37 research outputs found
A new GFP-tagged line reveals unexpected Otx2 protein localization in retinal photoreceptors
<p>Abstract</p> <p>Background</p> <p>Dynamic monitoring of protein expression and localization is fundamental to the understanding of biological processes. The paired-class homeodomain-containing transcription factor Otx2 is essential for normal head and brain development in vertebrates. Recent conditional knockout studies have pointed to multiple roles of this protein during late development and post-natal life. Yet, later expression and functions remain poorly characterized as specific reagents to detect the protein at any stage of development are still missing.</p> <p>Results</p> <p>We generated a new mouse line harbouring an insertion of the GFP gene within the Otx2 coding sequence to monitor the gene activity while preserving most of its functions. Our results demonstrate that this line represents a convenient tool to capture the dynamics of <it>Otx2 </it>gene expression from early embryonic stages to adulthood. In addition, we could visualize the intracellular location of Otx2 protein. In the retina, we reinterpret the former view of protein distribution and show a further level of regulation of intranuclear protein localization, which depends on the cell type.</p> <p>Conclusion</p> <p>The GFP-tagged <it>Otx2 </it>mouse line fully recapitulates previously known expression patterns and brings additional accuracy and easiness of detection of <it>Otx2 </it>gene activity. This opens up the way to live imaging of a highly dynamic actor of brain development and can be adapted to any mutant background to probe for genetic interaction between <it>Otx2 </it>and the mutated gene.</p
Otx2 Gene Deletion in Adult Mouse Retina Induces Rapid RPE Dystrophy and Slow Photoreceptor Degeneration
International audienceBACKGROUND: Many developmental genes are still active in specific tissues after development is completed. This is the case for the homeobox gene Otx2, an essential actor of forebrain and head development. In adult mouse, Otx2 is strongly expressed in the retina. Mutations of this gene in humans have been linked to severe ocular malformation and retinal diseases. It is, therefore, important to explore its post-developmental functions. In the mature retina, Otx2 is expressed in three cell types: bipolar and photoreceptor cells that belong to the neural retina and retinal pigment epithelium (RPE), a neighbour structure that forms a tightly interdependent functional unit together with photoreceptor cells. METHODOLOGY/PRINCIPAL FINDINGS: Conditional self-knockout was used to address the late functions of Otx2 gene in adult mice. This strategy is based on the combination of a knock-in CreERT2 allele and a floxed allele at the Otx2 locus. Time-controlled injection of tamoxifen activates the recombinase only in Otx2 expressing cells, resulting in selective ablation of the gene in its entire domain of expression. In the adult retina, loss of Otx2 protein causes slow degeneration of photoreceptor cells. By contrast, dramatic changes of RPE activity rapidly occur, which may represent a primary cause of photoreceptor disease. CONCLUSIONS: Our novel mouse model uncovers new Otx2 functions in adult retina. We show that this transcription factor is necessary for long-term maintenance of photoreceptors, likely through the control of specific activities of the RPE
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Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.
Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (nâ=â143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (nâ=â152), or no hydrocortisone (nâ=â108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (nâ=â137), shock-dependent (nâ=â146), and no (nâ=â101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707
Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19
IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19.
Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19.
DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 nonâcritically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022).
INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (nâ=â257), ARB (nâ=â248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; nâ=â10), or no RAS inhibitor (control; nâ=â264) for up to 10 days.
MAIN OUTCOMES AND MEASURES The primary outcome was organ supportâfree days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes.
RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ supportâfree days among critically ill patients was 10 (â1 to 16) in the ACE inhibitor group (nâ=â231), 8 (â1 to 17) in the ARB group (nâ=â217), and 12 (0 to 17) in the control group (nâ=â231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ supportâfree days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively).
CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes.
TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570
Thyroid Hormone Signaling in the Mouse Retina.
Thyroid hormone is a crucial regulator of gene expression in the developing and adult retina. Here we sought to map sites of thyroid hormone signaling at the cellular level using the transgenic FINDT3 reporter mouse model in which neurons express ÎČ-galactosidase (ÎČ-gal) under the control of a hybrid Gal4-TRα receptor when triiodothyronine (T3) and cofactors of thyroid receptor signaling are present. In the adult retina, nearly all neurons of the ganglion cell layer (GCL, ganglion cells and displaced amacrine cells) showed strong ÎČ-gal labeling. In the inner nuclear layer (INL), a minority of glycineric and GABAergic amacrine cells showed ÎČ-gal labeling, whereas the majority of amacrine cells were unlabeled. At the level of amacrine types, ÎČ-gal labeling was found in a large proportion of the glycinergic AII amacrines, but only in a small proportion of the cholinergic/GABAergic 'starburst' amacrines. At postnatal day 10, there also was a high density of strongly ÎČ-gal-labeled neurons in the GCL, but only few amacrine cells were labeled in the INL. There was no labeling of bipolar cells, horizontal cells and MĂŒller glia cells at both stages. Most surprisingly, the photoreceptor somata in the outer nuclear layer also showed no ÎČ-gal label, although thyroid hormone is known to control cone opsin expression. This is the first record of thyroid hormone signaling in the inner retina of an adult mammal. We hypothesize that T3 levels in photoreceptors are below the detection threshold of the reporter system. The topographical distribution of ÎČ-gal-positive cells in the GCL follows the overall neuron distribution in that layer, with more T3-signaling cells in the ventral than the dorsal half-retina
Double immunostaining of a vertical cryostat section of adult FINDT3 mouse retina for ÎČ-gal and cholinergic amacrine cells.
<p>ÎČ-gal label (<b>a</b>) colocalizes with the ChAT label (<b>b</b>) in a few somata in the INL and GCL (arrows), but the majority of cholinergic amacrine cells show no ÎČ-gal signal, as evident in the merge (<b>c</b>). Images were acquired with a Zeiss Axioplan 2 microscope.</p
Double immunostaining of a vertical cryostat section of adult FINDT3 mouse retina for ÎČ-gal and rod bipolar cells.
<p>ÎČ-gal label (<b>a</b>) does not colocalize with the PKCα label of rod bipolar cells (<b>b</b>), as shown in the merge (<b>c</b>). The magenta signal among the rod bipolar cell somata in the upper INL represents blood vessel staining. Images were acquired with a Zeiss Axioplan 2 microscope.</p
Higher power micrographs from wholemounted FINDT3 retinae stained with X-gal.
<p>For the adult mouse (left), the GCL and INL are shown separately as they are on different focal planes. For the p10 mouse (right) only the GCL is shown. Dorsal and ventral fields are from far peripheral retina. The scale bar applies to all images. Images were acquired with a Zeiss Axiophot 2 microscope.</p
Population densities of ganglion cell layer (GCL) neurons along the dorso-ventral axis of adult mouse retina intersecting the optic nerve head.
<p>The blue line gives the densities of X-gal-stained GCL cells in FINDT3 retina. The black line gives the densities of GCL neurons from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168003#pone.0168003.ref032" target="_blank">32</a>]. The grey area shows the density range of GCL neurons reported for three retinae by [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168003#pone.0168003.ref033" target="_blank">33</a>]. For details see text. Eccentricity â0â refers to the position of the optic nerve head.</p