The tax-inducible actin-bundling protein fascin is crucial for release and cell-to-cell transmission of human T-cell leukemia virus type 1 (HTLV-1)

The delta-retrovirus Human T-cell leukemia virus type 1 (HTLV-1) preferentially infects CD4(+) T-cells via cell-to-cell transmission. Viruses are transmitted by polarized budding and by transfer of viral biofilms at the virological synapse (VS). Formation of the VS requires the viral Tax protein and polarization of the host cytoskeleton, however, molecular mechanisms of HTLV-1 cell-to-cell transmission remain incompletely understood. Recently, we could show Tax-dependent upregulation of the actin-bundling protein Fascin (FSCN-1) in HTLV-1-infected T-cells. Here, we report that Fascin contributes to HTLV-1 transmission. Using single-cycle replication-dependent HTLV-1 reporter vectors, we found that repression of endogenous Fascin by short hairpin RNAs and by Fascin-specific nanobodies impaired gag p19 release and cell-to-cell transmission in 293T cells. In Jurkat T-cells, Tax-induced Fascin expression enhanced virus release and Fascin-dependently augmented cell-to-cell transmission to Raji/CD4(+) B-cells. Repression of Fascin in HTLV-1-infected T-cells diminished virus release and gag p19 transfer to co-cultured T-cells. Spotting the mechanism, flow cytometry and automatic image analysis showed that Tax-induced T-cell conjugate formation occurred Fascin-independently. However, adhesion of HTLV-1-infected MT-2 cells in co-culture with Jurkat T-cells was reduced upon knockdown of Fascin, suggesting that Fascin contributes to dissemination of infected T-cells. Imaging of chronically infected MS9 T-cells in co-culture with Jurkat T-cells revealed that Fascin's localization at tight cell-cell contacts is accompanied by gag polarization suggesting that Fascin directly affects the distribution of gag to budding sites, and therefore, indirectly viral transmission. In detail, we found gag clusters that are interspersed with Fascin clusters, suggesting that Fascin makes room for gag in viral biofilms. Moreover, we observed short, Fascin-containing membrane extensions surrounding gag clusters and clutching uninfected T-cells. Finally, we detected Fascin and gag in long-distance cellular protrusions. Taken together, we show for the first time that HTLV-1 usurps the host cell factor Fascin to foster virus release and cell-to-cell transmission

Intermediate progenitors support migration of neural stem cells into dentate gyrus outer neurogenic niches.

The hippocampal dentate gyrus (DG) is a unique brain region maintaining neural stem cells (NCSs) and neurogenesis into adulthood. We used multiphoton imaging to visualize genetically defined progenitor subpopulations in live slices across key stages of mouse DG development, testing decades old static models of DG formation with molecular identification, genetic-lineage tracing, and mutant analyses. We found novel progenitor migrations, timings, dynamic cell-cell interactions, signaling activities, and routes underlie mosaic DG formation. Intermediate progenitors (IPs, Tbr2+) pioneered migrations, supporting and guiding later emigrating NSCs (Sox9+) through multiple transient zones prior to converging at the nascent outer adult niche in a dynamic settling process, generating all prenatal and postnatal granule neurons in defined spatiotemporal order. IPs (Dll1+) extensively targeted contacts to mitotic NSCs (Notch active), revealing a substrate for cell-cell contact support during migrations, a developmental feature maintained in adults. Mouse DG formation shares conserved features of human neocortical expansion

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) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). 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 (n = 137), shock-dependent (n = 146), and no (n = 101) 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 non–critically 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 (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free 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 support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (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 support–free 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

Transfer of HTLV-1 p8 and Gag to target T-cells depends on VASP, a novel interaction partner of p8

The Human T-cell leukemia virus type 1 (HTLV-1) orf I-encoded accessory protein p8 is cleaved from its precursor p12, and both proteins contribute to viral persistence. p8 induces cellular protrusions, which are thought to facilitate transfer of p8 to target cells and virus transmission. Host factors interacting with p8 and mediating p8 transfer are unknown. Here, we report that vasodilator-stimulated phosphoprotein (VASP), which promotes actin filament elongation, is a novel interaction partner of p8 and important for p8 and HTLV-1 Gag cell-to-cell transfer. VASP contains an Ena/VASP homology 1 (EVH1) domain that targets the protein to focal adhesions. Bioinformatics identified a short stretch in p8 (amino acids (aa) 24–45) which may mediate interactions with the EVH1 domain of VASP. Co-immunoprecipitations confirmed interactions of VASP:p8 in 293T, Jurkat and HTLV-1-infected MT-2 cells. Co-precipitation of VASP:p8 could be significantly blocked by peptides mimicking aa 26–37 of p8. Mutational studies revealed that the EVH1-domain of VASP is necessary, but not sufficient for the interaction with p8. Further, deletion of the VASP G- and F-actin binding domains significantly diminished co-precipitation of p8. Imaging identified areas of partial co-localization of VASP with p8 at the plasma membrane and in protrusive structures, which was confirmed by proximity ligation assays. Co-culture experiments revealed that p8 is transferred between Jurkat T-cells via VASP-containing conduits. Imaging and flow cytometry revealed that repression of both endogenous and overexpressed VASP by RNA interference or by CRISPR/Cas9 reduced p8 transfer to the cell surface and to target Jurkat T-cells. Stable repression of VASP by RNA interference in chronically infected MT-2 cells impaired both p8 and HTLV-1 Gag transfer to target Jurkat T-cells, while virus release was unaffected. Thus, we identified VASP as a novel interaction partner of p8, which is important for transfer of HTLV-1 p8 and Gag to target T-cells

A novel positive feedback-loop between the HTLV-1 oncoprotein Tax and NF-κB activity in T-cells

Background!#!Human T-cell leukemia virus type 1 (HTLV-1) infects primarily CD4!##!Results!#!Here we found that Tax mutants which are defective in NF-κB signaling showed diminished protein expression levels compared to Tax wildtype in T-cells, whereas Tax transcript levels were comparable. Strikingly, constant activation of NF-κB signaling by the constitutive active mutant of inhibitor of kappa B kinase (IKK2, IKK-β), IKK2-EE, rescued protein expression of the NF-κB defective Tax mutants M22 and K1-10R and even increased protein levels of Tax wildtype in various T-cell lines while Tax transcript levels were only slightly affected. Using several Tax expression constructs, an increase of Tax protein occurred independent of Tax transcripts and independent of the promoter used. Further, Tax and M22 protein expression were strongly enhanced by 12-O-Tetradecanoylphorbol-13-Acetate [TPA; Phorbol 12-myristate 13-acetate (PMA)]/ ionomycin, inducers of NF-κB and cytokine signaling, but not by tumor necrosis factor alpha (TNF-α). On the other hand, co-expression of Tax with a dominant negative inhibitor of κB, IκBα-DN, or specific inhibition of IKK2 by the compound ACHP, led to a vast decrease in Tax protein levels to some extent independent of Tax transcripts in transiently transfected and Tax-transformed T-cells. Cycloheximide chase experiments revealed that co-expression of IKK2-EE prolongs the half-life of M22, and constant repression of NF-κB signaling by IκBα-DN strongly reduces protein stability of Tax wildtype suggesting that NF-κB activity is required for Tax protein stability. Finally, protein expression of Tax and M22 could be recovered by NH!##!Conclusions!#!Together, these findings suggest that Tax's capability to induce NF-κB is critical for protein expression and stabilization of Tax itself. Overall, identification of this novel positive feedback loop between Tax and NF-κB in T-cells improves our understanding of Tax-driven transformation

Quantified Interference for a While Language

We show how an information theoretic approach can quantify interference in a simple imperative language that includes a looping construct. In this paper we focus on a particular case of this definition of interference: leakage of information from private variables to public ones in While language programs. The major result of the paper is a quantitative analysis for this language that employs a use-definition graph to calculate bounds on the leakage into each variable

Collagen IV (COL4A1, COL4A2), a Component of the Viral Biofilm, Is Induced by the HTLV-1 Oncoprotein Tax and Impacts Virus Transmission

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Intermediate progenitors support migration of neural stem cells into dentate gyrus outer neurogenic niches.

The hippocampal dentate gyrus (DG) is a unique brain region maintaining neural stem cells (NCSs) and neurogenesis into adulthood. We used multiphoton imaging to visualize genetically defined progenitor subpopulations in live slices across key stages of mouse DG development, testing decades old static models of DG formation with molecular identification, genetic-lineage tracing, and mutant analyses. We found novel progenitor migrations, timings, dynamic cell-cell interactions, signaling activities, and routes underlie mosaic DG formation. Intermediate progenitors (IPs, Tbr2+) pioneered migrations, supporting and guiding later emigrating NSCs (Sox9+) through multiple transient zones prior to converging at the nascent outer adult niche in a dynamic settling process, generating all prenatal and postnatal granule neurons in defined spatiotemporal order. IPs (Dll1+) extensively targeted contacts to mitotic NSCs (Notch active), revealing a substrate for cell-cell contact support during migrations, a developmental feature maintained in adults. Mouse DG formation shares conserved features of human neocortical expansion