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Increasing LFA-1 Expression Enhances Immune Synapse Architecture and T Cell Receptor Signaling in Jurkat E6.1 Cells
The Jurkat E6.1 clone has been extensively used as a powerful tool for the genetic and biochemical dissection of the TCR signaling pathway. More recently, these cells have been exploited in imaging studies to identify key players in immunological synapse (IS) assembly in superantigen-specific conjugates and to track the dynamics of signaling molecules on glass surfaces coated with activating anti-CD3 antibodies. By comparison, Jurkat cells have been used only scantily for imaging on supported lipid bilayers (SLBs) incorporating laterally mobile TCR and integrin ligands, which allow to study synaptic rearrangements of surface molecules and the fine architecture of the mature IS, likely due to limitations in the assembly of immune synapses with well-defined architecture. Here we have explored whether upregulating the low levels of endogenous LFA-1 expression on Jurkat E6.1 cells through transduction with CD11a- and CD18-encoding lentiviruses can improve IS architecture. We show that, while forced LFA-1 expression did not affect TCR recruitment to the IS, E6.1 LFA-1high cells assembled better structured synapses, with a tighter distribution of signaling-competent TCRs at the center of the IS. LFA-1 upregulation enhanced protein phosphotyrosine signaling on SLBs but not at the IS formed in conjugates with SEE-pulsed APCs, and led to the constitutive formation of an intracellular phosphotyrosine pool co-localizing with endosomal CD3ζ. This was paralleled by an increase in the levels of p-ZAP-70 and p-Erk both under basal conditions and following activation, and in enhanced Ca2+ mobilization from intracellular stores. The enhancement in early signaling E6.1 LFA-1high cells did not affect expression of the early activation marker CD69 but led to an increase in IL-2 expression. Our results highlight a new role for LFA-1 in the core architecture of the IS that can be exploited to study the spatiotemporal redistribution of surface receptors on SLBs, thereby extending the potential of E6.1 cells and their derivatives for fine-scale imaging studies.
Casirivimab and imdevimab in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial
SUMMARYBackgroundREGEN-COV is a combination of 2 monoclonal antibodies (casirivimab and imdevimab) that bind to two different sites on the receptor binding domain of the SARS-CoV-2 spike protein. We aimed to evaluate the efficacy and safety of REGEN-COV in patients admitted to hospital with COVID-19.MethodsIn this randomised, controlled, open-label platform trial, several possible treatments were compared with usual care in patients hospitalised with COVID-19. Eligible and consenting patients were randomly allocated (1:1) to either usual standard of care alone (usual care group) or usual care plus a single dose of REGEN-COV 8g (casirivimab 4g and imdevimab 4g) by intravenous infusion (REGEN-COV group). The primary outcome was 28-day mortality assessed first among patients without detectable antibodies to SARS-CoV-2 at randomisation (seronegative) and then in the overall population. The trial is registered with ISRCTN (50189673) and clinicaltrials.gov (NCT04381936).FindingsBetween 18 September 2020 and 22 May 2021, 9785 patients were randomly allocated to receive usual care plus REGEN-COV or usual care alone, including 3153 (32%) seronegative patients, 5272 (54%) seropositive patients and 1360 (14%) patients with unknown baseline antibody status. In the primary efficacy population of seronegative patients, 396 (24%) of 1633 patients allocated to REGEN-COV and 451 (30%) of 1520 patients allocated to usual care died within 28 days (rate ratio 0·80; 95% CI 0·70-0·91; p=0·0010). In an analysis involving all randomised patients (regardless of baseline antibody status), 944 (20%) of 4839 patients allocated to REGEN-COV and 1026 (21%) of 4946 patients allocated to usual care died within 28 days (rate ratio 0·94; 95% CI 0·86-1·03; p=0·17). The proportional effect of REGEN-COV on mortality differed significantly between seropositive and seronegative patients (p value for heterogeneity = 0·001).InterpretationIn patients hospitalised with COVID-19, the monoclonal antibody combination of casirivimab and imdevimab (REGEN-COV) reduced 28-day mortality among patients who were seronegative at baseline.FundingUK Research and Innovation (Medical Research Council) and National Institute of Health Research (Grant ref: MC_PC_19056).
Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial.
BACKGROUND: Many patients with COVID-19 have been treated with plasma containing anti-SARS-CoV-2 antibodies. We aimed to evaluate the safety and efficacy of convalescent plasma therapy in patients admitted to hospital with COVID-19. METHODS: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]) is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. The trial is underway at 177 NHS hospitals from across the UK. Eligible and consenting patients were randomly assigned (1:1) to receive either usual care alone (usual care group) or usual care plus high-titre convalescent plasma (convalescent plasma group). The primary outcome was 28-day mortality, analysed on an intention-to-treat basis. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936. FINDINGS: Between May 28, 2020, and Jan 15, 2021, 11558 (71%) of 16287 patients enrolled in RECOVERY were eligible to receive convalescent plasma and were assigned to either the convalescent plasma group or the usual care group. There was no significant difference in 28-day mortality between the two groups: 1399 (24%) of 5795 patients in the convalescent plasma group and 1408 (24%) of 5763 patients in the usual care group died within 28 days (rate ratio 1·00, 95% CI 0·93-1·07; p=0·95). The 28-day mortality rate ratio was similar in all prespecified subgroups of patients, including in those patients without detectable SARS-CoV-2 antibodies at randomisation. Allocation to convalescent plasma had no significant effect on the proportion of patients discharged from hospital within 28 days (3832 [66%] patients in the convalescent plasma group vs 3822 [66%] patients in the usual care group; rate ratio 0·99, 95% CI 0·94-1·03; p=0·57). Among those not on invasive mechanical ventilation at randomisation, there was no significant difference in the proportion of patients meeting the composite endpoint of progression to invasive mechanical ventilation or death (1568 [29%] of 5493 patients in the convalescent plasma group vs 1568 [29%] of 5448 patients in the usual care group; rate ratio 0·99, 95% CI 0·93-1·05; p=0·79). INTERPRETATION: In patients hospitalised with COVID-19, high-titre convalescent plasma did not improve survival or other prespecified clinical outcomes. FUNDING: UK Research and Innovation (Medical Research Council) and National Institute of Health Research.
Aspirin in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial
SUMMARYBackgroundAspirin has been proposed as a treatment for COVID-19 on the basis of its antithrombotic properties.MethodsIn this randomised, controlled, open-label platform trial, several possible treatments were compared with usual care in patients hospitalised with COVID-19. Eligible and consenting adults were randomly allocated in a 1:1 ratio to either usual standard of care plus 150mg aspirin once daily until discharge or usual standard of care alone using web-based simple (unstratified) randomisation with allocation concealment. The primary outcome was 28-day mortality. The trial is registered with ISRCTN (50189673) and clinicaltrials.gov (NCT04381936).FindingsBetween 01 November 2020 and 21 March 2021, 7351 patients were randomly allocated to receive aspirin and 7541 patients to receive usual care alone. Overall, 1222 (17%) patients allocated to aspirin and 1299 (17%) patients allocated to usual care died within 28 days (rate ratio 0·96; 95% confidence interval [CI] 0·89-1·04; p=0·35). Consistent results were seen in all pre-specified subgroups of patients. Patients allocated to aspirin had a slightly shorter duration of hospitalisation (median 8 vs. 9 days) and a higher proportion were discharged from hospital alive within 28 days (75% vs. 74%; rate ratio 1·06; 95% CI 1·02-1·10; p=0·0062). Among those not on invasive mechanical ventilation at baseline, there was no significant difference in the proportion meeting the composite endpoint of invasive mechanical ventilation or death (21% vs. 22%; risk ratio 0·96; 95% CI 0·90-1·03; p=0·23). Aspirin use was associated with an absolute reduction in thrombotic events of 0.6% (SE 0.4%) and an absolute increase in major bleeding events of 0.6% (SE 0.2%).InterpretationIn patients hospitalised with COVID-19, aspirin was not associated with reductions in 28-day mortality or in the risk of progressing to invasive mechanical ventilation or death but was associated with a small increase in the rate of being discharged alive within 28 days.FundingUK Research and Innovation (Medical Research Council), National Institute of Health Research (Grant ref: MC_PC_19056), and the Wellcome Trust (Grant Ref: 222406/Z/20/Z) through the COVID-19 Therapeutics Accelerator.
Association Between Administration of IL-6 Antagonists and Mortality Among Patients Hospitalized for COVID-19: A Meta-analysis.
Importance: Clinical trials assessing the efficacy of IL-6 antagonists in patients hospitalized for COVID-19 have variously reported benefit, no effect, and harm. Objective: To estimate the association between administration of IL-6 antagonists compared with usual care or placebo and 28-day all-cause mortality and other outcomes. Data Sources: Trials were identified through systematic searches of electronic databases between October 2020 and January 2021. Searches were not restricted by trial status or language. Additional trials were identified through contact with experts. Study Selection: Eligible trials randomly assigned patients hospitalized for COVID-19 to a group in whom IL-6 antagonists were administered and to a group in whom neither IL-6 antagonists nor any other immunomodulators except corticosteroids were administered. Among 72 potentially eligible trials, 27 (37.5%) met study selection criteria. Data Extraction and Synthesis: In this prospective meta-analysis, risk of bias was assessed using the Cochrane Risk of Bias Assessment Tool. Inconsistency among trial results was assessed using the I2 statistic. The primary analysis was an inverse variance-weighted fixed-effects meta-analysis of odds ratios (ORs) for 28-day all-cause mortality. Main Outcomes and Measures: The primary outcome measure was all-cause mortality at 28 days after randomization. There were 9 secondary outcomes including progression to invasive mechanical ventilation or death and risk of secondary infection by 28 days. Results: A total of 10 930 patients (median age, 61 years [range of medians, 52-68 years]; 3560 [33%] were women) participating in 27 trials were included. By 28 days, there were 1407 deaths among 6449 patients randomized to IL-6 antagonists and 1158 deaths among 4481 patients randomized to usual care or placebo (summary OR, 0.86 [95% CI, 0.79-0.95]; P = .003 based on a fixed-effects meta-analysis). This corresponds to an absolute mortality risk of 22% for IL-6 antagonists compared with an assumed mortality risk of 25% for usual care or placebo. The corresponding summary ORs were 0.83 (95% CI, 0.74-0.92; P
Bone turnover in pregnancy, measured by urinary C-terminal telopeptide of type I collagen (CTX), is influenced by vitamin D supplementation and is associated with maternal bone health: Findings from the MAVIDOS trial.
BackgroundThe pattern of change in maternal bone turnover throughout pregnancy is poorly characterized.ObjectiveWe investigated changes across pregnancy in a marker of maternal bone resorption, urinary C-terminal telopeptide of type I collagen (CTX), the influence of gestational vitamin D supplementation, and associations between CTX and maternal postnatal bone indices.DesignThe Maternal Vitamin D Osteoporosis Study (MAVIDOS) is a randomized, double-blind, placebo-controlled trial of 1000 IU/day cholecalciferol vs placebo from 14 weeks' gestation to birth. Maternal second void urinary α- and β-CTX were measured (Enzyme Linked Immunosorbent Assay, ELISA) at 14 and 34 weeks' gestation; DXA was performed within 2 weeks post-partum. Mann-Whitney rank sum, Spearman's rank correlation and linear regression were used to compare median CTX values within and between groups from early to late pregnancy, and associations with maternal bone outcomes.Results372 women had CTX and 25(OH)-vitamin D measured in early and late pregnancy. CTX at 14 and 34 weeks' gestation were correlated in both placebo (r = 0.31) and cholecalciferol (r = 0.45) groups (P ConclusionsMaternal urinary CTX, a bone resorption marker, rises through pregnancy, though to a lesser degree with gestational cholecalciferol supplementation, and is inversely associated with maternal bone mass post-partum.Clinical Trial Registry number and website: ISRCTN:82927713; EUDRACT:2007-001716-23.
The Bardet-Biedl Syndrome complex component BBS1 regulates proteasome-dependent F-actin clearance from the centrosome to enable its translocation to the T cell immune synapse
ABSTRACT Components of the intraflagellar transport (IFT) system that regulates the assembly of the primary cilium are exploited by the non-ciliated T cell to orchestrate polarized endosome recycling to sustain signaling during immune synapse formation. Here we have investigated the potential role of BBS1, an essential core component of the Bardet-Biedl syndrome complex that cooperates with the IFT system in ciliary protein trafficking, in the assembly of the T cell synapse. We show that BBS1 allows for centrosome polarization towards the immune synapse by promoting its untethering from the nuclear envelope. This function is achieved through the clearance of centrosomal F-actin and its positive regulator WASH, a process that we demonstrate to be dependent on the proteasome. We show that BBS1 regulates this process by coupling the 19S proteasome regulatory subunit to the microtubule motor dynein for its transport to the centrosome. Our data identify the ciliopathy-related protein BBS1 as a new player in T cell synapse assembly that acts upstream of the IFT system to set the stage for polarized vesicular trafficking and sustained signaling.
SUMIC: A Simple Ultrafast Multicolor Immunolabelling and Clearing Approach for Whole-Organ and Large Tissue 3D Imaging
High-resolution whole-organ imaging of cleared tissues captures cellular and molecular insights within the intact tissue and tumour microenvironments. However, current immunolabelling and clearing methods are complicated and time-consuming; extending to several weeks. Here, we developed S imple U ltrafast M ulticolor I mmunolabelling and C learing or SUMIC , a method that enables multicolor immunolabelling and clearing of whole murine organs and human tissues within 2 to 2.5 days. Moreover, SUMIC is simple, robust, non-hazardous and versatile comprising antigen retrieval, permeabilization, collagenase-based digestion, immunolabelling, dehydration, and clearing. SUMIC permits quantitative and singlecell resolution analysis and detection of rare cells in whole organs, for example, round αSMA positive cells in the thymus. Upon volumetric imaging, SUMIC-processed samples retain normal tissue architecture and can be used for paraffin-embedding and histology. We employed the SUMIC method for whole-organ mapping of lymphatic vessels across different ages and organs. This analysis revealed the expansion of lymphatic vessels in endocrine tissues but not in any other organs with aging. Hence, SUMIC will accelerate discoveries compared to other whole organ imaging pipelines.
AbstractPre-existing T cell immunity to SARS-CoV-2 in individuals without prior exposure to SARS-CoV-2 has been reported in several studies. While emerging evidence hints toward prior exposure to common-cold human coronaviruses (HCoV), the extent of- and conditions for-cross-protective immunity between SARS-CoV-2 and HCoVs remain open. Here, by leveraging a comprehensive pool of publicly available functionally evaluated SARS-CoV-2 peptides, we report 126 immunogenic SARS-CoV-2 peptides with high sequence similarity to 285 MHC-presented target peptides from at least one of four HCoV, thus providing a map describing the landscape of SARS-CoV-2 shared and private immunogenic peptides with functionally validated T cell responses. Using this map, we show that while SARS-CoV-2 immunogenic peptides in general exhibit higher level of dissimilarity to both self-proteome and -microbiomes, there exist several SARS-CoV-2 immunogenic peptides with high similarity to various human protein coding genes, some of which have been reported to have elevated expression in severe COVID-19 patients. We then combine our map with a SARS-CoV-2-specific TCR repertoire data from COVID-19 patients and healthy controls and show that whereas the public repertoire for the majority of convalescent patients are dominated by TCRs cognate to private SARS-CoV-2 peptides, for a subset of patients, more than 50% of their public repertoires that show reactivity to SARS-CoV-2, consist of TCRs cognate to shared SARS-CoV-2-HCoV peptides. Further analyses suggest that the skewed distribution of TCRs cognate to shared and private peptides in COVID-19 patients is likely to be HLA-dependent. Finally, by utilising the global prevalence of HLA alleles, we provide 10 peptides with known cognate TCRs that are conserved across SARS-CoV-2 and multiple human coronaviruses and are predicted to be recognised by a high proportion of the global population. Overall, our work indicates the potential for HCoV-SARS-CoV-2 reactive CD8+ T cells, which is likely dependent on differences in HLA-coding genes among individuals. These findings may have important implications for COVID-19 heterogeneity and vaccine-induced immune responses as well as robustness of immunity to SARS-CoV-2 and its variants.
Test-retest reliability for HAQ-DI and SF-36 PF for the measurement of physical function in psoriatic arthritis.
OBJECTIVE: Due to no existing data, we aimed to derive evidence to support test-retest reliability for the Health Assessment Questionnaire-Disability Index (HAQ-DI) and Medical Outcome Survey Short-Form-36 item physical functioning domain (SF-36 PF) in psoriatic arthritis (PsA). METHODS: We identified datasets that collected relevant data for test-retest reliability for HAQ-DI and SF-36 PF; and evaluated them using OMERACT Filter 2.1 methodology. We calculated intra-class correlation coefficients (ICC) as a measure of test-retest reliability. We then conducted a quality assessment and evaluated the adequacy of test-retest reliability performance. RESULTS: Two datasets were identified for HAQ-DI and one for SF-36 PF in PsA. The quality of the datasets was good. The ICCs for HAQ-DI were excellent in both datasets: 0.94 (95% CI: 0.88 to 0.97) and 0.94 (95% CI: 0.89 to 0.97). The ICC of SF-36 PF was good (0.89, 95% CI: 0.76 to 0.95). The performance of test-retest reliability for both instruments was judged to be adequate. CONCLUSION: The new data derived support good and reasonable test-retest reliability for HAQ-DI and SF-36 PF in PsA.
Association between Markers of Synovial Inflammation, Matrix Turnover and Symptoms in Knee Osteoarthritis: A Cross-Sectional Study
To investigate the association between markers of synovial inflammation and matrix turnover (MRI-based and serum biomarkers) and knee symptoms in established knee osteoarthritis (KOA). This cross-sectional study utilised data from a randomised, multicentre placebo-controlled trial (UK-VIDEO) of vitamin D therapy in symptomatic KOA. Data on serum biomarkers, type III collagen degradation (C3M), metabolite of C-reactive protein (CRPM) and cartilage oligomeric matrix protein (COMP), were available at baseline whilst contrast-enhanced (CE) MRI data were acquired in a subsample at baseline and annually. Knee symptoms were assessed using WOMAC at all visits. We examined the cross-sectional association between knee symptoms and three MRI-based and three serum markers of synovitis and matrix turnover, respectively. A total of 447 participants were included in the serum and 136 participants in the MRI analyses. MRI-defined medial perimeniscal synovitis was positively associated with knee pain and, suprapatellar and medial perimeniscal synovitis with knee function in multivariate analysis. We observed a statistically significant, negative association between a higher concentration of serum C3M and CRPM and knee pain, respectively. Furthermore, the highest CRPM quartile was negatively associated with knee function. Our findings suggest that, in established painful radiographic KOA, MRI-defined medial perimeniscal and suprapatellar synovitis were positively associated with knee symptoms. Serum-based C3M and CRPM markers were negatively associated with knee symptoms. Pain fluctuations are common in KOA and a better understanding of the relationship between markers of synovitis and matrix turnover and knee symptoms would facilitate a more accurate assessment of temporal changes in disease progression.
Measuring physical function in psoriatic arthritis: comparing the Multi-Dimensional Health Assessment Questionnaire to the Health Assessment Questionnaire Disability Index.
OBJECTIVE: To compare physical function scales of the Multi-Dimensional Health Assessment Questionnaire (MDHAQ) to the Health Assessment Questionnaire Disability Index (HAQDI) in patients with psoriatic arthritis (PsA), and examine whether either questionnaire is less prone to 'floor effects'. METHODS: Data were collected prospectively from 2018 to 2019 across three UK hospitals. All patients completed physical function scales within the MDHAQ and HAQDI in a single clinic visit. Agreement was assessed using medians and the Bland-Altman method. Intraclass correlation coefficients (ICCs) were used to assess test-retest reliability. RESULTS: 210 patients completed the clinic visit; one withdrew consent thus 209 were analysed. 60.0% were male, with mean age of 51.7 years and median disease duration of 7 years. In clinic, median MDHAQ and HAQDI including/excluding aids scores were 0.30, 0.50 and 0.50 respectively. Although the median score for HAQDI is higher than MDHAQ, the difference between the two mostly lies within 1.96 standard deviations from the mean suggesting good agreement. The ICCs demonstrated excellent test-retest reliability for both HAQ questionnaires.Similar numbers of patients scored '0' on the MDHAQ and HAQDI including/excluding aids (48, 47, and 49 respectively). Using a score of ≤0.5 as a cut-off for minor functional impairment, 23 patients had a MDHAQ ≤0.5 when their HAQDI including aids >0.5. Conversely, 4 patients had a MDHAQ > 0.5 when the HAQDI including aids ≤0.5. CONCLUSION: Both HAQ questionnaires appear to be similar in detecting floor effects in patients with PsA.
SHG microscopy has been used to detect the modification of the collagen meshwork in the articular cartilage affected by osteoarthritis. Higher signal in the forward direction and polarisation sensitivity has been observed. © OSA 2012.