Nine dogs with hemophilia A were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII ...(AAV-cFVIII) corrected the FVIII deficiency to 1.9-11.3% of normal FVIII levels. In two of nine dogs, levels of FVIII activity increased gradually starting about 4 years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of the integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A, while emphasizing the importance of long-term monitoring for potential genotoxicity.
We present CO(1−0) and CO(3−2) Atacama Large Millimeter/submillimeter Array observations of the molecular gas in PKS 1549−79, as well as mm and very long baseline interferometry 2.3-GHz continuum ...observations of its radio jet. PKS 1549−79 is one of the closest young, radio-loud quasars caught in an on-going merger in which the active galactic nucleus (AGN) is in the first phases of its evolution. We detect three structures tracing the accretion and the outflow of molecular gas: kpc-scale tails of gas accreting onto PKS 1549−79 from a merger, a circumnuclear disc in the inner few hundred parsec, and a very broad (> 2300 km s−1) component detected in CO(1−0) at the position of the AGN. Thus, in PKS 1549−79 we see the co-existence of accretion and the ejection of gas. The line ratio CO(3−2)/CO(1−0) suggests that the gas in the circumnuclear-disc has both high densities and high kinetic temperatures. We estimate a mass outflow rate of at least 650 M⊙ yr−1. This massive outflow is confined to the inner region (r < 120 pc) of the galaxy, which suggests that the AGN drives the outflow. Considering the amount of molecular gas available in the central nuclear disc and the observed outflow rate, we estimate a time scale of ∼105 yr over which the AGN would be able to destroy the circumnuclear disc, although gas from the merger may come in from larger radii, rebuilding this disc at the same time. The AGN appears to self-regulate gas accretion to the centre and onto the super-massive black hole. Surprisingly, from a comparison with Hubble Space Telescope data, we find that the ionised gas outflow is more extended. Nevertheless, the warm outflow is about two orders of magnitude less massive than the molecular outflow. PKS 1549−79 does not seem to follow the scaling relation between bolometric luminosity and the relative importance of warm ionised and molecular outflows claimed to exist for other AGN. We argue that, although PKS 1549−79 hosts a powerful quasar nucleus and an ultra-fast outflow, the radio jet plays a significant role in producing the outflow, which creates a cocoon of disturbed gas that expands into the circumnuclear disc.
Clonal expansion of infected CD4+ T cells is a major mechanism of HIV-1 persistence and a barrier to achieving a cure. Potential causes are homeostatic proliferation, effects of HIV-1 integration, ...and interaction with antigens. Here, we show that it is possible to link antigen responsiveness, the full proviral sequence, the integration site, and the T cell receptor β-chain (TCRβ) sequence to examine the role of recurrent antigenic exposure in maintaining the HIV-1 reservoir. We isolated CMV- and Gag-responding CD4+ T cells from 10 treated individuals. Proviral populations in CMV-responding cells were dominated by large clones, including clones harboring replication-competent proviruses. TCRβ repertoires showed high clonality driven by converging adaptive responses. Although some proviruses were in genes linked to HIV-1 persistence (BACH2, STAT5B, MKL1), the proliferation of infected cells under antigenic stimulation occurred regardless of the site of integration. Paired TCRβ and integration site analysis showed that infection could occur early or late in the course of a clone's response to antigen and could generate infected cell populations too large to be explained solely by homeostatic proliferation. Together, these findings implicate antigen-driven clonal selection as a major factor in HIV-1 persistence, a finding that will be a difficult challenge to eradication efforts.
Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells
. We report the initial results of nine severely affected X-linked CGD (X-CGD) patients who received ex vivo ...autologous CD34
hematopoietic stem and progenitor cell-based lentiviral gene therapy following myeloablative conditioning in first-in-human studies (trial registry nos. NCT02234934 and NCT01855685). The primary objectives were to assess the safety and evaluate the efficacy and stability of biochemical and functional reconstitution in the progeny of engrafted cells at 12 months. The secondary objectives included the evaluation of augmented immunity against bacterial and fungal infection, as well as assessment of hematopoietic stem cell transduction and engraftment. Two enrolled patients died within 3 months of treatment from pre-existing comorbidities. At 12 months, six of the seven surviving patients demonstrated stable vector copy numbers (0.4-1.8 copies per neutrophil) and the persistence of 16-46% oxidase-positive neutrophils. There was no molecular evidence of either clonal dysregulation or transgene silencing. Surviving patients have had no new CGD-related infections, and six have been able to discontinue CGD-related antibiotic prophylaxis. The primary objective was met in six of the nine patients at 12 months follow-up, suggesting that autologous gene therapy is a promising approach for CGD patients.
BACKGROUNDHIV-1-infected CD4+ T cells contribute to latent reservoir persistence by proliferating while avoiding immune recognition. Integration features of intact proviruses in elite controllers ...(ECs) and people on long-term therapy suggest that proviruses in specific chromosomal locations can evade immune surveillance. However, direct evidence of this mechanism is missing.METHODSIn this case report, we characterized integration sites and full genome sequences of expanded T cell clones in an EC before and after chemoradiation. We identified the cognate peptide of infected clones to investigate cell proliferation and virus production induced by T cell activation, and susceptibility to autologous CD8+ T cells.RESULTSThe proviral landscape was dominated by 2 large clones with replication-competent proviruses integrated into zinc finger (ZNF) genes (ZNF470 and ZNF721) in locations previously associated with deeper latency. A third nearly intact provirus, with a stop codon in Pol, was integrated into an intergenic site. Upon stimulation with cognate Gag peptides, infected clones proliferated extensively and produced virus, but the provirus in ZNF721 was 200-fold less inducible. While autologous CD8+ T cells decreased the proliferation of cells carrying the intergenic provirus, they had no effect on cells with the provirus in the ZNF721 gene.CONCLUSIONSWe provide direct evidence that upon activation of infected clones by cognate antigen, the lower inducibility of intact proviruses in ZNF genes can result in immune evasion and persistence.FUNDINGOffice of the NIH Director and National Institute of Dental & Craniofacial Research; NIAID, NIH; Johns Hopkins University Center for AIDS Research.
Younger age is associated with increased revision incidence following primary total hip arthroplasty, though the association between age and repeat revision following revision total hip arthroplasty ...(rTHA) has not been described. This study aimed to describe the incidences and indications for subsequent revision (re-revision) following rTHA based on age.
Patients undergoing aseptic rTHA from 2011 to 2021 with minimum 1-year follow-up were retrospectively reviewed. Patients were stratified into 3 groups based on age at the time of index rTHA (ie, <55 years, 55 to 74 years, and >74 years). Perioperative characteristics, complications, and re-revisions were compared between groups.
Of 694 included rTHAs, those in the >74 age group were more likely to undergo rTHA for periprosthetic fracture (P < .001) while those in the <55 age group were more likely to undergo rTHA for metallosis/taper corrosion (P = .028). Readmissions (P = .759) and emergency department visits (P = .498) within 90 days were comparable across ages. Rates of re-revision were comparable at 90 days (P = .495), 1 year (P = .443), and 2 years (P = .204). Kaplan–Meier analysis of all-cause re-revision at latest follow-up showed a nonstatistically significant trend toward increasing re-revisions in the <55 and 55 to 74 age groups. Using logistic regressions, smoking and index rTHA for instability were independently associated with re-revision, while age at index surgery was not.
While indications for rTHA differ across age groups, rates of 2-year re-revision are statistically comparable between groups. Further studies are warranted to understand the association between age, activity, and re-revision rates after 5 years postoperatively.
Despite the extensive literature on racial disparities in care and outcomes after total knee arthroplasty (TKA), data on manipulation under anesthesia (MUA) is lacking. We aimed to determine (1) the ...relationship between race and rate of (and time to) MUA after TKA, and (2) annual trends in racial differences in MUA from 2013 to 2018.
This retrospective cohort study (using 2013-2018 Medicare Limited Data Set claims data) included 836,054 primary TKA patients. The primary outcome was MUA <1 year after TKA; time from TKA to MUA in days was also recorded. A mixed-effects multivariable model measured the association between race (White, Black, Other) and odds of MUA. Odds ratios (OR) and 95% confidence intervals (CI) were reported. A Cochran Armitage Trend test was conducted to assess MUA trends over time, stratified by race.
MUA after TKA occurred in 1.7%, 3.2% and 2.1% of White, Black, and Other race categories, respectively (SMD = 0.07). After adjustment for covariates, (Black vs White) patients had increased odds of requiring an MUA after TKA: odds ratio (OR) 1.97, 95% confidence intervals (CI) 1.86-2.10, P < .0001. Moreover, White (compared to Black) patients had significantly shorter time to MUA after TKA: 60 days (interquartile range IQR 46-88) versus 64 days (interquartile range IQR 47-96); P < .0001. These disparities persisted from 2013 through 2018.
Continued racial differences exist for rates and timing of MUA following TKA signifying the continued need for efforts aimed toward understanding and eliminating inequalities that exist in total joint arthroplasty (TJA) care.
The influenza A M2 protein is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle. The monomeric subunits of the M2 homotetramer ...consist of an N-terminal ectodomain, a transmembrane domain, and a C-terminal cytoplasmic domain. The transmembrane domain forms a four-helix proton channel that promotes uncoating of virions upon host cell entry. The membrane-proximal region of the C-terminal domain forms a surface-associated amphipathic helix necessary for viral budding. The structure of the remaining ~34 residues of the distal cytoplasmic tail has yet to be fully characterized despite the functional significance of this region for influenza infectivity. Here, we extend structural and dynamic studies of the poorly characterized M2 cytoplasmic tail. We used SDSL-EPR to collect site-specific information on the mobility, solvent accessibility, and conformational properties of residues 61–70 of the full-length, cell-expressed M2 protein reconstituted into liposomes. Our analysis is consistent with the predominant population of the C-terminal tail dynamically extending away from the membranes surface into the aqueous medium. These findings provide insight into the hypothesis that the C-terminal domain serves as a sensor that regulates how M2 protein participates in critical events in the viral infection cycle.
Display omitted
•Dominant population of the C-terminal tail of M2 is highly mobile.•C-terminal tail of M2 extends away from the membrane surface into the aqueous phase.•Mobility and accessibility report on length of membrane-associated amphipathic helix.
Introduction. Dual-mobility (DM) implants for total hip arthroplasty (THA) have gained popularity due to their potential to reduce hip instability and dislocation events that may lead to revision ...surgery. These implants consist of a femoral head articulated within a polyethylene liner, which articulates within an outer acetabular shell, creating a dual-bearing surface. Our study aimed to report our observations on the survivorship of a novel DM implant for primary total hip arthroplasty at two years. Methods. We conducted a retrospective, multicenter study to assess the clinical outcomes of patients undergoing a THA with a novel DM implant (OR3O acetabular system™, Smith & Nephew, Inc., Memphis, TN) from January 2020 to September 2021. Patient demographics, surgical information, and survivorship data were collected from medical records for patients with a minimum of two years of follow-up. Primary outcomes included overall implant survivorship at two years as well as aseptic survivorship, revision rates of the DM acetabular shell, and average time to revision. Patient-reported outcomes were collected in the form of HOOS JR. Results. A total of 250 hips in 245 patients had a minimum two-year follow-up. Primary osteoarthritis (80%) was the most common indication for index THA. The average aseptic survivorship of the DM acetabular components at two years for the cohort was 98.4% and survivorship of the acetabular implants overall was 97.6%. There were a total of four (1.6%) aseptic revisions of the DM acetabular component. Reasons for aseptic acetabular revision included one case of instability, one intraprosthetic dislocation, one periprosthetic acetabular fracture, and one malpositioned acetabular cup resulting in impingement. The mean time of follow-up was 893.9 days. Eighty-seven patients had preoperative and two-year HOOS JR available. HOOS JR improved by an average of 38.5 points. Conclusion. This novel DM acetabular implant demonstrates excellent survivorship at two years follow-up with low rates of instability and intraprosthetic dislocation and no episodes of metal-on-metal corrosion. Use of the DM implant demonstrated clinically relevant improvements in patient-reported outcomes at two years.
Chimeric antigen receptor (CAR) T cells have demonstrated promising efficacy, particularly in hematologic malignancies. One challenge regarding CAR T cells in solid tumors is the immunosuppressive ...tumor microenvironment (TME), characterized by high levels of multiple inhibitory factors, including transforming growth factor (TGF)-β. We report results from an in-human phase 1 trial of castration-resistant, prostate cancer-directed CAR T cells armored with a dominant-negative TGF-β receptor (NCT03089203). Primary endpoints were safety and feasibility, while secondary objectives included assessment of CAR T cell distribution, bioactivity and disease response. All prespecified endpoints were met. Eighteen patients enrolled, and 13 subjects received therapy across four dose levels. Five of the 13 patients developed grade ≥2 cytokine release syndrome (CRS), including one patient who experienced a marked clonal CAR T cell expansion, >98% reduction in prostate-specific antigen (PSA) and death following grade 4 CRS with concurrent sepsis. Acute increases in inflammatory cytokines correlated with manageable high-grade CRS events. Three additional patients achieved a PSA reduction of ≥30%, with CAR T cell failure accompanied by upregulation of multiple TME-localized inhibitory molecules following adoptive cell transfer. CAR T cell kinetics revealed expansion in blood and tumor trafficking. Thus, clinical application of TGF-β-resistant CAR T cells is feasible and generally safe. Future studies should use superior multipronged approaches against the TME to improve outcomes.
PDF
