHAMM hyaluronan (HA) receptors have been studied in several systemic diseases such as osteoarthritis and cancer. However, not too much is known about their role in ocular surface disorders. The purpose of this research was to determine if CD44 and RHAMM are implicated in human ocular surface inflammation. METHODS: Upper tarsal conjunctival epithelial samples from patients with active ocular surface inflammation (n = 17) and healthy controls (n = 14) were recovered by brush cytology. Patients were evaluated by an ophthalmologist and classified in different groups according to the etiology (immune atopic diseases or immune non-atopic diseases) and inflammation intensity (mild/moderate or severe). CD44, RHAMM, and p53 mRNAs were measured using real-time PCR. RESULTS: CD44, RHAMM, and p53 mRNAs were detected in all samples. In immune atopic diseases, higher levels of CD44 and RHAMM mRNAs were present, reaching a 300 % increase for RHAMM in severe inflammation (p < 0.001). In contrast, in immune non-atopic diseases, the HA receptors were downregulated. CD44 tended to decrease up to 30 % in severe patients (p = 0.06), and RHAMM decreased 40 % in severe inflammation (p = 0.021). CONCLUSIONS: RHAMM may be implicated in severe ocular surface inflammation affecting the upper tarsal conjunctiva.

Involvement of corneal epithelial cells in the Th17 response in an in vitro bacterial inflammation model.

PURPOSE: Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) are frequent causes of bacterial keratitis, an inflammatory process that can lead to vision loss. We used a human corneal epithelial (HCE) cell line to study the Th17 inflammatory pathway, including interleukin (IL-) 6, IL-17, and associated receptors, in response to stimulation by SA and PA culture supernatants. METHODS: Cells of the HCE cell line were exposed to either SA or PA supernatants in dilutions of 1:100 or 1:50, or to human recombinant IL-17A (20 ng/ml). Cell culture supernatants were collected at 6, 24, and 72 h, and protein and RNA were isolated. Expression of cytokine (IL-6, IL-17A), receptor (sIL-6R, IL-17RA), and mediator (soluble glycoprotein [sgp] 130, MIP3α) proteins and mRNAs were determined with enzyme-linked immunosorbent assay, immunohistochemistry, western blotting, and real-time, reverse-transcription quantitative PCR. In addition, IL-17RA was localized by transmission electron microscopy after immunogold labeling. RESULTS: Basal secretion of IL-6 and IL-17A by HCE cells occurred in a time-dependent manner. Expression of IL-6 was significantly enhanced by SA stimulation, but not by PA stimulation. IL-6 mRNA expression was higher in the control and SA-stimulated cells at 6 and 24 h, but not at 72 h. In the PA-stimulated cells, mRNA levels were significantly lower than the controls at 6 and 24 h. Expression of sIL-6R was not altered by SA or PA supernatants, but sgp130 expression was greater than controls at 6 h, less than controls at 24 h, and the same as controls at 72 h. HCE cells secreted IL-17A in a time-dependent manner that was not altered by stimulation; however, the IL-17A mRNA levels were lower than those of the controls at 6 h. With immunohistochemistry, IL-17RA was localized in perinuclear vesicles and in the cytosol and membranes of HCE cells. IL-17RA was also present in the epithelial cells from human ocular surface tissues. As quantified with western blotting, expression of IL-17RA was unchanged in HCE cells stimulated by SA or PA supernatants. CONCLUSIONS: HCE cells react to bacterial inflammation by enhancing the secretion of IL-6 and by regulating the proinflammatory response with differential secretion of sgp130. Under normal conditions, HCE cells and ocular surface tissues express IL-17RA. Additionally, HCE cells express IL-17RA after bacterial stimulation. All of these molecules are involved in the Th17 differentiation pathway, suggesting that corneal epithelial cells may act as indirect participants in the Th17 signaling pathway.

[Ruptured cerebral artery blister aneurysm]. / Aneurisma cerebral roto tipo «blíster¼

We report the case of a young patient with subarachnoid haemorrhage secondary to a ruptured blister-like aneurysm. Since this kind of aneurysms have fragile walls without a well-defined neck, their treatment is difficult. We initially planned the deployment of a flow-diverter stent, but an angiogram obtained after 10 days revealed a morphological change of the aneurysm. Therefore, we finally deployed a conventional stent and introduced 2 micro coils into the point of rupture, obtaining a good morphological result without rebleeding. Follow-up at 1 and 6 months did not observe regrowth of the aneurysm. We offer a brief introduction and discussion of this pathology and its treatment.

Hyaluronan receptors in the human ocular surface: a descriptive and comparative study of RHAMM and CD44 in tissues, cell lines and freshly collected samples.

The purpose of this study was to demonstrate the presence of the receptor for hyaluronan-mediated motility (RHAMM) in human conjunctival epithelium and in two widely used cell lines from human corneal (HCE) and conjunctival (IOBA-NHC) epithelia. We compared the distribution of RHAMM proteins and mRNAs in human ocular surface tissues (corneal, limbal and conjunctival), HCE and IOBA-NHC cell lines, and corneal and conjunctival epithelia primary samples from healthy donors with the previously identified hyaluronan receptor CD44. We also aimed to determine if soluble CD44 (sCD44) was present in human tears, as it could have a role in the interaction of the tear fluid with hyaluronan. Protein expression was evaluated by Western blots and immunofluorescence microscopy. mRNA expression was evaluated by RT-PCR and Q-PCR. sCD44 was analyzed by ELISA in culture supernatants and in human tears. We describe the expression of RHAMM in human healthy conjunctiva and in HCE and IOBA-NHC cells at both protein and mRNA levels, and the presence of sCD44 in human tears. Furthermore, we detected CD44 and sCD44 expression variations in in vitro inflammatory conditions. This study also focused on the necessary caution with which the conclusions extracted from cell lines should be made, and in the great value of using primary samples as often as possible.

Intracellular trafficking of hyaluronic acid-chitosan oligomer-based nanoparticles in cultured human ocular surface cells.

PURPOSE: Nanoparticles are a promising alternative for ocular drug delivery, and our group has proposed that they are especially suited for ocular mucosal disorders. The goal of the present study was to determine which internalization pathway is used by cornea-derived and conjunctiva-derived cell lines to take up hyaluronic acid (HA)-chitosan oligomer (CSO)-based nanoparticles (HA-CSO NPs). We also determined if plasmids loaded onto the NPs reached the cell nucleus. METHODS: HA-CSO NPs were made of fluoresceinamine labeled HA and CSO by ionotropic gelation and were conjugated with a model plasmid DNA for secreted alkaline phosphatase. Human epithelial cell lines derived from the conjunctiva and the cornea were exposed to HA-CSO NPs for 1 h and the uptake was investigated in living cells by fluorescence microscopy. The influence of temperature and metabolic inhibition, the effect of blocking hyaluronan receptors, and the inhibition of main endocytic pathways were studied by fluorometry. Additionally, the metabolic pathways implicated in the degradation of HA-CSO NPs were evaluated by lysosome identification. RESULTS: There was intracellular localization of plasmid-loaded HACSO NPs in both corneal and conjunctival cells. The intracellular presence of NPs diminished with time. HA-CSO NP uptake was significantly reduced by inhibition of active transport at 4 °C and by sodium azide. Uptake was also inhibited by blocking hyaluronan receptors with anti-CD44 Hermes-1 antibody, by excess HA, and by filipin, an inhibitor of caveolin-dependent endocytosis. HA-CSO NPs had no effect on cell viability. The transfection efficiency of the model plasmid was significantly higher in NP treated cells than in controls. CONCLUSIONS: HA-CSO NPs were internalized by two different ocular surface cell lines by an active transport mechanism. The uptake was mediated by hyaluronan receptors through a caveolin-dependent endocytic pathway, yielding remarkable transfection efficiency. Most of HA-CSO NPs were metabolized within 48 h. This uptake did not compromise cell viability. These findings further support the potential use of HA-CSO NPs to deliver genetic material to the ocular surface.

Expression of MUC5AC in ocular surface epithelial cells using cationized gelatin nanoparticles.

Decreased production of the mucin MUC5AC in the eye is related to several pathological conditions, including dry eye syndrome. A specific strategy for increasing the ocular levels of MUC5AC is not yet available. Using a plasmid specially designed to encode human MUC5AC, we evaluated the ability of hybrid cationized gelatin nanoparticles (NPs) containing polyanions (chondroitin sulfate or dextran sulfate) to transfect ocular epithelial cells. NPs were developed using the ionic gelation technique and characterized by a small size (<200 nm), positive zeta potential (+20/+30 mV), and high plasmid association efficiency (>95%). MUC5AC mRNA and protein were detected in conjunctival cells after in vitro transfection of the NPs. The in vivo administration of the NPs resulted in significantly higher MUC5AC expression in the conjunctiva compared to untreated control and naked plasmid. These results provide a proof-of-concept that these NPs are effective vehicles for gene therapy and candidates for restoring the MUC5AC concentration in the ocular surface.

Histological and immunohistochemical characterization of the porcine ocular surface.

The ocular surface of the white domestic pig (Sus scrofa domestica) is used as a helpful model of the human ocular surface; however, a complete histological description has yet to be published. In this work, we studied porcine eyeballs with intact eyelids to describe and characterize the different structures that form the ocular surface, including the cornea and conjunctiva that covers the bulbar sclera, tarsi, and the nictitating membrane. We determined the distribution of goblet cells of different types over the conjunctiva and analyzed the conjunctival-associated lymphoid tissue (CALT). Porcine eyeballs were obtained from a local slaughterhouse, fixed, processed, and embedded in paraffin blocks. Tissue sections (4 µm) were stained with hematoxylin/eosin, Alcian blue/Periodic Acid Schiff, and Giemsa. Slides were also stained with lectins from Arachis hypogaea (PNA) and Helix pomatia (HPA) agglutinins and immunostained with rabbit anti-CD3. We found that the porcine cornea was composed of 6-8 epithelial cell layers, stroma, Descemet's membrane, and an endothelial monolayer. The total corneal thickness was 1131.0±87.5 µm (mean±standard error of the mean) in the center and increased to 1496.9±138.2 µm at the limbus. The goblet cell density was 71.25±12.29 cells/mm, ranging from the highest density (113.04±37.21 cells/mm) in the lower palpebral conjunctiva to the lowest density (12.69±4.29 cells/mm) in the bulbar conjunctiva. The CALT was distributed in the form of intraepithelial lymphocytes and subepithelial diffuse lymphoid tissue. Lenticular-shaped lymphoid follicles, about 8 per histological section, were also present within the conjunctival areas. In conclusion, we demonstrated that the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.

Sorbitan ester nanoparticles (SENS) as a novel topical ocular drug delivery system: Design, optimization, and in vitro/ex vivo evaluation.

We explored the potential of two types of sorbitan ester nanoparticles (SENS) as novel tools for topical ocular drug delivery. The optimized SENS formulation (SENS-OPT) consisted of nanoparticles (NPs) of 170.5 nm, zeta potential +33.9 mV, and cyclosporine loading of 19.66%. After hyaluronic acid (HA) coating, the resulting SENS-OPT-HA NPs had a particle size of 177.6 nm and zeta potential of -20.6 mV. The NPs were stable during 3 months of storage at different temperatures and did not aggregate in the presence of protein-enriched simulated lacrimal fluid. There was no toxicity to cultured human corneal epithelial (HCE) cells when exposed to NPs up to 0.4% (w/v). Both NPs were effectively internalized by HCE cells through active mechanisms. Endocytosis of SENS-OPT NPs was caveolin-dependent whereas SENS-OPT-HA NP endocytosis was mediated by HA receptors. HA-receptor-mediated endocytosis may be responsible for the higher cellular uptake of SENS-OPT-HA NPs. After cyclosporine incorporation into the NPs, corneal penetration of this immunosuppressive drug by loaded SENS-OPT NPs was 1.3-fold higher than the commercial reference formulation Sandimmun®. For cyclosporine-loaded SENS-OPT-HA NPs, the penetration was 2.1-fold higher than for Sandimmun®. In ex vivo stimulated lymphocytes, both formulations demonstrated the same reduction in IL-2 levels as Sandimmun®.

An engineered human conjunctival-like tissue to study ocular surface inflammatory diseases.

The aim of this study was to develop a three-dimensional model of the human conjunctiva that can be used to perform physiology and pathophysiology experiments. Fibrin-based matrices (derived from human plasma or plasma cryoprecipitate) were used as scaffolds, and primary cells were obtained from conjunctival tissue. Conjunctival constructs were analyzed by immunofluorescent staining and scanning electron microscopy and cell proliferation was measured with alamarBlue® assay. After characterizing the constructs, four different experimental conditions were analyzed in cryoprecipitate matrices: controls, air-lifted cultures (to increase cell stratification), partially desiccated cultures (to mimic dry eye disease), and IL-13-treated cultures (to mimic allergy). Constructs were stained with hematoxylin/eosin to observe changes in morphology. High molecular weight glycoconjugates were identified by HPA staining. MUC5AC and IL-6 secretion was evaluated by ELISA. The fibrin-based matrices supported conjunctival cell growth. Epithelial cells grew on the surface of the scaffolds and underwent stratification that increased over time. These cells had microvilli, which suggests cell polarization and functionality. Fibroblasts were integrated in the scaffold and showed elongated shape. Compared to controls, air-lifted construct had increased epithelial stratification and upregulated MUC5AC secretion. Increased MUC5AC secretion also occurred in partially desiccated and IL-13-treated cultures. The inflammatory status of cells was evaluated by IL-6 levels which were increased in air-lifted and partially desiccated cultures, but not in IL-13-treated ones. In conclusion, we have developed a new three-dimensional model of human conjunctiva that can be used to study ocular surface inflammatory diseases.

Histologic Characterization of Retina Neuroglia Modifications in Diabetic Zucker Diabetic Fatty Rats.

Purpose: The purpose of this study was to characterize retinal degenerative morphologic modifications in Zucker Diabetic Fatty (ZDF) rats, a genetic model of type 2 diabetes, by histologic and immunohistochemical evaluation. Methods: Male lean (?/+; n = 10) and ZDF (fa/fa; n = 20) rats were used. At 24 weeks of age, body weights and blood glucose levels were determined. Eyes were removed and processed for paraffin wax embedding. Sections through the optic disc were stained for hematoxylin and eosin or immunostained for TUNEL, advanced glycation end products (AGEs), glial fibrillary acidic protein (GFAP), glutamate/aspartate transporter (GLAST), isolectin B4, recoverin, retinal pigment epithelium-specific 65-kDa protein, rhodopsin, vimentin, and zonula occludens protein 1. Retinal morphometry, cell counts, glial activation degree and immunoreactivity of AGEs and GLAST were also determined. Results: ZDF rats were observed to be diabetic from week 9 and by week 24. These animals showed retinal morphologic degenerative changes, increased neuroretinal thickness, and decreased number of nuclei. Glial cells activation with massive GFAP upregulation was present. Cellular morphologic modifications were also observed. GLAST immunofluorescence was decreased, whereas AGEs were increased in comparison with lean rats. Conclusions: Spontaneous development of diabetes in ZDF rats results in neuroglia morphologic degenerative changes at 24 weeks of age. This animal model may be useful to understand the pathogenesis of diabetic retinopathy and to screen neuroprotective drugs in diabetes.

Meningioma Associated with Brain Aneurysm: Report of Two Cases.

The occurrence of brain tumors together with aneurysms has been considered as an uncommon phenomenon. However, its incidence may be underestimated. This coexistence is a diagnostic challenge, but also a therapeutic one as no consensus has been reached. We report two cases of a 71 and 67 years old patient with a meningioma and aneurysm: one noticed and treated before with good outcome and the other without treatment before surgery with fatal outcome. The different outcome of these patients shows the importance of vascular study in surgical planning. Treatments options are changing and although some authors think the pathology that causes symptoms should be treated first, endovascular treatment of the aneurysm is a safe option to prevent aneurysm rupture during surgery.

Analysis of endovascular treatment of ruptured aneurysms of the middle cerebral artery compared to other anatomical locations.

OBJECTIVE: The objective of this article is to compare the results of endovascular treatment of ruptured middle cerebral artery (MCA) aneurysms with ruptured aneurysms of other anatomic locations. METHODS: Fifty consecutive ruptured aneurysms of the MCA and 209 aneurysms at other anatomical locations were selected retrospectively. We compared epidemiological, clinical and radiological variables, prognosis and complications. RESULTS: The MCA aneurysms had a greater size and a poor dome/neck ratio. There were no significant differences in endovascular technique complications, occlusion rate or rebleeding between the two groups (p > 0.1). There were no significant differences in the mortality and number of dependent patients after one month. CONCLUSION: The endovascular treatment of ruptured MCA aneurysms without hematoma is as safe and effective as other aneurysm localizations. Complication rates, occlusion rates and rebleeding of ruptured MCA aneurysms are comparable to other locations.

Inflammatory Cytokine-Mediated Regulation of Thrombospondin-1 and CD36 in Conjunctival Cells.

PURPOSE: Increased expression of transforming growth factor-ß2 (TGF-ß2) is reported in the conjunctiva of dry eye patients with no increase of anti-inflammatory activity of TGF-ß2. Our aim was to compare the expression of molecules involved in TGF-ß2 activation, thrombospondin-1 (TSP-1) and CD36, during murine and human conjunctival inflammation. METHODS: Human conjunctival tissue from cadaveric donors, human conjunctival epithelial primary cells and fibroblasts, and murine conjunctivas were immunostained for TSP-1, CD36, or TGF-ß2. Inflamed conjunctival tissues were obtained from C57BL/6 wild-type (WT) mice induced to develop experimental dry eye (EDE) with 10 days of desiccating conditions and scopolamine injections and TSP-1-deficient (TSP1(-/-)) mice, which spontaneously develop Sjögren's syndrome-associated conjunctival inflammation with age. Immunostaining intensities were compared using ImageJ software. Cultures of human conjunctival fibroblasts were stimulated with IL-1ß and both secreted protein and message levels of TSP-1, CD36, and TGF-ß2 were analyzed. RESULTS: TSP-1 and CD36 were detectable in human and murine conjunctival tissues as well as primary conjunctival epithelial cells and fibroblasts. Increased conjunctival immunostaining of TGF-ß2 and reduced CD36 were detected in EDE mice compared with WT mice. Interestingly, increased TGF-ß2 and CD36 conjunctival immunostaining was detected in TSP1(-/-) mice. The expression of TSP-1 and CD36 was downregulated in IL-1ß-stimulated conjunctival fibroblasts at both the protein and message level, while active TGF-ß2 was undetected. CONCLUSIONS: The absence or reduced expression of either of the molecules involved in TGF-ß2 activation supports proinflammatory conditions in the conjunctiva. Changes in TSP-1 and CD36 may serve as potential biomarkers of conjunctival inflammation.