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Mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) cause Blau syndrome, an inflammatory disorder characterized by uveitis. The antimicrobial functions of Nod2 are well-established, yet the cellular mechanisms by which dysregulated Nod2 causes uveitis remain unknown. Here, we report a non-conventional, T cell-intrinsic function for Nod2 in suppression of Th17 immunity and experimental uveitis. Reconstitution of lymphopenic hosts with Nod2−/− CD4+ T cells or retina-specific autoreactive CD4+ T cells lacking Nod2 reveals a T cell-autonomous, Rip2-independent mechanism for Nod2 in uveitis. In naive animals, Nod2 operates downstream of TCR ligation to suppress activation of memory CD4+ T cells that associate with an autoreactive-like profile involving IL-17 and Ccr7. Interestingly, CD4+ T cells from two Blau syndrome patients show elevated IL-17 and increased CCR7. Our data define Nod2 as a T cell-intrinsic rheostat of Th17 immunity, and open new avenues for T cell-based therapies for Nod2-associated disorders such as Blau syndrome., How mutations in the microbial receptor NOD2 induce Blau syndrome in humans and related uveitis is unclear. Here the authors show, using Nod2-deficient mice and experimental uveitis, that Nod2 negatively regulates T cell activation and transcription of autoimmunity-related genes to suppress Th17 responses and uveitis.

The landscape of fear is an important driver of prey space use. However, prey can navigate the landscape of fear by exploiting temporal refuges from predation risk. We hypothesized that diel patterns of predator and prey movement and space use would be inversely correlated due to temporal constraints on predator habitat domain. Specifically, we evaluated habitat selection and activity of the vicuña and its only predator, the puma, during three diel periods: day, dawn/dusk, and night. Pumas selected the same habitats regardless of diel period-vegetated and rugged areas that feature stalking cover for pumas-but increased their activity levels during dawn/dusk and night when they benefit from reduced detection by prey. Vicuñas avoided areas selected by pumas and reduced activity at night, but selected vegetated areas and increased activity by day and dawn/dusk. Vicuña habitat selection and movement strategies appeared to reduce the risk of encountering pumas; movement rates of pumas and vicuñas were negatively correlated across the diel cycle, and habitat selection was negatively correlated during dawn/dusk and night. Our study shows that an ambush predator's temporal activity and space use patterns interact to create diel refugia and shape the antipredator behaviors of its prey. Importantly, it is likely the very nature of ambush predators' static habitat specificity that makes predator activity important to temporally varying perceptions of risk. Prey which depend on risky habitats for foraging appear to mitigate risk by feeding when they can more easily detect predators and when predators are least active.

Human activity has rapidly transformed the planet, leading to declines of animal populations around the world through a range of direct and indirect pathways. Humans have strong numerical effects on wild animal populations, as highly efficient hunters and through unintentional impacts of human activity and development. Human disturbance also induces costly non-lethal effects by changing the behavior of risk-averse animals. Here, we suggest that the unique strength of these lethal and non-lethal effects is amplified by mismatches between the nature of risk associated with anthropogenic stimuli and the corresponding response by wild animals. We discuss the unique characteristics of cues associated with anthropogenic stimuli in the context of animal ecology and evolutionary history to explore why and when animals fail to appropriately (a) detect, (b) assess, and (c) respond to both benign and lethal stimuli. We then explore the costs of over-response to a benign stimulus (Type I error) and under-response to a lethal stimulus (Type II error), which can scale up to affect individual fitness and ultimately drive population dynamics and shape ecological interactions. Finally, we highlight avenues for future research and discuss conservation measures that can better align animal perception and response with risk to mitigate unintended consequences of human disturbance.

Species assemblages often have a non-random nested organization, which in vertebrate scavenger (carrion-consuming) assemblages is thought to be driven by facilitation in competitive environments. However, not all scavenger species play the same role in maintaining assemblage structure, as some species are obligate scavengers (i.e., vultures) and others are facultative, scavenging opportunistically. We used a database with 177 vertebrate scavenger species from 53 assemblages in 22 countries across five continents to identify which functional traits of scavenger species are key to maintaining the scavenging network structure. We used network analyses to relate ten traits hypothesized to affect assemblage structure with the “role” of each species in the scavenging assemblage in which it appeared. We characterized the role of a species in terms of both the proportion of monitored carcasses on which that species scavenged, or scavenging breadth (i.e., the species “normalized degree”), and the role of that species in the nested structure of the assemblage (i.e., the species “paired nested degree”), therefore identifying possible facilitative interactions among species. We found that species with high olfactory acuity, social foragers, and obligate scavengers had the widest scavenging breadth. We also found that social foragers had a large paired nested degree in scavenger assemblages, probably because their presence is easier to detect by other species to signal carcass occurrence. Our study highlights differences in the functional roles of scavenger species and can be used to identify key species for targeted conservation to maintain the ecological function of scavenger assemblages., ESG, ZMR, JMB and LNA were supported by Generalitat Valenciana (SEJI/2018/024, APOSTD/2019/016, CIDEGENT/2020/030 and ACIF/2019/056, respectively), JMB, JMPG and CGC by Juan de la Cierva contracts (MEC; IJCI-2017-32149, FJCI-2015-25632 and IJC2018-036642-I, respectively), ACA by the Govern de les Illes Balears (PD/039/2017) and ESG and MM by Ramón y Cajal contracts (MEC; RYC-2019-027216-I, RYC-2015-19231). EA was supported by La Caixa-Severo Ochoa International PhD Program 2015. NS was partly supported by the National Science Centre in Poland (2013/08/M/NZ9/00469 and 2016/22/Z/NZ8/00). MK and KJ were supported by the Slovenian Research Agency (P4-0059). Contributions of HG, KLT, EFA, OER, TLD, and JCB were partially supported through funding from U.S. Department of Agriculture and the U.S. Department of Energy under (DE-EM0004391) to the University of Georgia Research Foundation. HG was also supported by the Institute of Environmental Radioactivity at Fukushima University. ALB and JDA were partially supported by Queens College and the Graduate Center at the City University of New York. JDA is currently supported by a Ramón y Cajal contract (RYC-2017-22783). ERB and EF were supported by the USA National Science Foundation Graduate Research Fellowship (1256065). CK completed study with support from Hawk Mountain Sanctuary, The Peregrine Fund, and via Pompeo M. Maresi Memorial Fund via Princeton University. JAS and CCW were supported by the USA National Science Foundation #1255913, the American Association for University Women, and the Gordon and Betty Moore Foundation. HUW acknowledges funding from the California Department of Fish & Wildlife (P0880013). PLP was supported by the Rufford Foundation and University of Wisconsin-Madison. JB and JdT thank the Percy Sladen Memorial Fund and Mr Rodney Fuhr. Several authors were funded by funds from the MEC (CGL2012-40013-C02-01/02, CGL2015-66966-C2-1-R, CGL2015-66966-C2-1-R2, CGL2017-89905-R, RTI2018-099609-B-C21 and RTI2018-099609-B-C22) and from the Junta de Andalucía (RNM-1925). POV was supported by a research contract by the University of Lleida. EES and TMN were funded and supported by Australian Geographic, Bush Heritage Australia, Australian Academy of Sciences, Ecological Society of Australia, NSW Office of Environment and Heritage, NESP Threatened Species Recovery Hub, Emirates Wolgan Valley One and Only Resort. EP, YA, MS and UY completed study under research permit by The Republic of Turkey Ministry of Agriculture and Forestry. SAL thanks PICT (BID) 0725/2014, and IAATE. ALB and JDA would like to thank the Edmund Niles Huyck Preserve, Lyme Adirondack Forest Company, and LandVest Timberland.

During recent Zika epidemics, adults infected with Zika virus (ZIKV) have developed organ-specific inflammatory complications. The most serious Zika-associated inflammatory eye disease is uveitis, which is commonly anterior in type, affecting both eyes and responding to corticosteroid eye drops. Mechanisms of Zika-associated anterior uveitis are unknown, but ZIKV has been identified in the aqueous humor of affected individuals. The iris pigment epithelium is a target cell population in viral anterior uveitis, and it acts to maintain immune privilege within the anterior eye. Interactions between ZIKV and human iris pigment epithelial cells were investigated with infectivity assays and RNA-sequencing. Primary cell isolates were prepared from eyes of 20 cadaveric donors, and infected for 24 hours with PRVABC59 strain ZIKV or incubated uninfected as control. Cytoimmunofluorescence, RT-qPCR on total cellular RNA, and focus-forming assays of culture supernatant showed cell isolates were permissive to infection, and supported replication and release of infectious ZIKV. To explore molecular responses of cell isolates to ZIKV infection at the whole transcriptome level, RNA was sequenced on the Illumina NextSeq 500 platform, and results were aligned to the human GRCh38 genome. Multidimensional scaling showed clear separation between transcriptomes of infected and uninfected cell isolates. Differential expression analysis indicated a vigorous molecular response of the cell to ZIKV: 7,935 genes were differentially expressed between ZIKV-infected and uninfected cells (FDR < 0.05), and 99% of 613 genes that changed at least two-fold were up-regulated. Reactome and KEGG pathway and Gene Ontology enrichment analyses indicated strong activation of viral recognition and defense, in addition to biosynthesis processes. A CHAT network included 6275 molecular nodes and 24 contextual hubs in the cell response to ZIKV infection. Receptor-interacting serine/threonine kinase 1 (RIPK1) was the most significantly connected contextual hub. Correlation of gene expression with read counts assigned to the ZIKV genome identified a negative correlation between interferon signaling and viral load across isolates. This work represents the first investigation of mechanisms of Zika-associated anterior uveitis using an in vitro human cell model. The results suggest the iris pigment epithelium mounts a molecular response that limits intraocular pathology in most individuals.

Understanding the distribution of biodiversity across the Earth is one of the most challenging questions in biology. Much research has been directed at explaining the species latitudinal pattern showing that communities are richer in tropical areas; however, despite decades of research, a general consensus has not yet emerged. In addition, global biodiversity patterns are being rapidly altered by human activities. Here, we aim to describe large-scale patterns of species richness and diversity in terrestrial vertebrate scavenger (carrion-consuming) assemblages, which provide key ecosystem functions and services. We used a worldwide dataset comprising 43 sites, where vertebrate scavenger assemblages were identified using 2,485 carcasses monitored between 1991 and 2018. First, we evaluated how scavenger richness (number of species) and diversity (Shannon diversity index) varied among seasons (cold vs. warm, wet vs. dry). Then, we studied the potential effects of human impact and a set of macroecological variables related to climatic conditions on the scavenger assemblages. Vertebrate scavenger richness ranged from species-poor to species rich assemblages (4–30 species). Both scavenger richness and diversity also showed some seasonal variation. However, in general, climatic variables did not drive latitudinal patterns, as scavenger richness and diversity were not affected by temperature or rainfall. Rainfall seasonality slightly increased the number of species in the community, but its effect was weak. Instead, the human impact index included in our study was the main predictor of scavenger richness. Scavenger assemblages in highly human-impacted areas sustained the smallest number of scavenger species, suggesting human activity may be overriding other macroecological processes in shaping scavenger communities. Our results highlight the effect of human impact at a global scale. As species-rich assemblages tend to be more functional, we warn about possible reductions in ecosystem functions and the services provided by scavengers in human-dominated landscapes in the Anthropocene., SG, JMPG, and ACA were supported by Juan de la Cierva contracts (Ministerio de Economía y Competitividad, MEC; IJCI‐2015‐24947, FJCI‐2015‐25632, and IJCI‐2014‐20744, respectively). ESG was also supported by Generalitat Valenciana (SEJI/2018/024), ACA by the Govern de les Illes Balears (PD/039/2017), and MM by a Ramón y Cajal contract (MEC; RYC‐2015‐19231). EA was supported by La Caixa‐Severo Ochoa International PhD Program 2015. NS was partly supported by the National Science Centre in Poland (2013/08/M/NZ9/00469). SAL thanks PICT (BID) 0725/2014. MK and KJ were supported by the Slovenian Research Agency (P4‐0059). Contributions of KT and JCB were partially supported through funding from U.S. Department of Agriculture, the U.S. Department of Energy under (DE‐EM0004391) to the University of Georgia Research Foundation. EB and EF were supported by the USA National Science Foundation. CK completed study under research permit NCST/5/002/R/448 with support from Hawk Mountain Sanctuary, The Peregrine Fund, and via Pompeo M. Maresi Memorial Fund via Princeton University. JAS and CCW were supported by the USA National Science Foundation #1255913 and the Gordon and Betty Moore Foundation. Several authors were funded by funds from the MEC (CGL2012‐40013‐C02‐01/02, CGL2015‐66966‐C2‐1‐R and CGL2017‐89905‐R) and from the Junta de Andalucía (RNM‐1925).

Ocular toxoplasmosis is a retinitis -almost always accompanied by vitritis and choroiditis- caused by intraocular infection with Toxoplasma gondii. Depending on retinal location, this condition may cause substantial vision impairment. T. gondii is an obligate intracellular protozoan parasite, with both sexual and asexual life cycles, and infection is typically contracted orally by consuming encysted bradyzoites in undercooked meat, or oocysts on unwashed garden produce or in contaminated water. Presently available anti-parasitic drugs cannot eliminate T. gondii from the body. In vitro studies using T. gondii tachyzoites, and human retinal cells and tissue have provided important insights into the pathogenesis of ocular toxoplasmosis. T. gondii may cross the vascular endothelium to access human retina by at least three routes: in leukocyte taxis; as a transmigrating tachyzoite; and after infecting endothelial cells. The parasite is capable of navigating the human neuroretina, gaining access to a range of cell populations. Retinal Muller glial cells are preferred initial host cells. T. gondii infection of the retinal pigment epithelial cells alters the secretion of growth factors and induces proliferation of adjacent uninfected epithelial cells. This increases susceptibility of the cells to parasite infection, and may be the basis of the characteristic hyperpigmented toxoplasmic retinal lesion. Infected epithelial cells also generate a vigorous immunologic response, and influence the activity of leukocytes that infiltrate the retina. A range of T. gondii genotypes are associated with human ocular toxoplasmosis, and individual immunogenetics -including polymorphisms in genes encoding innate immune receptors, human leukocyte antigens and cytokines- impacts the clinical manifestations. Research into basic pathogenic mechanisms of ocular toxoplasmosis highlights the importance of prevention and suggests new biological drug targets for established disease.

Diseases that involve retinal or choroidal vascular endothelial cells are leading causes of vision loss: age-related macular degeneration, retinal ischemic vasculopathies, and noninfectious posterior uveitis. Proteins differentially expressed by these endothelial cell populations are potential drug targets. We used deep proteomic profiling to define the molecular phenotype of human retinal and choroidal endothelial cells at the protein level.Retinal and choroidal vascular endothelial cells were separately isolated from 5 human eye pairs by selection on CD31. Total protein was extracted and digested, and peptide fractions were analyzed by reverse-phase liquid chromatography tandem mass spectrometry. Peptide sequences were assigned to fragment ion spectra, and proteins were inferred from openly accessible protein databases. Protein abundance was determined by spectral counting. Publicly available software packages were used to identify proteins that were differentially expressed between human retinal and choroidal endothelial cells, and to classify proteins that were highly abundant in each endothelial cell population.Human retinal and/or choroidal vascular endothelial cells expressed 5042 nonredundant proteins. Setting the differential expression false discovery rate at 0.05, 498 proteins of 3454 quantifiable proteins (14.4%) with minimum mean spectral counts of 2.5 were differentially abundant in the 2 cell populations. Retinal and choroidal endothelial cells were enriched in angiogenic proteins, and retinal endothelial cells were also enriched in immunologic proteins.This work describes the different protein expression profiles of human retinal and choroidal vascular endothelial cells, and provides multiple candidates for further study as novel treatments or drug targets for posterior eye diseases. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Vitreoretinal lymphoma, which most commonly is diffuse large B-cell non-Hodgkin in type, is a rare cancer with high morbidity and high mortality. Making a tissue diagnosis of vitreoretinal lymphoma is a major challenge for clinicians due to biological and technical factors. Yet, the delay in start of treatment may have vision- and life-threatening consequences, and there is considerable interest in the application of molecular assays to improve the accuracy of the diagnostic process: detection of a clonal immunoglobulin heavy-chain rearrangements in lymphoma cells by polymerase chain reaction; measurement of vitreous or aqueous interleukin-10 protein levels in ocular fluids; and identification of mutations in the myeloid differentiation primary response gene 88 in tumour cells. In this article, we review the historical development and current application of each of these molecular methods. We also discuss future opportunities for the molecular diagnosis of vitreoretinal lymphoma through next-generation sequencing technologies.

BACKGROUND: Human retinal endothelial cells are employed increasingly for investigations of retinal vascular diseases. Analysis of gene expression response to disease-associated stimuli by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is common. However, most reported work does not follow the minimum information for publication of qPCR experiments (MIQE) recommendation that multiple, stably expressed reference genes be used for normalization. METHODS: Two human retinal endothelial cell lines were treated with medium alone or containing stimuli that included: glucose at supraphysiological concentration, dimethyloxalyl-glycine, vascular endothelial growth factor, tumor necrosis factor-α, lipopolysaccharide and Toxoplasma gondii tachyzoites. Biological response of cells was confirmed by measuring significant increase in a stimulus-relevant transcript. Total RNA was reverse transcribed and analyzed by commercial PCR arrays designed to detect 28 reference genes. Stability of reference gene expression, for each and both cell lines, and for each and all conditions, was judged on gene-stability measure (M-value) less than 0.2 and coefficient of variation (CV-value) less than 0.1. RESULTS: Reference gene expression varied substantially across stimulations and between cell lines. Of 27 detectable reference genes, 11–21 (41–78%) maintained expression stability across stimuli and cell lines. Ranking indicated substantial diversity in the most stable reference genes under different conditions, and no reference gene was expressed stably under all conditions of stimulation and for both cell lines. Four reference genes were expressed stably under 5 conditions: HSP90AB1, IPO8, PSMC4 and RPLPO. CONCLUSIONS: We observed variation in stability of reference gene expression with different stimuli and between human retinal endothelial cell lines. Our findings support adherence to MIQE recommendations regarding normalization in RT-qPCR studies of human retinal endothelial cells.

Background Over-production of reactive oxygen species (ROS) and resulting oxidative stress contribute to retinal damage in vascular diseases that include diabetic retinopathy, retinopathy of prematurity and major retinal vessel occlusions. NADPH oxidase (Nox) proteins are professional ROS-generating enzymes, and therapeutic targeting in these diseases has strong appeal. Pharmacological inhibition of Nox4 reduces the severity of experimental retinal vasculopathy. We investigated the potential application of this drug approach in humans. Methods Differential Nox enzyme expression was studied by real-time-quantitative polymerase chain reaction in primary human retinal endothelial cell isolates and a characterized human retinal endothelial cell line. Oxidative stress was triggered chemically in endothelial cells, by treatment with dimethyloxalylglycine (DMOG; 100 μM); Nox4 and vascular endothelial growth factor (VEGFA) transcript were measured; and production of ROS was detected by 2',7'-dichlorofluorescein. DMOG-stimulated endothelial cells were treated with two Nox1/Nox4 inhibitors, GKT136901 and GKT137831; cell growth was monitored by DNA quantification, in addition to VEGFA transcript and ROS production. Results Nox4 (isoform Nox4A) was the predominant Nox enzyme expressed by human retinal endothelial cells. Treatment with DMOG significantly increased endothelial cell expression of Nox4 over 72 h, accompanied by ROS production and increased VEGFA expression. Treatment with GKT136901 or GKT137831 significantly reduced DMOG-induced ROS production and VEGFA expression by endothelial cells, and the inhibitory effect of DMOG on cell growth. Conclusions Our findings in experiments on activated human retinal endothelial cells provide translational corroboration of studies in experimental models of retinal vasculopathy and support the therapeutic application of Nox4 inhibition by GKT136901 and GKT137831 in patients with retinal vascular diseases.

Summary Uveitis (intraocular inflammation) is a leading cause of loss of vision. Although its aetiology is largely speculative, it is thought to arise from complex genetic–environmental interactions that break immune tolerance to generate eye-specific autoreactive T cells. Experimental autoimmune uveitis (EAU), induced by immunization with the ocular antigen, interphotoreceptor retinoid binding protein (IRBP), in combination with mycobacteria-containing complete Freund's adjuvant (CFA), has many clinical and histopathological features of human posterior uveitis. Studies in EAU have focused on defining pathogenic CD4+ T cell effector responses, such as those of T helper type 17 (Th17) cells, but the innate receptor pathways precipitating development of autoreactive, eye-specific T cells remain poorly defined. In this study, we found that fungal-derived antigens possess autoimmune uveitis-promoting function akin to CFA in conventional EAU. The capacity of commensal fungi such as Candida albicans or Saccharomyces cerevisae to promote IRBP-triggered EAU was mediated by Card9. Because Card9 is an essential signalling molecule of a subgroup of C-type lectin receptors (CLRs) important in host defence, we evaluated further the proximal Card9-activating CLRs. Using single receptor-deficient mice we identified Dectin-2, but not Mincle or Dectin-1, as a predominant mediator of fungal-promoted uveitis. Conversely, Dectin-2 activation by α-mannan reproduced the uveitic phenotype of EAU sufficiently, in a process mediated by the Card9-coupled signalling axis and interleukin (IL)-17 production. Taken together, this report relates the potential of the Dectin-2/Card9-coupled pathway in ocular autoimmunity. Not only does it contribute to understanding of how innate immune receptors orchestrate T cell-mediated autoimmunity, it also reveals a previously unappreciated ability of fungal-derived signals to promote autoimmunity.

Summary How animals respond to anthropogenic disturbances is a core component of conservation biology and how they respond to predators and competitors is equally of central importance to wildlife ecology. Camera traps have rapidly become a critical tool in wildlife research by providing a fully automated means of observing animals without needing an observer present, permitting data to be collected on rare or elusive species and infrequent events. Snapshots from camera traps revealing a species’ presence have been the principal data used to date to gauge behaviour; but, lacking experimental controls, such data permit only correlational analyses potentially open to confounding effects. Playback experiments provide a powerful means to directly test the behavioural responses of animals, enabling strong inferences and rigorous conclusions not subject to the potential confounds affecting the interpretation of snapshot data; the principal factor to date limiting the use of playback experiments being the need to have an observer present. We developed an Automated Behavioural Response system (ABR) comprising a custom-built motion-sensitive speaker system that can be paired with any commercially available camera trap, providing the means to conduct playback experiments directly testing the behavioural responses of any species that can be ‘caught’ on a camera trap. We describe field tests in Uganda, Canada and the USA, experimentally testing the effects of anthropogenic disturbances and interactions among large carnivores, in species as diverse as elephants, black bears, chimpanzees and cougars; experiments that would be completely infeasible without the ABR. We evaluate factors affecting the rate of successful data collection in the experiments in Uganda and Canada, and detail how we maximized the system's performance in the USA experiment. By integrating the power playback experiments provide to directly and rigorously test behavioural responses with the capacity camera trapping affords to study virtually any animal anywhere, the ABR can both greatly expand the range of research questions addressed by conservation biologists and wildlife ecologists and qualitatively improve the rigour of the resulting conclusions. We discuss various ways to optimize the ABR's performance in any circumstance, and the many novel research opportunities made available by this new methodology.

Domestic dogs are the most abundant large carnivore on the planet, and their ubiquity has led to concern regarding the impacts of dogs as predators of and competitors with native wildlife. If native large carnivores perceive dogs as threatening, impacts could extend to the community level by altering interactions between large carnivores and their prey. Dog impacts may be further exacerbated if these human-associated predators are also perceived as indicators of risk from humans. However, observational approaches used to date have led to ambiguity regarding the effects of dog presence on wildlife. We experimentally quantified dog impacts on the behavior of a native large carnivore, presenting playbacks of dog vocalizations to pumas in central California. We show that the perceived presence of dogs has minimal impacts on puma behavior at their kill sites, and is no more likely to affect total feeding time at kills than non-threatening controls. We previously demonstrated that pumas exhibit strong responses to human cues, and here show that perceived risk from human presence far exceeds that from dogs. Our results suggest that protected areas management policies that restrict dogs but permit human access may in some cases be of limited value for large carnivores.

Dengue virus (DENV) infection is associated with clinical ocular presentations and here DENV infection of the eye was assessed in mice. In an AG129 mouse model of antibody-dependent enhancement of DENV infection, DENV RNA was detected in the eye and vascular changes were present in the retinae. Intraocular CD8 and IFN-γ mRNA were increased in mice born to DENV-naive, but not DENV-immune mothers, while TNF-α mRNA was induced and significantly higher in mice born to DENV-immune than DENV-naive mothers. DENV RNA was detected in the eye following intracranial DENV infection and CD8 mRNA but not IFN-γ nor TNF-α were induced. In all models, viperin was increased following DENV infection. Thus, DENV in the circulation or the brain can infect the eye and stimulate innate immune responses, with induction of viperin as one response that consistently occurs in multiple DENV eye-infection models in both an IFN-dependent and independent manner.

The most common human disease caused by infection with Toxoplasma gondii is ocular toxoplasmosis, which typically is manifest as recurrent attacks of necrotizing retinal inflammation with subsequent scarring. The multilayered retina contains specialized cell populations, including endothelial cells, epithelial cells, neurons and supporting cells, all of which may be involved in this condition. In vitro investigations of basic mechanisms operating in human ocular toxoplasmosis use cellular and molecular methods that are common to the study of many pathological processes, and the novel aspect of this research is the use of human retinal cell subsets. Most in vivo research on ocular toxoplasmosis is conducted in the laboratory mouse. Experimental models involve local or systemic inoculation of parasites to induce acute disease, or sequential systemic and local parasite inoculations to trigger recurrent disease. We present methods for in vitro and in vivo studies of ocular toxoplasmosis, including dissection of the human eye, and culture and infection of differentiated cell populations from the retina, as well as induction of mouse ocular toxoplasmosis by intraocular, or sequential systemic and intraocular, inoculations, and imaging of toxoplasmic retinal lesions.

Purpose Retinal damage in ocular toxoplasmosis reflects Toxoplasma gondii-induced cell lysis and reactive inflammation. Human retinal histopathology demonstrates the presence of neutrophils, but activities of this leukocyte subset are unstudied. We conducted in vitro experiments to evaluate roles for neutrophils as retinal taxis for T. gondii and as contributors to the inflammation. Methods Human neutrophils were isolated from peripheral blood. Migration to disease-relevant chemokines was evaluated in transwells, seeded with human retinal endothelial cells for some assays, using neutrophils infected with GT-1 strain T. gondii tachyzoites. Neutrophils were cocultured with T. gondii-infected ARPE-19 and primary human retinal pigment epithelial cells, and production of reactive oxygen species (ROS) was estimated by dihydroethidium reaction. Proteins produced by T. gondii-infected ARPE-19 cells were profiled by immunoarray, and candidate neutrophil-activating proteins were targeted with specific blocking antibody in coculture assays. Results Infection with T. gondii arrested neutrophil migration across retinal endothelium regardless of the presence of CXCL8. Migration to CXCL1, CXCL2, and CXCL8 also was significantly inhibited in infected neutrophils. Neutrophils generated more ROS when cocultured with infected versus uninfected ARPE-19 cells and three of four primary retinal pigment epithelial cell isolates. Infected ARPE-19 cells augmented the synthesis of 12 neutrophil-activating proteins also expressed by primary retinal pigment epithelial cells. Antibody blockade of granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6) and IL-18 significantly reduced ROS production by neutrophils cocultured with T. gondii-infected ARPE-19 cells. Conclusions Our findings support involvement of neutrophils in retinal inflammation, but not parasite transport, in the setting of ocular toxoplasmosis.

Predator-prey games emerge when predators and prey dynamically respond to the behavior of one another, driving the outcomes of predator-prey interactions. Predation success is a function of the combined probabilities of encountering and capturing prey, which are influenced by both prey behavior and environmental features. While the relative importance of encounter and capture probabilities have been evaluated in a spatial framework, temporal variation in prey behavior and intrinsic catchability are likely to also affect the distribution of predation events. Using a single-predator-single-prey (puma-vicuna) system, we evaluated which factors predict predation events across both temporal and spatial dimensions of the components of predation by testing the prey-abundance hypothesis (predators select for high encounter probability) and the prey-catchability hypothesis (predators select for high relative capture probability) in time and space. We found that for both temporal and spatial analyses, neither the prey-abundance hypothesis nor the prey-catchability hypothesis alone predicted kill frequency or distribution; puma kill frequency was static throughout the diel cycle and pumas consistently selected a single habitat type when hunting, despite temporal and spatial variation in encounter rates and intrinsic catchability. Our integrated spatiotemporal analysis revealed that an interaction between time of day and habitat influences kill probability, suggesting that trade-offs in the temporal and spatial components of predation drive the probability of predation events. These findings reinforce the importance of examining both the temporal and spatial patterns of the components of predation, rather than unidimensional measures of predator or prey behavior, to comprehensively describe the feedbacks between predator and prey in the predator-prey game.

Retinal infection with Toxoplasma gondii&mdash, ocular toxoplasmosis&mdash, is a common cause of vision impairment worldwide. Pathology combines parasite-induced retinal cell death and reactive intraocular inflammation. M&uuml, ller glial cells, which represent the supporting cell population of the retina, are relatively susceptible to infection with T. gondii. We investigated expression of long non-coding RNAs (lncRNAs) with immunologic regulatory activity in M&uuml, ller cells infected with virulent T. gondii strains&mdash, GT1 (haplogroup 1, type I) and GPHT (haplogroup 6). We first confirmed expression of 33 lncRNA in primary cell isolates. MIO-M1 human retinal M&uuml, ller cell monolayers were infected with T. gondii tachyzoites (multiplicity of infection = 5) and harvested at 4, 12, 24, and 36 h post-infection, with infection being tracked by the expression of parasite surface antigen 1 (SAG1). Significant fold-changes were observed for 31 lncRNAs at one or more time intervals. Similar changes between strains were measured for BANCR, CYTOR, FOXD3-AS1, GAS5, GSTT1-AS1, LINC-ROR, LUCAT1, MALAT1, MIR22HG, MIR143HG, PVT1, RMRP, SNHG15, and SOCS2-AS1. Changes differing between strains were measured for APTR, FIRRE, HOTAIR, HOXD-AS1, KCNQ1OT1, LINC00968, LINC01105, lnc-SGK1, MEG3, MHRT, MIAT, MIR17HG, MIR155HG, NEAT1, NeST, NRON, and PACER. Our findings suggest roles for lncRNAs in regulating retinal M&uuml, ller cell immune responses to T. gondii, and encourage future studies on lncRNA as biomarkers and/or drug targets in ocular toxoplasmosis.

Objective Survivors of Ebola virus disease (EVD) are at risk of developing blinding intraocular inflammation—or uveitis—which is associated with retinal pigment epithelial (RPE) scarring and persistence of live Zaire ebolavirus (EBOV) within the eye. As part of a large research project aimed at defining the human RPE cell response to being infected with EBOV, this work focused on the microRNAs (miRNAs) associated with the infection. Results Using RNA-sequencing, we detected 13 highly induced and 2 highly repressed human miRNAs in human ARPE-19 RPE cells infected with EBOV, including hsa-miR-1307-5p, hsa-miR-29b-3p and hsa-miR-33a-5p (up-regulated), and hsa-miR-3074-3p and hsa-miR-27b-5p (down-regulated). EBOV-miR-1-5p was also found in infected RPE cells. Through computational identification of putative miRNA targets, we predicted a broad range of regulatory activities, including effects on innate and adaptive immune responses, cellular metabolism, cell cycle progression, apoptosis and autophagy. The most highly-connected molecule in the miR-target network was leucine-rich repeat kinase 2, which is involved in neuroinflammation and lysosomal processing. Our findings should stimulate new studies on the impact of miRNA changes in EBOV-infected RPE cells to further understanding of intraocular viral persistence and the pathogenesis of uveitis in EVD survivors.

Ocular toxoplasmosis is the commonest clinical manifestation of infection with obligate intracellular parasite, Toxoplasma gondii. Active ocular toxoplasmosis is characterized by replication of T. gondii tachyzoites in the retina, with reactive inflammation. The multifunctional retinal pigment epithelium is a key target cell population for T. gondii. Since the global gene expression profile is germane to understanding molecular involvements of retinal pigment epithelial cells in ocular toxoplasmosis, we performed RNA-Sequencing (RNA-Seq) of human cells following infection with T. gondii tachyzoites. Primary cell isolates from eyes of cadaveric donors (n = 3), and the ARPE-19 human retinal pigment epithelial cell line, were infected for 24 h with GT-1 strain T. gondii tachyzoites (multiplicity of infection = 5) or incubated uninfected as control. Total and small RNA were extracted from cells and sequenced on the Illumina NextSeq 500 platform; results were aligned to the human hg19 reference sequence. Multidimensional scaling showed good separation between transcriptomes of infected and uninfected primary cell isolates, which were compared in edgeR software. This differential expression analysis revealed a sizeable response in the total RNA transcriptome—with significantly differentially expressed genes totaling 7,234 (28.9% of assigned transcripts)—but very limited changes in the small RNA transcriptome—totaling 30 (0.35% of assigned transcripts) and including 8 microRNA. Gene ontology and pathway enrichment analyses of differentially expressed total RNA in CAMERA software, identified a strong immunologic transcriptomic signature. We conducted RT-qPCR for 26 immune response-related protein-coding and long non-coding transcripts in epithelial cell isolates from different cadaveric donors (n = 3), extracted by a different isolation protocol but similarly infected with T. gondii, to confirm immunological activity of infected cells. For microRNA, increases in miR-146b and miR-212 were detected by RT-qPCR in 2 and 3 of these independent cell isolates. Biological network analysis in the InnateDB platform, including 735 annotated differentially expressed genes plus 2,046 first-order interactors, identified 10 contextural hubs and 5 subnetworks in the transcriptomic immune response of cells to T. gondii. Our observations provide a solid base for future studies of molecular and cellular interactions between T. gondii and the human retinal pigment epithelium to illuminate mechanisms of ocular toxoplasmosis.

When a person becomes infected with Toxoplasma gondii, ocular toxoplasmosis is the most common clinical presentation. The medical literature describes retinitis with surrounding hyperpigmentation secondary to proliferative changes in the retinal pigment epithelium, which is sufficiently characteristic that investigation often is not needed to make the diagnosis. We aimed to establish the frequency of &ldquo, typical&rdquo, ocular toxoplasmosis and delineate its molecular basis. Among 263 patients presenting consecutively with ocular toxoplasmosis to Ribeir&atilde, o Preto General Hospital in Brazil, where T. gondii infection is endemic, 94.2% of 345 eyes had retinal hyperpigmentation. In ARPE-19 and primary human retinal pigment epithelial cell monolayers exposed to minimal numbers of T. gondii tachyzoites, the proliferation marker&ndash, KI-67&ndash, was increased in uninfected cells, which also were rendered more susceptible to infection. RT-qPCR and ELISA detected increased expression of vascular endothelial growth factor A (VEGF) and insulin-like growth factor (IGF)1, and decreased expression of thrombospondin (TSP)1 by infected cells. Blockade of VEGF and IGF1&mdash, or supplementation of TSP1&mdash, reversed the proliferation phenotype in uninfected cells. Our findings confirm that hyperpigmentation is a characteristic feature of retinitis in ocular toxoplasmosis, and demonstrate that T. gondii-infected human retinal pigment epithelial cells secrete VEGF and IGF1, and reduce production of TSP1, to promote proliferation of adjacent uninfected cells and create this disease-specific appearance.

B cells participate in diverse retinal immunopathologies. Endothelial adhesion molecules and chemokines direct leukocyte trafficking. We examined the involvement of three molecular signals in retinal transendothelial migration of human B cells: ICAM-1, VCAM-1, and CXCL13.Peripheral blood B cells were isolated by negative selection. Migration was studied in transwells populated with human retinal endothelial monolayers, using antibody to block ICAM-1 or VCAM-1. Retinal expression of CXCL13 was investigated.B cells crossed retinal endothelium. ICAM-1 blockade significantly reduced migration when results for all subjects were combined, and for a majority when results were analyzed by individual. This effect was irrespective of the presence or absence of CXCL13, although CXCL13 increased migration. CXCL13 was detected in neural retina and retinal pigment epithelium. Endothelial cells of some retinal vessels presented CXCL13 protein.ICAM-1 blockade may be an effective treatment in some patients with retinal diseases that involve B cells.

Understanding how anthropogenic development affects food webs is essential to implementing sustainable growth measures, yet little is known about how the spatial configuration of residential development affects the foraging behavior and prey habits of top predators. We examined the influence of the spatial characteristics of residential development on prey composition in the puma (Puma concolor). We located the prey remains of kills from 32 pumas fitted with global positioning system (GPS) satellite collars to determine the housing characteristics most influencing prey size and species composition. We examined how differences in housing density, proximity, and clustering influenced puma prey size and diversity. We found that at both local (150 m) and regional (1 km) spatial scales surrounding puma kill sites, housing density (but not the clustering of housing) was the greatest contributor to puma consumption of small prey (

Recently recognized forms of uveitis include intraocular inflammations that occur during or following one of several emerging infectious diseases: chikungunya fever, dengue, Zika virus disease and Ebola virus disease. Anterior, intermediate, posterior and pan-uveitis have been described in individuals infected with chikungunya virus. Persons who contract dengue or Zika viruses also may develop different types of uveitis in the course of the infection: maculopathy is a common manifestation of dengue eye disease, and Zika eye disease may cause hypertensive anterior uveitis or mimic a white dot syndrome. Up to one-third of Ebola survivors develop aggressive uveitis, which is frequently associated with vision loss and complicated by cataract. There are no specific anti-viral drugs for these forms of uveitis, and thus treatment is largely supportive. In this article, we summarize the systemic infectious diseases and virology, and describe the clinical presentations, outcomes and management of emerging viral forms of uveitis.

Severe dengue virus (DENV) infection is associated with overactivity of the complement alternative pathway (AP) in patient studies. Here, the molecular changes in components of the AP during DENV infection in vitro were investigated. mRNA for factor H (FH), a major negative regulator of the AP, was significantly increased in DENV-infected endothelial cells (EC) and macrophages, but, in contrast, production of extracellular FH protein was not. This discord was not seen for the AP activator factor B (FB), with DENV induction of both FB mRNA and protein, nor was it seen with Toll-like receptor 3 or 4 stimulation of EC and macrophages, which induces both FH and FB mRNA and protein. Surface-bound and intracellular FH protein was, however, induced by DENV, but only in DENV antigen-positive cells, while in two other DENV-susceptible immortalized cell lines (ARPE-19 and human retinal endothelial cells), FH protein was induced both intracellularly and extracellularly by DENV infection. Regardless of the cell type, there was an imbalance in AP components and an increase in markers of complement AP activity associated with DENV-infected cells, with lower FH relative to FB protein, an increased ability to promote AP-mediated lytic activity, and increased deposition of complement component C3b on the surface of DENV-infected cells. For EC in particular, these changes are predicted to result in higher complement activity in the local cellular microenvironment, with the potential to induce functional changes that may result in increased vascular permeability, a hallmark of dengue disease. IMPORTANCE Dengue virus (DENV) is a significant human viral pathogen with a global medical and economic impact. DENV may cause serious and life-threatening disease, with increased vascular permeability and plasma leakage. The pathogenic mechanisms underlying these features remain unclear; however, overactivity of the complement alternative pathway has been suggested to play a role. In this study, we investigate the molecular events that may be responsible for this observed alternative pathway overactivity and provide novel findings of changes in the complement system in response to DENV infection in primary cell types that are a major target for DENV infection (macrophages) and pathogenesis (endothelial cells) in vivo . Our results suggest a new dimension of cellular events that may influence endothelial cell barrier function during DENV infection that could expand strategies for developing therapeutics to prevent or control DENV-mediated vascular disease.

Objective Regulation of intercellular adhesion molecule (ICAM)-1 in retinal endothelial cells is a promising druggable target for retinal vascular diseases. The ICAM-1-related (ICR) long non-coding RNA stabilizes ICAM-1 transcript, increasing protein expression. However, studies of ICR involvement in disease have been limited as the promoter is uncharacterized. To address this issue, we undertook a comprehensive in silico analysis of the human ICR gene promoter region. Results We used genomic evolutionary rate profiling to identify a 115 base pair (bp) sequence within 500 bp upstream of the transcription start site of the annotated human ICR gene that was conserved across 25 eutherian genomes. A second constrained sequence upstream of the orthologous mouse gene (68 bp; conserved across 27 Eutherian genomes including human) was also discovered. Searching these elements identified 33 matrices predictive of binding sites for transcription factors known to be responsive to a broad range of pathological stimuli, including hypoxia, and metabolic and inflammatory proteins. Five phenotype-associated single nucleotide polymorphisms (SNPs) in the immediate vicinity of these elements included four SNPs (i.e. rs2569693, rs281439, rs281440 and rs11575074) predicted to impact binding motifs of transcription factors, and thus the expression of ICR and ICAM-1 genes, with potential to influence disease susceptibility. We verified that human retinal endothelial cells expressed ICR, and observed induction of expression by tumor necrosis factor-α. Electronic supplementary material The online version of this article (10.1186/s13104-018-3384-8) contains supplementary material, which is available to authorized users.

Large carnivores' fear of the human ‘super predator’ has the potential to alter their feeding behaviour and result in human-induced trophic cascades. However, it has yet to be experimentally tested if large carnivores perceive humans as predators and react strongly enough to have cascading effects on their prey. We conducted a predator playback experiment exposing pumas to predator (human) and non-predator control (frog) sounds at puma feeding sites to measure immediate fear responses to humans and the subsequent impacts on feeding. We found that pumas fled more frequently, took longer to return, and reduced their overall feeding time by more than half in response to hearing the human ‘super predator’. Combined with our previous work showing higher kill rates of deer in more urbanized landscapes, this study reveals that fear is the mechanism driving an ecological cascade from humans to increased puma predation on deer. By demonstrating that the fear of humans can cause a strong reduction in feeding by pumas, our results support that non-consumptive forms of human disturbance may alter the ecological role of large carnivores.

Purpose Success of Ebola virus (EBOV) as a human pathogen relates at the molecular level primarily to blockade the host cell type I interferon (IFN) antiviral response. Most individuals who survive Ebola virus disease (EVD) develop a chronic disease syndrome: approximately one-quarter of survivors suffer from uveitis, which has been associated with presence of EBOV within the eye. Clinical observations of post-Ebola uveitis indicate involvement of retinal pigment epithelial cells. Methods We inoculated ARPE-19 human retinal pigment epithelial cells with EBOV, and followed course of infection by immunocytochemistry and measurement of titer in culture supernatant. To interrogate transcriptional responses of infected cells, we combined RNA sequencing with in silico pathway, gene ontology, transcription factor binding site, and network analyses. We measured infection-induced changes of selected transcripts by reverse transcription-quantitative polymerase chain reaction. Results Human retinal pigment epithelial cells were permissive to infection with EBOV, and supported viral replication and release of virus in high titer. Unexpectedly, 28% of 560 upregulated transcripts in EBOV-infected cells were type I IFN responsive, indicating a robust type I IFN response. Following EBOV infection, cells continued to express multiple immunomodulatory molecules linked to ocular immune privilege. Conclusions Human retinal pigment epithelial cells may serve as an intraocular reservoir for EBOV, and the molecular response of infected cells may contribute to the persistence of live EBOV within the human eye. Translational relevance This bedside-to-bench research links ophthalmic findings in survivors of EVD who suffer from uveitis with interactions between retinal pigment epithelial cells and EBOV.

Human development strongly influences large carnivore survival and persistence globally. Behavior changes are often the first measureable responses to human disturbances, and can have ramifications on animal populations and ecological communities. We investigated how a large carnivore responds to anthropogenic disturbances by measuring activity, movement behavior, and energetics in pumas along a housing density gradient. We used log-linear analyses to examine how habitat, time of day, and proximity to housing influenced the activity patterns of both male and female pumas in the Santa Cruz Mountains. We used spatial GPS location data in combination with Overall Dynamic Body Acceleration measurements recorded by onboard accelerometers to quantify how development density affected the average distances traveled and energy expended by pumas. Pumas responded to development differently depending on the time of day; at night, they were generally more active and moved further when they were in developed areas, but these relationships were not consistent during the day. Higher nighttime activity in developed areas increased daily caloric expenditure by 10.1% for females and 11.6% for males, resulting in increases of 3.4 and 4.0 deer prey required annually by females and males respectively. Our results support that pumas have higher energetic costs and resource requirements in human-dominated habitats due to human-induced behavioral change. Increased energetic costs for pumas are likely to have ramifications on prey species and exacerbate human-wildlife conflict, especially as exurban growth continues. Future conservation work should consider the consequences of behavioral shifts on animal energetics, individual fitness, and population viability.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited., Recent clinical reports indicate that infection with dengue virus (DENV) commonly has ocular manifestations. The most serious threat to vision is dengue retinopathy, including retinal vasculopathy and macular edema. Mechanisms of retinopathy are unstudied, but observations in patients implicate retinal pigment epithelial cells and retinal endothelial cells. Human retinal cells were inoculated with DENV-2 and monitored for up to 72 hours. Epithelial and endothelial cells supported DENV replication and release, but epithelial cells alone demonstrated clear cytopathic effect, and infection was more productive in those cells. Infection induced type I interferon responses from both cells, but this was stronger in epithelial cells. Endothelial cells increased expression of adhesion molecules, with sustained overexpression of vascular adhesion molecule-1. Transcellular impedance decreased for epithelial monolayers, but not endothelial monolayers, coinciding with cytopathic effect. This reduction was accompanied by disorganization of intracellular filamentous-actin and decreased expression of junctional molecules, zonula occludens 1, and catenin-β1. Changes in endothelial expression of adhesion molecules are consistent with the retinal vasculopathy seen in patients infected with DENV; decreases in epithelial junctional protein expression, paralleling loss of integrity of the epithelium, provide a molecular basis for DENV-associated macular edema. These molecular processes present potential therapeutic targets for vision-threatening dengue retinopathy.

Caching decisions have been studied for many species, but large-scale variation of selective preferences due to environmental heterogeneity has rarely been examined. We investigated large-scale patterns of selective caching behavior in the American pika (Ochotona princeps), a non-hibernating generalist herbivore that caches vegetation in haypiles to serve as winter food. At 13 sites throughout the southern Rocky Mountains, we identified the three most common available and cached plant species and analyzed them for dietary quality. Selectivity at each site was measured as the difference between average quality of the most common cached and available vegetation. Pikas consistently cached materials of higher quality than the most common available vegetation. Selectivity for high nitrogen corresponded to quality of available plants and site summer temperature, whereas selectivity for high water content was negatively correlated with elevation. Our results indicate that pikas cache a variety of plant ...

Retinal endothelial cells line the arborizing microvasculature that supplies and drains the neural retina. The anatomical and physiological characteristics of these endothelial cells are consistent with nutritional requirements and protection of a tissue critical to vision. On the one hand, the endothelium must ensure the supply of oxygen and other nutrients to the metabolically active retina, and allow access to circulating cells that maintain the vasculature or survey the retina for the presence of potential pathogens. On the other hand, the endothelium contributes to the blood-retinal barrier that protects the retina by excluding circulating molecular toxins, microorganisms, and pro-inflammatory leukocytes. Features required to fulfill these functions may also predispose to disease processes, such as retinal vascular leakage and neovascularization, and trafficking of microbes and inflammatory cells. Thus, the retinal endothelial cell is a key participant in retinal ischemic vasculopathies that include diabetic retinopathy and retinopathy of prematurity, and retinal inflammation or infection, as occurs in posterior uveitis. Using gene expression and proteomic profiling, it has been possible to explore the molecular phenotype of the human retinal endothelial cell and contribute to understanding of the pathogenesis of these diseases. In addition to providing support for the involvement of well-characterized endothelial molecules, profiling has the power to identify new players in retinal pathologies. Findings may have implications for the design of new biological therapies. Additional progress in this field is anticipated as other technologies, including epigenetic profiling methods, whole transcriptome shotgun sequencing, and metabolomics, are used to study the human retinal endothelial cell.

Targeting the endothelial adhesion molecules that control leukocyte trafficking into a tissue has been explored as a biological therapy for inflammatory diseases. However, these molecules also participate in leukocyte migration for immune surveillance, and inhibiting the physiological level of an adhesion molecule might promote infection or malignancy. We explored the concept of targeting endothelial adhesion molecule transcription during inflammation in a human system. Intercellular adhesion molecule 1 (ICAM-1) mediates leukocyte migration across the retinal endothelium in noninfectious posterior uveitis. We observed an increase in the transcription factor, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NF-κB1), in parallel with ICAM-1, in human retinal endothelial cells treated with tumor necrosis factor-alpha (TNF-α), and identified putative binding sites for NF-κB1 within theICAM-1regulatory region. We targeted induced NF-κB1 expression in endothelial cells with small interfering (si)RNA. Knockdown of NF-κB1 significantly decreased cell surface expression of ICAM-1 protein induced by TNF-αbut did not reduce constitutive ICAM-1 expression. Consistently, NF-κB1 knockdown significantly reduced leukocyte binding to cell monolayers in the presence of TNF-αbut did not impact baseline binding. Findings of this proof-of-concept study indicate that induced transcription of endothelial adhesion molecules might be targeted therapeutically for inflammatory disease in humans.

Activated leukocyte cell adhesion molecule (ALCAM; CD166) is an immunoglobulin superfamily member that has been described in several non-ocular endothelial populations, but not in relation to endothelium within the eye. Studies in extraocular systems have implicated ALCAM in angiogenesis and leukocyte transendothelial migration, which are key processes in retinal vascular diseases. We investigated the expression of ALCAM in human retinal endothelium, and studied the regulation of expression by established angiogenic and inflammatory stimuli. Retinal endothelial expression of ALCAM was detected in primary retinal endothelial cultures isolated from human cadavers by RT-PCR (n = 4 donors) and Western blot (n = 4 donors), and in intact human retina by immunohistochemistry (n = 3 donors). In the 4 donors studied by RT-PCR, transcript encoding the truncated soluble isoform, sALCAM, was also detected. Quantitative real-time RT-PCR demonstrated significant up-regulation of ALCAM and sALCAM in response to stimulation with master cytokine, tumor necrosis factor (TNF)-α. However, general inflammatory stimulus, lipopolysaccharide (LPS), and the prototype Th1, Th2 and Th17 cytokines, interferon (IFN)-γ, interleukin (IL)-4 and IL-17A, respectively, did not impact ALCAM or sALCAM expression. In contrast, expression of ALCAM was significantly up-regulated by vascular endothelial growth factor (VEGF)(165). Up-regulation in the presence of VEGF and TNF-α, but not LPS, IFN-γ, IL-4 and IL-17A, suggests a potential role for ALCAM in human retinal angiogenesis in some settings.

The term, ocular toxoplasmosis, refers to eye disease related to infection with the parasite, Toxoplasma gondii. Recurrent posterior uveitis is the typical form of this disease, characterized by unilateral, necrotizing retinitis with secondary choroiditis, occurring adjacent to a pigmented retinochoroidal scar and associated with retinal vasculitis and vitritis. Multiple atypical presentations are also described, and severe inflammation is observed in immunocompromised patients. Histopathological correlations demonstrate focal coagulative retinal necrosis, and early in the course of the disease, this inflammation is based in the inner retina. For typical ocular toxoplasmosis, a diagnosis is easily made on clinical examination. In atypical cases, ocular fluid testing to detect parasite DNA by polymerase chain reaction or to determine intraocular production of specific antibody may be extremely helpful for establishing aetiology. Given the high seroprevalence of toxoplasmosis in most communities, serological testing for T. gondii antibodies is generally not useful. Despite a lack of published evidence for effectiveness of current therapies, most ophthalmologists elect to treat patients with ocular toxoplasmosis that reduces or threatens to impact vision. Classic therapy consists of oral pyrimethamine and sulfadiazine, plus systemic corticosteroid. Substantial toxicity of this drug combination has spurred interest in alternative antimicrobials, as well as local forms of drug delivery. At this time, however, no therapeutic approach is curative of ocular toxoplasmosis.

Ocular toxoplasmosis results from retinal infection with the protozoan, Toxoplasma gondii. This parasite, which exists as multiple clonal subpopulations and in three stages, is capable of replication in any nucleated cell of its primary feline or multiple paratenic hosts. Human seroprevalence of toxoplasmosis is high across the globe, but with geographic variation. While prevalence of ocular toxoplasmosis is not well documented, toxoplasmic retinochoroiditis is the commonest form of posterior uveitis in many countries. Correlation of parasite genotype with disease is an important area of new research. Ocular infection with T. gondii often follows ingestion of bradyzoites in undercooked infected meat. Oocysts may survive for an extended period in the environment, and water contaminated with oocysts is an important source in toxoplasmosis epidemics. Ocular toxoplasmosis is preventable by a combination of community activities and personal measures. Public health action is well justified by the considerable burden of congenital and postnatal infections.

Retinal infection is the most common clinical manifestation of toxoplasmosis. The route by which circulating Toxoplasma gondii tachyzoites cross the vascular endothelium to enter the human retina is unknown. Convincing studies using murine encephalitis models have strongly implicated leukocyte taxis as one pathway used by the parasite to access target organs. To establish whether tachyzoites might also interact directly with vascular endothelium, we populated a transwell system with human ocular endothelial cells. Human retinal endothelial monolayers permitted transmigration of tachyzoites of RH and three natural isolate strains. Antibody blockade of intercellular adhesion molecule-1 significantly reduced this migration, but did not impact tachyzoite movement across an endothelial monolayer derived from the choroid, which lies adjacent to the retina within the eye. In demonstrating that tachyzoites are capable of independent migration across human vascular endothelium in vitro, this study carries implications for the development of therapeutics aimed at preventing access of tachyzoites to the retina.

Endothelial cells form the lining of the vasculature. Despite the continuity of this layer throughout the body, endothelial cells exhibit remarkable heterogeneity in structure, molecular composition and activity: between sites; and in response to different exposures. One important consequence of endothelial diversity is the localized nature of many vascu- lar disorders. To date a limited number of studies have attempted to define unique phenotypic features of the different in- traocular endothelial subpopulations, which include the endothelial cells of vascular beds in the iris, the choroid and the retina. Differences that distinguish endothelial cells in the circulations of the choroid and the retina, in particular, are be- lieved to be major etiological factors controlling the specific involvement of the two tissues in some of the most common blinding diseases. Age-related macular degeneration involves choroid, and diabetic retinopathy and posterior uveitis are primarily diseases of the retina. Development of effective targeted therapies for these ocular disorders will require a de- tailed understanding of the heterogeneity of ocular endothelia. Our review summarizes the existing literature relating to diversity of the ocular endothelial cells. We highlight structural, metabolic and functional characteristics that distinguish intraoocular endothelial subtypes from each other and from extraocular endothelial cells, and we consider the implications of these differences for the design of novel biological therapeutics for eye diseases.

Cultured endothelial cells of the human retina and choroid demonstrate distinct patterns of gene expression. We hypothesized that differential gene expression reflected differences in the interactions of transcription factors and respective cis-regulatory motifs(s) in these two endothelial cell subpopulations, recognizing that motifs often exist as modules. We tested this hypothesis in silico by using TRANSFAC Professional and CisModule to identify cis-regulatory motifs and modules in genes that were differentially expressed by human retinal versus choroidal endothelial cells, as identified by analysis of a microarray data set. Motifs corresponding to eight transcription factors were significantly (p

The abundance and distribution of Rainbow (Trichoglossus haematodus) and Musk (Glossopsitta concinna) Lorikeets in Melbourne have increased markedly in recent years. We studied the feeding ecology ...

We show that inhibitory effect of interleukin-13 on endotoxin-induced uveitis in the Lewis rat is dependent on signaling activity of protein kinase Cζ (PKCζ). To understand the effect of interleukin-13 or PKCζ inhibitor treatment, the activation status of rat bone marrow-derived macrophages was studied in vitro. At 6 hours, lipopolysaccharide-stimulated macrophages produced tumor necrosis factor-α (TNF-α) with nuclear factor κB (NF-κB)/p65 expression. Treatment led to absence of NF-κB/p65 expression and low levels of TNF-α, suggesting accelerated inactivation of macrophages. At 24 hours after lipopolysaccharide stimulation, nuclear NF-κB/p65 decreased and nuclear NF-κB/p50 increased, associated with nuclear BCL-3 and a low level of TNF-α, indicating onset of spontaneous resolution. Treatment limited PKCζ cleavage, with expression of nuclear NF-κB/p50 and BCL-3 and low nuclear NF-κB/p65 promoting macrophage survival, as evidenced by Bcl-2 expression. At 24 hours, intraocular treatment decreased membranous expression of PKCζ by ocular cells, reduced vascular leakage with low nitric-oxide synthase-2 expression in vascular endothelial cells, and limited inflammatory cell infiltration with decreased intraocular TNF-α, interleukin-6, and nitric-oxide synthase-2 mRNA. Importantly, treatment decreased nuclear NF-κB/p65, increased transforming growth factor-β2, and reduced caspase 3 expression in infiltrating macrophages, implying a change of their phenotype within ocular microenvironment. Treatment accelerated endotoxin-induced uveitis resolution through premature apoptosis of neutrophils related to high expression of toll-like receptor 4 and caspase 3.

Non-infectious uveitis—or intraocular inflammatory disease—causes substantial visual morbidity and reduced quality of life amongst affected individuals. To date, research of pathogenic mechanisms has largely been focused on processes involving T lymphocyte and/or myeloid leukocyte populations. Involvement of B lymphocytes has received relatively little attention. In contrast, B-cell pathobiology is a major field within general immunological research, and large clinical trials have showed that treatments targeting B cells are highly effective for multiple systemic inflammatory diseases. B cells, including the terminally differentiated plasma cell that produces antibody, are found in the human eye in different forms of non-infectious uveitis; in some cases, these cells outnumber other leukocyte subsets. Recent case reports and small case series suggest that B-cell blockade may be therapeutic for patients with non-infectious uveitis. As well as secretion of antibody, B cells may promote intraocular inflammation by presentation of antigen to T cells, production of multiple inflammatory cytokines and support of T-cell survival. B cells may also perform various immunomodulatory activities within the eye. This translational review summarizes the evidence for B-cell involvement in non-infectious uveitis, and considers the potential contributions of B cells to the development and control of the disease. Manipulations of B cells and/or their products are promising new approaches to the treatment of non-infectious uveitis.

Mate selection influences individual fitness, is often based on complex cues and behaviours, and can be difficult to study in solitary species including carnivores. We used motion-triggered cameras at 29 community scrapes (i.e. scent marking locations used by multiple individuals) and home range data from 39 GPS-collared pumas (Puma concolor) to assess the relevance of communication behaviours for mate selection by female pumas in California. Female pumas visited community scrapes irregularly and visitation bouts appeared to be correlated with oestrus. Female pumas on average selected from 1.7 collared males, and selection was based on multiple cues that varied among the different time periods measured (i.e. the female’s visitation bout and in 90 days previous to the consorting event). Female mate selection over the course of a visitation bout was based on frequency of the male visitation, mass, and age. In the 90 days previous to consorting, the number of scrapes a male created was the most important contributor to selection, which was likely related to his residency status. We also found that at least 14% of females mated with multiple males, thus possibly confusing paternity. Our findings provide a mechanistic understanding of how female pumas use scent and auditory communication at community scrapes to select dominant resident males to mate with.

The fear induced by predators on their prey is well known to cause behavioural adjustments by prey that can ripple through food webs. Little is known, however, about the analogous impacts of humans as perceived top predators on the foraging behaviour of carnivores. Here, we investigate the influence of human-induced fear on puma foraging behaviour using location and prey consumption data from 30 tagged individuals living along a gradient of human development. We observed strong behavioural responses by female pumas to human development, whereby their fidelity to kill sites and overall consumption time of prey declined with increasing housing density by 36 and 42%, respectively. Females responded to this decline in prey consumption time by increasing the number of deer they killed in high housing density areas by 36% over what they killed in areas with little residential development. The loss of food from declines in prey consumption time paired with increases in energetic costs associated with killing more prey may have consequences for puma populations, particularly with regard to reproductive success. In addition, greater carcass availability is likely to alter community dynamics by augmenting food resources for scavengers. In light of the extensive and growing impact of habitat modification, our study emphasizes that knowledge of the indirect effects of human activity on animal behaviour is a necessary component in understanding anthropogenic impacts on community dynamics and food web function.

In the human host, infection with the protozoan parasite, Toxoplasma gondii, most commonly involves the eye and/or the brain. Previous work indicates a relative susceptibility of the human retinal vascular endothelium to infection with the T. gondii tachyzoite, which may contribute to this tissue localization. To facilitate the investigation of potential adhesive interactions between parasite and endothelium in the retina, we have modified the Woodruff-Stamper assay, originally described to study lymphocytic-endothelial binding. Vascular endothelium was identified in sections of human retina by Alexa Fluor 594-tagged anti-von Willebrand factor antibody. Binding of yellow fluorescent protein-expressing tachyzoites to endothelium under conditions of flow, simulated by rotation on an orbital shaker, was quantified in a masked fashion using imaging software. We observed multiple yellow spots in contact with Alexa Fluor 594-positive retina, indicating binding of T. gondii tachyzoites to retinal vascular endothelium. This modification of the Woodruff-Stamper assay provides an opportunity to evaluate potential host receptors for T. gondii on the retinal vascular endothelium. In addition, the assay suggests a methodology that could be used to examine adhesion of other microbes to microvasculature in different tissues.

Objective Uveitis or intraocular inflammation is a major cause of visual loss. Acute anterior uveitis (AAU) affects ∼40% of patients with ankylosing spondylitis (AS) but also affects patients with no evidence of spondylarthritis. We sought to determine whether a unique genetic region could be implicated in a specific manifestation—AAU—of a multisystem, inflammatory, genetically complex disease, AS. Methods Individuals from families multiplex for AAU were genotyped at 400 markers representing the ABI PRISM linkage map MD-10, and at the HLA–B, DRB1, DQA1, DQB1, and DPB1 alleles. Among the family members with AAU, 76 affected sibpairs were analyzed (6 without concomitant AS, 12 discordant for AS, and 58 concordant for AS). Two-point and multipoint nonparametric linkage analyses were performed, and 1-parameter allele-sharing model logarithm of odds (LOD) scores were determined. Results As previously reported for AS, linkage at the major histocompatibility complex region (chromosome 6p21) was evident, exhibiting the highest multipoint LOD score (4.96 at marker HLA–B). Strong linkage was seen at a region on chromosome 9p21–9p24, with a LOD score of 3.72 at marker D9S157. When compared with a companion cohort of AS families, the linkage at this region was found in association with AAU but not with AS. A third region on chromosome 1q23–1q31 was observed to have suggestive linkage (LOD 2.05 at marker D1S238), which overlaps with a region associated with AS. Conclusion This is the first study in which a genetic region for AAU has been identified by genome-wide scan. Even though AS was highly prevalent in this cohort of families, a locus at chromosome 9p21–9p24 was identified that uniquely associates with AAU. Identifying the genetic perturbation at this region may advance our understanding of the mechanisms involved in tissue-specific pathology of complex inflammatory diseases.