A Case for Below-Ground Dispersal? Insights into the Biology, Ecology and Conservation of Blind Cave Spiders in the Genus Troglodiplura (Mygalomorphae: Anamidae).

Previously described from only fragments of exoskeleton and juvenile specimens, the cave spider genus Troglodiplura (Araneae: Anamidae), endemic to the Nullarbor Plain, is the only troglomorphic member of the infraorder Mygalomorphae recorded from Australia. We investigated the distribution of Troglodiplura in South Australia, collecting and observing the first (intact) mature specimens, widening the number of caves it has been recorded in, and documenting threats to conservation. Phylogenetic analyses support the placement of Troglodiplura as an independent lineage within the subfamily Anaminae (the 'Troglodiplura group') and provide unequivocal evidence that populations from apparently isolated cave systems are conspecifics of T. beirutpakbarai Harvey & Rix, 2020, with extremely low or negligible inter-population mitochondrial divergences. This is intriguing evidence for recent or contemporary subterranean dispersal of these large, troglomorphic spiders. Observations of adults and juvenile spiders taken in the natural cave environment, and supported by observations in captivity, revealed the use of crevices within caves as shelters, but no evidence of silk use for burrow construction, contrasting with the typical burrowing behaviours seen in other Anamidae. We identify a range of threats posed to the species and to the fragile cave ecosystem, and provide recommendations for further research to better define the distribution of vulnerable taxa within caves and identify actions needed to protect them.

ICAM-1-related long non-coding RNA: promoter analysis and expression in human retinal endothelial cells.

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.

Molecular Signals Involved in Human B Cell Migration into the Retina: In Vitro Investigation of ICAM-1, VCAM-1, and CXCL13.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: ICAM-1 blockade may be an effective treatment in some patients with retinal diseases that involve B cells.

Involvement of B cells in non-infectious uveitis.

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.

Bilateral giant retinal tears in a pediatric patient with leukemia inhibitory factor receptor deficiency (Stuve-Wiedemann syndrome).

PURPOSE: To describe a case of retinal detachment in a patient with Stuve-Wiedemann syndrome. METHODS: This report is a retrospective observational case report. The patient's demographics include age, gender, and race, as well as visual acuity, ophthalmic examination, and surgical intervention were extracted from the medical record. For immunohistochemistry studies, a sample of normal human retina from an enucleated specimen was obtained from the Pathology laboratory. A leukemia inhibitory factor receptor/CD118 antibody was obtained from Santa Cruz Biotechnology. RESULTS: A 13-year-old Hispanic boy with known history of Stuve-Wiedemann syndrome (confirmed by genetic testing) presented with bilateral rhegmatogenous retinal detachments secondary to bilateral giant retinal tears. He underwent multiple surgical repairs in both eyes, resulting in successful reattachment in the right eye and an intractable closed funnel detachment in the left eye. CONCLUSION: This is the first case of vitreoretinal pathology reported in Stuve-Wiedemann syndrome. Using immunohistochemistry staining, the authors found ubiquitous expression of leukemia inhibitory factor receptor protein in the normal human retina. They hypothesize that leukemia inhibitory factor receptor mutation may cause intrinsic weakness of the neurosensory retina predisposing it to injury.

Simultaneous visualization and cell-specific confirmation of RNA and protein in the mouse retina.

PURPOSE: Simultaneous dual labeling to visualize specific RNA and protein content within the same formalin-fixed paraffin embedded (FFPE) section can be technically challenging and usually impossible, because of variables such as tissue fixation time and pretreatment methods to access the target RNA or protein. Within a specific experiment, ocular tissue sections can be a precious commodity. Thus, the ability to easily and consistently detect and localize cell-specific expression of RNA and protein within a single slide would be advantageous. In this study, we describe a simplified and reliable method for combined in situ hybridization (ISH) and immunohistochemistry (IHC) for detection of mRNA and protein, respectively, within the same FFPE ocular tissue. METHODS: Whole mouse eyes were prepared for 5 micron FFPE sections after fixation for 3, 24, 48 or 72 h. Customized probes from Advanced Cell Diagnostics to detect mRNA for vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1-alpha (HIF-1α), and hypoxia-inducible factor 2-alpha (HIF-2α) were used for ISH. Various parameters were tested using the novel RNAscope method for ISH and optimized for compatibility with subsequent IHC for glial fibrillary acidic protein (GFAP) or GS-lectin within the same tissue section. Dual fluorescent visualization of Fast Red ISH and Alexa Fluor 488 IHC signal was observed with confocal microscopy. RESULTS: A fixation time of 72 h was found to be optimal for ISH and subsequent IHC. The RNAscope probes for VEGF, HIF-1α, and HIF-2α mRNA all gave a strong Fast Red signal with both 48 h and 72 h fixed tissue, but the optimal IHC signal for either GFAP or GS-lectin within a retinal tissue section after ISH processing was observed with 72 h fixation. A pretreatment boiling time of 15 min and a dilution factor of 1:15 for the pretreatment protease solution were found to be optimal and necessary for successful ISH visualization with 72 h FFPE ocular tissue. CONCLUSIONS: The protocol presented here provides a simple and reliable method to simultaneously detect mRNA and protein within the same paraffin-embedded ocular tissue section. The procedure, after preparation of FFPE sections, can be performed over a 2-day or 4-day period. We provide an optimization strategy that may be adapted for any RNAscope probe set and antibody for determining retinal or ocular cell-specific patterns of expression.

Role of the retinal vascular endothelial cell in ocular disease.

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.

Imaging Retinal Vascular Changes in the Mouse Model of Oxygen-Induced Retinopathy.

PURPOSE: Oxygen-induced retinopathy in the mouse is the standard experimental model of retinopathy of prematurity. Assessment of the pathology involves in vitro analysis of retinal vaso-obliteration and retinal neovascularization. The authors studied the clinical features of oxygen-induced retinopathy in vivo using topical endoscopy fundus imaging (TEFI), in comparison to standard investigations, and evaluated a system for grading these features. METHODS: Postnatal day (P)7 mice were exposed to 75% oxygen for five days to induce retinopathy or maintained in room air as controls. Retinal vascular competence was graded against standard photographs by three masked graders. Retinal photographs were obtained at predetermined ages using TEFI. Postmortem, retinal vaso-obliteration was measured in whole mounts with labeled vasculature, and retinal neovascularization was quantified in hematoxylin- and eosin-stained ocular cross sections. RESULTS: Fundus photography by TEFI was possible from P15, when retinal vascular incompetence, including dilatation and tortuosity, was significant in mice with oxygen-induced retinopathy in comparison to controls. Vascular incompetence peaked in severity at P17 and persisted through P25. Comparison with in vitro analyses indicated that vascular changes were most severe after retinal avascularity had begun to decrease in area, and coincident with the maximum of retinal neovascularization. A weighted Fleiss-Cohen kappa indicated good intra- and interobserver agreement for a 5-point grading system. CONCLUSIONS: Topical endoscopy fundus imaging demonstrates retinal vascular incompetence in mice with oxygen-induced retinopathy. The technique complements standard postmortem analysis for following the course of the model. TRANSLATIONAL RELEVANCE: Topical endoscopy fundus imaging has application in the evaluation of novel biologic drugs for retinopathy of prematurity.

Altered vascular expression of EphrinB2 and EphB4 in a model of oxygen-induced retinopathy.

EphrinB2 ligands and EphB4 receptors are expressed on endothelial cells (EC) of arteries and veins, respectively, and are essential for vascular development. To understand how these molecules regulate retinal neovascularization (NV), we evaluated their expression in a model of oxygen-induced retinopathy (OIR). EphrinB2 and EphB4 were expressed on arterial and venous trunks, respectively, and on a subset of deep capillary vessels. EphB4 expression was reduced following hyperoxia, while ephrinB2 expression remained unaltered. In addition, a subset of EphB4-positive veins regressed in a caspase-3-dependent manner during hyperoxia. Arteriovenous malformations were also observed with loss of arterial-venous boundaries. Finally, both ephrinB2 and EphB4 were expressed on a subset of neovascular tufts following hyperoxia. These data confirm the contribution of ECs from both venous and arterial origins to the development of retinal NV.

TRAIL-deficient mice exhibit delayed regression of retinal neovascularization.

While it is well established that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various cell types, the role of TRAIL in regulation of retinal neovascularization (NV) has not been described. Here we determined the role of TRAIL in retinal NV during oxygen-induced retinopathy using TRAIL deficient ((-/-)) mice. TRAIL and its receptor, DR5, were expressed in wild-type retinas at all time points evaluated (postnatal days 12, 17, 21, 24) during oxygen-induced retinopathy and in age-matched room air control animals. Localization of TRAIL(+) cells within the neovascular tufts of hyperoxia- exposed wild-type mice suggested TRAIL plays a role in oxygen-induced retinopathy. Retinal vascular development appeared normal in the TRAIL(-/-) mice, except for a small but significant difference in the capillary-free zone surrounding major arteries. A minimal difference in avascularity was observed at postnatal day 12 in the retinas of TRAIL(-/-) mice after hyperoxia-exposure compared with wild-type mice, suggesting that TRAIL does not play a major role in the vaso-obliterative phase of oxygen-induced retinopathy. However, at the peak of NV, TRAIL(-/-) mice had a significant increase in retinal neovascularization. In addition, when NV naturally regresses in wild-type mice, TRAIL(-/-) mice continued to display significantly high levels of NV. This was attributed to a significant decrease in neovascular tuft cells undergoing apoptosis in TRAIL(-/-) mice. Together, these data strongly suggest that TRAIL plays a role in the control of retinal NV.

MCP-1 deficiency delays regression of pathologic retinal neovascularization in a model of ischemic retinopathy.

PURPOSE: The present study investigates whether retinal neovascularization (NV) and apoptosis are altered in MCP-1-deficient ((-/-)) mice in the OIR model. METHODS: Postnatal day (P) 7 MCP-1(-/-) and C57BL/6 (B6) mice were exposed to 75% oxygen for 5 days and then recovered in room air. Immunostaining was performed to localize macrophages/microglia within retinal whole mounts and cross-sections. Retinopathy was qualitatively assessed in FITC-dextran-perfused retinas, and preretinal NV was quantified on P17, P21, and P24. TUNEL analysis was used to compare apoptosis between B6 and MCP-1(-/-) mice. RESULTS: MCP-1(-/-) and B6 mice revealed normal vascular development in room air controls and similar vaso-obliteration in oxygen-exposed mice on P12. MCP-1(-/-) mice exhibited significantly reduced vascular tuft-associated F4/80(+) cells compared with B6 mice. FITC-dextran-perfused retinas exhibited prominent neovascular tufts on P17, and quantification of preretinal nuclei revealed no significant differences between MCP-1(-/-) and B6 mice. In contrast, on P21 and P24, MCP-1(-/-) mice exhibited significant increases in preretinal neovascular nuclei compared with B6 controls. These increases in NV in the MCP-1(-/-) mice were associated with a significant reduction in vascular tuft apoptosis. CONCLUSIONS: The results demonstrate that the absence of MCP-1 does not alter normal retinal vascular development. Furthermore, MCP-1(-/-) mice exhibit a similar neovascular response on P17. However, the reduction in tuft-associated macrophages/microglia in the MCP-1(-/-) mice correlates with reduced vascular tuft apoptosis and delayed regression of retinal NV. These findings suggest that macrophages/microglia may contribute to tuft regression through their proapoptotic properties.