Photoreceptor replacement by transplantation is proposed as a treatment for blindness. Transplantation of healthy photoreceptor precursor cells into diseased murine eyes leads to the presence of ...functional photoreceptors within host retinae that express an array of donor-specific proteins. The resulting improvement in visual function was understood to be due to donor cells integrating within host retinae. Here, however, we show that while integration occurs the majority of donor-reporter-labelled cells in the host arises as a result of material transfer between donor and host photoreceptors. Material transfer does not involve permanent donor-host nuclear or cell-cell fusion, or the uptake of free protein or nucleic acid from the extracellular environment. Instead, RNA and/or protein are exchanged between donor and host cells in vivo. These data require a re-evaluation of the mechanisms underlying rescue by photoreceptor transplantation and raise the possibility of material transfer as a strategy for the treatment of retinal disorders.
To establish the mature neuroepithelial tissue architecture, newborn neurons often migrate from their place of birth, usually at the apical neuroepithelial limit, towards their target destination. ...Even before neurons are born, the nucleus of the mitotic neuronal progenitor cell migrates within the apico-basal cellular extent in synchrony with the cell cycle (interkinetic nuclear migration or IKNM) – a migratory pattern so far only observed in mitotic epithelial progenitor cells. Rod photoreceptors, too, are born at the apical limit of the retinal neuroepithelium. They then populate a layer directly adjacent (outer nuclear layer or ONL), but not the more basal retinal layers. How rod photoreceptors become enriched specifically within the ONL is presently ill-defined. Here, it was identified that rod photoreceptor somata of the developing mouse retina are constantly pushed basally (presumably caused by proximal progenitor IKNM events). To become enriched apically within the (presumptive) ONL despite this, the post-mitotic rod photoreceptors utilised an IKNM-like migratory behaviour more typically associated with dividing cells: rod somata actively migrated apically, driven by microtubule-associated dynein I motors. Another microtubule-associated motor protein, KIF1A, acts as a molecular brake during basal displacement, preventing ectopic basal positions. Rod somata oscillate between apical and basal motions at least from P1 up until ~P10. Whether this involves a component of glial-guided migration could not be established beyond reasonable doubt. Nonetheless, this is the first report of an oscillatory, IKNM-like migration behaviour occurring within a post-mitotic neuronal cell population. Rod photoreceptors have also been assumed to migrate into the adult neural retina following sub-retinal transplantation for cell-replacement therapeutic purposes, although this has not been directly observed. Here, time-lapse footage for the first time showed rod photoreceptors migrating from the sub-retinal space into the host retina. This supports the notion that photoreceptor cell replacement therapy could become a clinically viable treatment option.
To establish the mature neuroepithelial tissue architecture, newborn neurons often migrate from their place of birth, usually at the apical neuroepithelial limit, towards their target destination. ...Even before neurons are born, the nucleus of the mitotic neuronal progenitor cell migrates within the apico-basal cellular extent in synchrony with the cell cycle (interkinetic nuclear migration or IKNM) – a migratory pattern so far only observed in mitotic epithelial progenitor cells. Rod photoreceptors, too, are born at the apical limit of the retinal neuroepithelium. They then populate a layer directly adjacent (outer nuclear layer or ONL), but not the more basal retinal layers. How rod photoreceptors become enriched specifically within the ONL is presently ill-defined. Here, it was identified that rod photoreceptor somata of the developing mouse retina are constantly pushed basally (presumably caused by proximal progenitor IKNM events). To become enriched apically within the (presumptive) ONL despite this, the post-mitotic rod photoreceptors utilised an IKNM-like migratory behaviour more typically associated with dividing cells: rod somata actively migrated apically, driven by microtubule-associated dynein I motors. Another microtubule-associated motor protein, KIF1A, acts as a molecular brake during basal displacement, preventing ectopic basal positions. Rod somata oscillate between apical and basal motions at least from P1 up until ~P10. Whether this involves a component of glial-guided migration could not be established beyond reasonable doubt. Nonetheless, this is the first report of an oscillatory, IKNM-like migration behaviour occurring within a post-mitotic neuronal cell population. Rod photoreceptors have also been assumed to migrate into the adult neural retina following sub-retinal transplantation for cell-replacement therapeutic purposes, although this has not been directly observed. Here, time-lapse footage for the first time showed rod photoreceptors migrating from the sub-retinal space into the host retina. This supports the notion that photoreceptor cell replacement therapy could become a clinically viable treatment option.
In development, almost all stratified neurons must migrate from their birthplace to the appropriate neural layer. Photoreceptors reside in the most apical layer of the retina, near their place of ...birth. Whether photoreceptors require migratory events for fine-positioning and/or retention within this layer is not well understood. Here, we show that photoreceptor nuclei of the developing mouse retina cyclically exhibit rapid, dynein-1-dependent translocation toward the apical surface, before moving more slowly in the basal direction, likely due to passive displacement by neighboring retinal nuclei. Attenuating dynein 1 function in rod photoreceptors results in their ectopic basal displacement into the outer plexiform layer and inner nuclear layer. Synapse formation is also compromised in these displaced cells. We propose that repeated, apically directed nuclear translocation events are necessary to ensure retention of post-mitotic photoreceptors within the emerging outer nuclear layer during retinogenesis, which is critical for correct neuronal lamination.
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•Photoreceptor nuclei exhibit apico-basal motility in the developing mouse retina•Rapid apically directed nuclear translocation is powered by dynein 1•Dynein 1 disruption impedes rapid apical nuclear translocation, causing displacement•Synapse formation is disrupted in basally displaced photoreceptors
Photoreceptors occupy a defined layer in the retina. Aghaizu et al. show that mammalian rod and cone photoreceptors use repeated, dynein-1-driven apically directed nuclear translocations for fine-positioning and retention within this layer. Dynein 1 disruption in photoreceptors results in ectopic displacement beyond the photoreceptor layer and impaired synapse formation.
In the adult central nervous system, endothelial and neuronal cells engage in tight cross-talk as key components of the so-called neurovascular unit. Impairment of this important relationship ...adversely affects tissue homeostasis, as observed in neurodegenerative conditions including Alzheimer's and Parkinson's disease. In development, the influence of neuroprogenitor cells on angiogenesis is poorly understood. Here, we show in mouse that these cells interact intimately with the growing retinal vascular network, and we identify a novel regulatory mechanism of vasculature development mediated by hypoxia-inducible factor 2a (Hif2a). By
gene excision, we show that Hif2a in retinal neuroprogenitor cells upregulates the expression of the pro-angiogenic mediators vascular endothelial growth factor and erythropoietin, whereas it locally downregulates the angiogenesis inhibitor endostatin. Importantly, absence of
in retinal neuroprogenitor cells causes a marked reduction of proliferating endothelial cells at the angiogenic front. This results in delayed retinal vascular development, fewer major retinal vessels and reduced density of the peripheral deep retinal vascular plexus. Our findings demonstrate that retinal neuroprogenitor cells are a crucial component of the developing neurovascular unit.
•Cholinergic and adrenergic neurons can readily be isolated from the intracardiac ganglia and superior cervical ganglia, respectively.•Dissociated neurons of the autonomic nervous system can be ...cryopreserved as long as a month.•Preserved neurons exhibit similar neurite outgrowth as freshly isolated neurons for both populations.•Preserved neurons possess similar electrophysiological characteristics as freshly isolated controls.•Persevered neurons for on-demand use is paramount for inclusion of the autonomic nervous system in new in vitro platforms.
Generally, primary neurons are isolated and seeded within hours of isolation, but cryopreservation, documented for a small number of central and peripheral neuronal subtypes, can contribute to improved utility and reduce the cost of developing new in vitro models. The preservation of cells of the autonomic nervous system (ANS), specifically sympathetic and parasympathetic neurons, has not been explored.
In this work, we establish a method for preserving cardiac ANS neurons as well as evaluating the phenotypical changes of dissociated superior cervical ganglia (sympathetic neurons) and intracardiac ganglia (parasympathetic neurons) for up to a month of storage in liquid nitrogen.
Neuron populations maintained a viability of at least 35%, and the extent of neurite outgrowth was not different from fresh cells, regardless of the storage duration studied. Expression of tyrosine hydroxylase and choline acetyl transferase were maintained over one month of cryopreservation in sympathetic and parasympathetic populations, respectively. Electrophysiological recordings for both neuron types indicate sustained characteristic resting potentials, excitability, and action potentials after more than one month in liquid nitrogen.
Primary cultures of the autonomic nervous system have been previously established for in vitro investigations. This is the first example of preserving primary ANS neuron cultures for long-term on-demand use.
This report describes a readily implemented method for cryopreserving sympathetic and parasympathetic neurons that does not alter neither morphological nor electrophysiological characteristics. This methodology expands the utility of ANS cultures for use in morphological and functional assays.
In 2009, Public Health England (PHE) introduced the routine application of a recent infection testing algorithm (RITA) to new HIV diagnoses, where a positive RITA result indicates likely acquisition ...of infection in the previous six months. Laboratories submit serum specimens to PHE for testing using the HIV 1/2gO AxSYM assay modified for the determination of HIV antibody avidity. Results are classified according to avidity index and data on CD₄ count, antiretroviral treatment and the presence of an AIDS-defining illness. Between 2009 and 2011, 38.4% (6,966/18,134) of new HIV diagnoses in England, Wales and Northern Ireland were tested. Demographic characteristics of those tested were similar to all persons with diagnosed HIV. Overall, recent infection was 14.7% (1,022/6,966) and higher among men who have sex with men (MSM) (22.3%, 720/3,223) compared with heterosexual men and women (7.8%, 247/3,164). Higher proportions were among persons aged 15-24 years compared with those ≥50 years (MSM 31.2% (139/445) vs 13.6% (42/308); heterosexual men and women 17.3% (43/249) vs 6.2% (31/501)). Among heterosexual men and women, black Africans were least likely to have recent infection compared with whites (4.8%, 90/1,892 vs 13.3%, 97/728; adjusted odds ratio: 0.6; 95% CI: 0.4-0.9). Our results indicate evidence of ongoing HIV transmission during the study period, particularly among MSM.
Objectives
In order to estimate HIV incidence among high‐risk groups, in January 2009 the Health Protection Agency introduced the Recent Infection Testing Algorithm (RITA) in England and Northern ...Ireland (E&NI), currently the only regions to inform patients of RITA results. This survey of HIV specialists aimed to investigate the role of RITA in patient management and explore clinicians' views on its role in clinical practice and during partner notification.
Methods
An online questionnaire was distributed to HIV specialists via the British HIV Association membership email list in February 2011.
Results
Forty‐two HIV specialists from 32 HIV centres responded to the survey among 90 centres enrolled in the programme (response rate 36%). Testing for recent infection was considered standard of care by 83% of respondents, 80% felt confident in interpreting results and 92% discussed results with patients, particularly in the context of a possible HIV seroconversion illness (96%) or when deciding when to start antiretroviral therapy (70%). A third (36%) of specialists were initially concerned that RITA results may cause additional anxiety among patients; however, no adverse events were reported. The majority (90%) felt that results could assist with contact tracing by prioritizing patients with likely recent infection. However, only a few centres have currently incorporated RITA into their HIV partner notification protocols.
Conclusions
RITA has been introduced into clinical practice with no reported patient adverse events. Access to results at centre level should be improved. National guidance regarding use of RITA as a tool for contact tracing is required.
The prevalence of HIV-1 transmitted drug resistance (TDR) among men-who-have-sex-with-men (MSM) in the UK peaked in 2000 at ~14%. It then declined to a nadir of 9% in 2007 and has since remained ...relatively stable. This was estimated using sequences from treatment-naive patients. Here, we describe a method for enhanced surveillance of TDR using samples from recently infected MSM. The recent infection testing algorithm (RITA) is currently applied to 52% of all newly diagnosed HIV infections in the national reference laboratory for England. We amplified the HIV-1 pol gene from 96 MSM sampled between July 2011 and September 2012, ascertained by RITA as incident infections. These were sequenced by Sanger and Illumina next-generation sequencing (NGS). The clinical significance of low frequency TDR mutations identified by NGS requires further investigation as they affect susceptibility to first-line PIs used in the UK. The data also reveal an appreciable current transmission of drug resistant HIV among recently infected UK MSM.
Objectives
In order to estimate
HIV
incidence among high‐risk groups, in
J
anuary 2009 the
H
ealth
P
rotection
A
gency introduced the
R
ecent
I
nfection
T
esting
A
lgorithm (
RITA
) in
E
ngland and
N
...orthern
I
reland (
E&NI
), currently the only regions to inform patients of
RITA
results. This survey of
HIV
specialists aimed to investigate the role of
RITA
in patient management and explore clinicians' views on its role in clinical practice and during partner notification.
Methods
An online questionnaire was distributed to
HIV
specialists via the
B
ritish
HIV A
ssociation membership email list in
F
ebruary 2011.
Results
Forty‐two
HIV
specialists from 32
HIV
centres responded to the survey among 90 centres enrolled in the programme (response rate 36%). Testing for recent infection was considered standard of care by 83% of respondents, 80% felt confident in interpreting results and 92% discussed results with patients, particularly in the context of a possible
HIV
seroconversion illness (96%) or when deciding when to start antiretroviral therapy (70%). A third (36%) of specialists were initially concerned that
RITA
results may cause additional anxiety among patients; however, no adverse events were reported. The majority (90%) felt that results could assist with contact tracing by prioritizing patients with likely recent infection. However, only a few centres have currently incorporated
RITA
into their
HIV
partner notification protocols.
Conclusions
RITA
has been introduced into clinical practice with no reported patient adverse events. Access to results at centre level should be improved. National guidance regarding use of
RITA
as a tool for contact tracing is required.
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