Flow cytometric analysis of inflammatory and resident myeloid populations in mouse ocular inflammatory models

Myeloid cells make a pivotal contribution to tissue homeostasis during inflammation. Both tissue-specific resident populations and infiltrating myeloid cells can cause tissue injury through aberrant activation and/or dysregulated activity. Reliable identification and quantification of myeloid cells within diseased tissues is important to understand pathological inflammatory processes. Flow cytometry is a valuable technique for leukocyte analysis, but a standardized flow cytometric method for myeloid cell populations in the eye is lacking. Here, we validate a reproducible flow cytometry gating approach to characterize myeloid cells in several commonly used models of ocular inflammation. We profile and quantify myeloid subsets across these models, and highlight the value of this strategy in identifying phenotypic differences using Ccr2-deficient mice. This method will aid standardization in the field and facilitate future investigations into the roles of myeloid cells during ocular inflammation

Early-Onset Progressive Degeneration of the Area Centralis in RPE65-Deficient Dogs.

PURPOSE: Retinal epithelium-specific protein 65 kDa (RPE65)-deficient dogs are a valuable large animal model species that have been used to refine gene augmentation therapy for Leber congenital amaurosis type-2 (LCA2). Previous studies have suggested that retinal degeneration in the dog model is slower than that observed in humans. However, the area centralis of the dog retina is a cone and rod photoreceptor rich region comparable to the human macula, and the effect of RPE65 deficiency specifically on this retinal region, important for high acuity vision, has not previously been reported. METHODS: Spectral-domain optical coherence tomography, fundus photography, and immunohistochemistry of retinal wholemounts and sagittal frozen sections were used to define the time-course and cell-types affected in degeneration of the area centralis in affected dogs. RESULTS: Area centralis photoreceptor degeneration was evident from 6 weeks of age, and progressed to involve the inner retina. Immunohistochemistry showed that RPE65-deficient dogs developed early loss of S-cone outer segments, with slower loss of L/M-cone outer segments and rods. CONCLUSIONS: Early-onset severe photoreceptor degeneration in the area centralis of dogs with RPE65-deficiency offers a model of the early foveal/perifoveal degeneration in some patients with LCA2. This model could be used to refine interventions aiming to improve function and halt the progression of foveal/perifoveal photoreceptor degeneration

Assessment and in vivo scoring of murine experimental autoimmune uveoretinitis using optical coherence tomography

Despite advances in clinical imaging and grading our understanding of retinal immune responses and their morphological correlates in experimental autoimmune uveoretinitis (EAU), has been hindered by the requirement for post-mortem histology. To date, monitoring changes occurring during EAU disease progression and evaluating the effect of therapeutic intervention in real time has not been possible. We wanted to establish whether optical coherence tomography (OCT) could detect intraretinal changes during inflammation and to determine its utility as a tool for accurate scoring of EAU. EAU was induced in C57BL/6J mice and animals evaluated after 15, 26, 36 and 60 days. At each time-point, contemporaneous Spectralis-OCT scanning, topical endoscopic fundal imaging (TEFI), fundus fluorescein angiography (FFA) and CD45-immunolabelled histology were performed. OCT features were further characterised on retinal flat-mounts using immunohistochemistry and 3D reconstruction. Optic disc swelling and vitreous opacities detected by OCT corresponded to CD45+ cell infiltration on histology. Vasculitis identified by FFA and OCT matched perivascular myeloid and T-cell infiltrates and could be differentiated from unaffected vessels. Evolution of these changes could be followed over time in the same eye. Retinal folds were visible and found to encapsulate mixed populations of activated myeloid cells, T-cells and microglia. Using these features, an OCT-based EAU scoring system was developed, with significant correlation to validated histological (Pearson r(2) = 0.6392, P<0.0001, n = 31 eyes) and TEFI based scoring systems (r(2) = 0.6784, P<0.0001). OCT distinguishes the fundamental features of murine EAU in vivo, permits dynamic assessment of intraretinal changes and can be used to score disease severity. As a result, it allows tissue synchronisation with subsequent cellular and functional assessment and greater efficiency of animal usage. By relating OCT signals with immunohistochemistry in EAU, our findings offer the opportunity to inform the interpretation of OCT changes in human uveitis

Development of an optimized AAV2/5 gene therapy vector for Leber congenital amaurosis owing to defects in RPE65

Leber congenital amaurosis is a group of inherited retinal dystrophies that cause severe sight impairment in childhood; RPE65-deficiency causes impaired rod photoreceptor function from birth and progressive impairment of cone photoreceptor function associated with retinal degeneration. In animal models of RPE65 deficiency, subretinal injection of recombinant adeno-associated virus (AAV) 2/2 vectors carrying RPE65 cDNA improves rod photoreceptor function, and intervention at an early stage of disease provides sustained benefit by protecting cone photoreceptors against retinal degeneration. In affected humans, administration of these vectors has resulted to date in relatively modest improvements in photoreceptor function, even when retinal degeneration is comparatively mild, and the duration of benefit is limited by progressive retinal degeneration. We conclude that the demand for RPE65 in humans is not fully met by current vectors, and predict that a more powerful vector will provide more durable benefit. With this aim we have modified the original AAV2/2 vector to generate AAV2/5-OPTIRPE65. The new configuration consists of an AAV vector serotype 5 carrying an optimized hRPE65 promoter and a codon-optimized hRPE65 gene. In mice, AAV2/5-OPTIRPE65 is at least 300-fold more potent than our original AAV2/2 vector

Early vitrectomy for exogenous endophthalmitis following surgery

This is a protocol for a Cochrane Review (intervention). The objectives are as follows: To assess the potential role of combined pars plana vitrectomy and intravitreal antibiotics in the acute management of exogenous endophthalmitis, versus the standard of care, defined as vitreous tap and intravitreal antibiotics

Surgery for idiopathic epiretinal membrane

BACKGROUND: Epiretinal membrane is an abnormal sheet of avascular fibrocellular tissue that develops on the inner surface of the retina. Epiretinal membrane can cause impairment of sight as a consequence of progressive distortion of retinal architecture. OBJECTIVES: To determine the effects of surgery compared to no intervention for epiretinal membrane. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE Ovid, Embase Ovid, ISRCTN registry, US National Institutes of Health Ongoing Trials Register ClinicalTrials.gov, and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP). There were no restrictions to language or year of publication. The databases were last searched on 20 May 2020. SELECTION CRITERIA: We included randomised controlled trials (RCTs) assessing surgical removal of idiopathic epiretinal membrane compared to placebo, no treatment or sham treatment. Paired or within-person studies were included, as well as those where both eyes of a single participant were treated. DATA COLLECTION AND ANALYSIS: We used standard methods expected by Cochrane, and assessed certainty using the GRADE system. We considered the following five outcome measures: mean change in best corrected visual acuity (BCVA) in the study eye between baseline (before randomisation), 6 months and 12 months later; proportion of people with a gain of 0.3 logMAR or more of visual acuity in the study eye as measured by a logMAR chart at a starting distance of 4 m at 6 months and 12 months after randomisation; proportion of people with a loss of 0.3 logMAR or more of visual acuity in the study eye as measured by a logMAR chart at a starting distance of 4 m at 6 months and 12 months after randomisation; mean quality of life score at 6 months and 12 months following surgery, measured using a validated questionnaire; and any harm identified during follow-up. MAIN RESULTS: We included one study in the review. This was a RCT including 53 eyes of 53 participants with mild symptomatic epiretinal membrane and BCVA of 65 or more Early Treatment Diabetic Retinopathy Study (ETDRS) letters. Participants were randomly allocated to immediate surgery or to watchful waiting with deferred surgery if indicated by evidence of disease progression. The study was limited by imprecision owing to the small number of participants and was at some risk of bias owing to inconsistencies in the time points for outcome assessment and in the management of lens opacity. At 12 months, the visual acuity in the immediate surgery group was higher by a mean of 2.1 (95% confidence interval (CI) -2.0 to 6.2 ETDRS letters; 53 participants; low-certainty evidence) than the watchful waiting/deferred surgery group. The evidence of the effect of immediate surgery on gains of 0.3 logMAR or more of visual acuity is very uncertain (risk ratio (RR) 0.55, 95% CI 0.06 to 4.93; 53 participants; very low-certainty evidence). At 12 months, no participant in either group sustained a loss of 0.3 logMAR or more of visual acuity (53 participants; low-certainty evidence). The included study did not measure quality of life. At 12 months, no serious adverse event was identified in any participant. One participant developed chronic minimal cystoid macular oedema following immediate surgery (53 participants; low-certainty evidence). AUTHORS' CONCLUSIONS: We found no RCT that directly investigated the effect of surgery compared to no intervention. For severe disabling epiretinal membrane, the lack of a RCT comparing surgery to no intervention may reflect evidence from non-randomised studies in favour of surgery; a RCT may be considered unnecessary and ethically unacceptable because a superior effect of surgery is widely accepted. For mild symptomatic epiretinal membrane, however, the value of surgery is uncertain. Low-certainty evidence from this review suggests that watchful waiting or deferred surgery may offer outcomes as favourable as immediate surgery. However, this finding needs to be confirmed in further RCTs with appropriate statistical power, masking of treatment allocation, consistent management of cataract, and measurement of outcomes including patient-reported quality of life over a more extended time frame

Comparison of 5-year progression of retinitis pigmentosa involving the posterior pole among siblings by means of SD-OCT: a retrospective study

The blockchain technology promises to transform finance, money and evengovernments. However, analyses of blockchain applicability and robustness typicallyfocus on isolated systems whose actors contribute mainly by running the consensusalgorithm. Here, we highlight the importance of considering trustless platformswithin the broader ecosystem that includes social and communication networks. Asan example, we analyse the flash-crash observed on 21st June 2017 in the Ethereumplatform and show that a major phenomenon of social coordination led to acatastrophic cascade of events across several interconnected systems. We proposethe concept of “emergent centralisation” to describe situations where a single systembecomes critically important for the functioning of the whole ecosystem, and arguethat such situations are likely to become more and more frequent in interconnectedsocio-technical systems. We anticipate that the systemic approach we propose willhave implications for future assessments of trustless systems and call for the attentionof policy-makers on the fragility of our interconnected and rapidly changing world

Conformational adaptation of Asian macaque TRIMCyp directs lineage specific antiviral activity

TRIMCyps are anti-retroviral proteins that have arisen independently in New World and Old World primates. All TRIMCyps comprise a CypA domain fused to the tripartite domains of TRIM5α but they have distinct lentiviral specificities, conferring HIV-1 restriction in New World owl monkeys and HIV-2 restriction in Old World rhesus macaques. Here we provide evidence that Asian macaque TRIMCyps have acquired changes that switch restriction specificity between different lentiviral lineages, resulting in species-specific alleles that target different viruses. Structural, thermodynamic and viral restriction analysis suggests that a single mutation in the Cyp domain, R69H, occurred early in macaque TRIMCyp evolution, expanding restriction specificity to the lentiviral lineages found in African green monkeys, sooty mangabeys and chimpanzees. Subsequent mutations have enhanced restriction to particular viruses but at the cost of broad specificity. We reveal how specificity is altered by a scaffold mutation, E143K, that modifies surface electrostatics and propagates conformational changes into the active site. Our results suggest that lentiviruses may have been important pathogens in Asian macaques despite the fact that there are no reported lentiviral infections in current macaque populations

Does \u2018bigger\u2019mean \u2018better\u2019? Pitfalls and shortcuts associated with big data for social research

\u2018Big data is here to stay.\u2019 This key statement has a double value: is an assumption as well as the reason why a theoretical reflection is needed. Furthermore, Big data is something that is gaining visibility and success in social sciences even, overcoming the division between humanities and computer sciences. In this contribution some considerations on the presence and the certain persistence of Big data as a socio-technical assemblage will be outlined. Therefore, the intriguing opportunities for social research linked to such interaction between practices and technological development will be developed. However, despite a promissory rhetoric, fostered by several scholars since the birth of Big data as a labelled concept, some risks are just around the corner. The claims for the methodological power of bigger and bigger datasets, as well as increasing speed in analysis and data collection, are creating a real hype in social research. Peculiar attention is needed in order to avoid some pitfalls. These risks will be analysed for what concerns the validity of the research results \u2018obtained through Big data. After a pars distruens, this contribution will conclude with a pars construens; assuming the previous critiques, a mixed methods research design approach will be described as a general proposal with the objective of stimulating a debate on the integration of Big data in complex research projecting

Assisted evolution enables HIV-1 to overcome a high trim5α-imposed genetic barrier to rhesus macaque tropism

Diversification of antiretroviral factors during host evolution has erected formidable barriers to cross-species retrovirus transmission. This phenomenon likely protects humans from infection by many modern retroviruses, but it has also impaired the development of primate models of HIV-1 infection. Indeed, rhesus macaques are resistant to HIV-1, in part due to restriction imposed by the TRIM5α protein (rhTRIM5α). Initially, we attempted to derive rhTRIM5α-resistant HIV-1 strains using two strategies. First, HIV-1 was passaged in engineered human cells expressing rhTRIM5α. Second, a library of randomly mutagenized capsid protein (CA) sequences was screened for mutations that reduced rhTRIM5α sensitivity. Both approaches identified several individual mutations in CA that reduced rhTRIM5α sensitivity. However, neither approach yielded mutants that were fully resistant, perhaps because the locations of the mutations suggested that TRIM5α recognizes multiple determinants on the capsid surface. Moreover, even though additive effects of various CA mutations on HIV-1 resistance to rhTRIM5α were observed, combinations that gave full resistance were highly detrimental to fitness. Therefore, we employed an 'assisted evolution' approach in which individual CA mutations that reduced rhTRIM5α sensitivity without fitness penalties were randomly assorted in a library of viral clones containing synthetic CA sequences. Subsequent passage of the viral library in rhTRIM5α-expressing cells resulted in the selection of individual viral species that were fully fit and resistant to rhTRIM5α. These viruses encoded combinations of five mutations in CA that conferred complete or near complete resistance to the disruptive effects of rhTRIM5α on incoming viral cores, by abolishing recognition of the viral capsid. Importantly, HIV-1 variants encoding these CA substitutions and SIVmac239 Vif replicated efficiently in primary rhesus macaque lymphocytes. These findings demonstrate that rhTRIM5α is difficult to but not impossible to evade, and doing so should facilitate the development of primate models of HIV-1 infection