MicroRNA-223 Dampens Pulmonary Inflammation during Pneumococcal Pneumonia

Community-acquired pneumonia remains a major contributor to global communicable disease-mediated mortality. Neutrophils play a leading role in trying to contain bacterial lung infection, but they also drive detrimental pulmonary inflammation, when dysregulated. Here we aimed at understanding the role of microRNA-223 in orchestrating pulmonary inflammation during pneumococcal pneumonia. Serum microRNA-223 was measured in patients with pneumococcal pneumonia and in healthy subjects. Pulmonary inflammation in wild-type and microRNA-223-knockout mice was assessed in terms of disease course, histopathology, cellular recruitment and evaluation of inflammatory protein and gene signatures following pneumococcal infection. Low levels of serum microRNA-223 correlated with increased disease severity in pneumococcal pneumonia patients. Prolonged neutrophilic influx into the lungs and alveolar spaces was detected in pneumococci-infected microRNA-223-knockout mice, possibly accounting for aggravated histopathology and acute lung injury. Expression of microRNA-223 in wild-type mice was induced by pneumococcal infection in a time-dependent manner in whole lungs and lung neutrophils. Single-cell transcriptome analyses of murine lungs revealed a unique profile of antimicrobial and cellular maturation genes that are dysregulated in neutrophils lacking microRNA-223. Taken together, low levels of microRNA-223 in human pneumonia patient serum were associated with increased disease severity, whilst its absence provoked dysregulation of the neutrophil transcriptome in murine pneumococcal pneumonia

Efficient clinical-grade γ-retroviral vector purification by high-speed centrifugation for CAR T cell manufacturing

γ-Retroviral vectors (γ-RV) are powerful tools for gene therapy applications. Current clinical vectors are produced from stable producer cell lines which require minimal further downstream processing, while purification schemes for γ-RV produced by transient transfection have not been thoroughly investigated. We aimed to develop a method to purify transiently produced γ-RV for early clinical studies. Here, we report a simple one-step purification method by high-speed centrifugation for γ-RV produced by transient transfection for clinical application. High-speed centrifugation enabled the concentration of viral titers in the range of 107-108 TU/mL with >80% overall recovery. Analysis of research-grade concentrated vector revealed sufficient reduction in product- and process-related impurities. Furthermore, product characterization of clinical-grade γ-RV by BioReliance demonstrated two-logs lower impurities per transducing unit compared with regulatory authority-approved stable producer cell line vector for clinical application. In terms of CAR T cell manufacturing, clinical-grade γ-RV produced by transient transfection and purified by high-speed centrifugation was similar to γ-RV produced from a clinical-grade stable producer cell line. This method will be of value for studies using γ-RV to bridge vector supply between early- and late-stage clinical trials

Intratumoral pan-ErbB targeted CAR-T for head and neck squamous cell carcinoma: interim analysis of the T4 immunotherapy study

Background: Locally advanced/recurrent head and neck squamous cell carcinoma (HNSCC) is associated with significant morbidity and mortality. To target upregulated ErbB dimer expression in this cancer, we developed an autologous CD28-based chimeric antigen receptor T-cell (CAR-T) approach named T4 immunotherapy. Patient-derived T-cells are engineered by retroviral transduction to coexpress a panErbB-specific CAR called T1E28ζ and an IL-4-responsive chimeric cytokine receptor, 4αβ, which allows IL-4-mediated enrichment of transduced cells during manufacture. These cells elicit preclinical antitumor activity against HNSCC and other carcinomas. In this trial, we used intratumoral delivery to mitigate significant clinical risk of on-target off-tumor toxicity owing to low-level ErbB expression in healthy tissues. // Methods: We undertook a phase 1 dose-escalation 3+3 trial of intratumoral T4 immunotherapy in HNSCC (NCT01818323). CAR T-cell batches were manufactured from 40 to 130 mL of whole blood using a 2-week semiclosed process. A single CAR T-cell treatment, formulated as a fresh product in 1–4 mL of medium, was injected into one or more target lesions. Dose of CAR T-cells was escalated in 5 cohorts from 1×107−1×109 T4+ T-cells, administered without prior lymphodepletion. // Results: Despite baseline lymphopenia in most enrolled subjects, the target cell dose was successfully manufactured in all cases, yielding up to 7.5 billion T-cells (67.5±11.8% transduced), without any batch failures. Treatment-related adverse events were all grade 2 or less, with no dose-limiting toxicities (Common Terminology Criteria for Adverse Events V.4.0). Frequent treatment-related adverse events were tumor swelling, pain, pyrexias, chills, and fatigue. There was no evidence of leakage of T4+ T-cells into the circulation following intratumoral delivery, and injection of radiolabeled cells demonstrated intratumoral persistence. Despite rapid progression at trial entry, stabilization of disease (Response Evaluation Criteria in Solid Tumors V.1.1) was observed in 9 of 15 subjects (60%) at 6 weeks post-CAR T-cell administration. Subsequent treatment with pembrolizumab and T-VEC oncolytic virus achieved a rapid complete clinical response in one subject, which was durable for over 3 years. Median overall survival was greater than for historical controls. Disease stabilization was associated with the administration of an immunophenotypically fitter, less exhausted, T4 CAR T-cell product. // Conclusions: These data demonstrate the safe intratumoral administration of T4 immunotherapy in advanced HNSCC

High spatial overlap but diverging age-related trajectories of cortical magnetic resonance imaging markers aiming to represent intracortical myelin and microstructure

ABSTRACT: Statistical effects of cortical metrics derived from standard T1- and T2-weighted magnetic resonance imaging (MRI) images, such as gray–white matter contrast (GWC), boundary sharpness coefficient (BSC), T1-weighted/T2-weighted ratio (T1w/T2w), and cortical thickness (CT), are often interpreted as representing or being influenced by intracortical myelin content with little empirical evidence to justify these interpretations. We first examined spatial correspondence with more biologically specific microstructural measures, and second compared between-marker age-related trends with the underlying hypothesis that different measures primarily driven by similar changes in myelo- and microstructural underpinnings should be highly related. Cortical MRI markers were derived from MRI images of 127 healthy subjects, aged 18–81, using cortical surfaces that were generated with the CIVET 2.1.0 pipeline. Their gross spatial distributions were compared with gene expression-derived cell-type densities, histology-derived cytoarchitecture, and quantitative R1 maps acquired on a subset of participants. We then compared between-marker age-related trends in their shape, direction, and spatial distribution of the linear age effect. The gross anatomical distributions of cortical MRI markers were, in general, more related to myelin and glial cells than neuronal indicators. Comparing MRI markers, our results revealed generally high overlap in spatial distribution (i.e., group means), but mostly divergent age trajectories in the shape, direction, and spatial distribution of the linear age effect. We conclude that the microstructural properties at the source of spatial distributions of MRI cortical markers can be different from microstructural changes that affect these markers in aging

Selective targeting of pro-inflammatory Th1 cells by microRNA-148a-specific antagomirs in vivo.

In T lymphocytes, expression of miR-148a is induced by T-bet and Twist1, and is specific for pro-inflammatory Th1 cells. In these cells, miR-148a inhibits the expression of the pro-apoptotic protein Bim and promotes their survival. Here we use sequence-specific cholesterol-modified oligonucleotides against miR-148a (antagomir-148a) for the selective elimination of pro-inflammatory Th1 cells in vivo. In the murine model of transfer colitis, antagomir-148a treatment reduced the number of pro-inflammatory Th1 cells in the colon of colitic mice by 50% and inhibited miR-148a expression by 71% in the remaining Th1 cells. Expression of Bim protein in colonic Th1 cells was increased. Antagomir-148a-mediated reduction of Th1 cells resulted in a significant amelioration of colitis. The effect of antagomir-148a was selective for chronic inflammation. Antigen-specific memory Th cells that were generated by an acute immune reaction to nitrophenylacetyl-coupled chicken gamma globulin (NP-CGG) were not affected by treatment with antagomir-148a, both during the effector and the memory phase. In addition, antibody titers to NP-CGG were not altered. Thus, antagomir-148a might qualify as an effective drug to selectively deplete pro-inflammatory Th1 cells of chronic inflammation without affecting the protective immunological memory

Percutaneous revascularization for ischemic left ventricular dysfunction: Cost-effectiveness analysis of the REVIVED-BCIS2 trial

BACKGROUND: Percutaneous coronary intervention (PCI) is frequently undertaken in patients with ischemic left ventricular systolic dysfunction. The REVIVED (Revascularization for Ischemic Ventricular Dysfunction)-BCIS2 (British Cardiovascular Society-2) trial concluded that PCI did not reduce the incidence of all-cause death or heart failure hospitalization; however, patients assigned to PCI reported better initial health-related quality of life than those assigned to optimal medical therapy (OMT) alone. The aim of this study was to assess the cost-effectiveness of PCI+OMT compared with OMT alone. METHODS: REVIVED-BCIS2 was a prospective, multicenter UK trial, which randomized patients with severe ischemic left ventricular systolic dysfunction to either PCI+OMT or OMT alone. Health care resource use (including planned and unplanned revascularizations, medication, device implantation, and heart failure hospitalizations) and health outcomes data (EuroQol 5-dimension 5-level questionnaire) on each patient were collected at baseline and up to 8 years post-randomization. Resource use was costed using publicly available national unit costs. Within the trial, mean total costs and quality-adjusted life-years (QALYs) were estimated from the perspective of the UK health system. Cost-effectiveness was evaluated using estimated mean costs and QALYs in both groups. Regression analysis was used to adjust for clinically relevant predictors. RESULTS: Between 2013 and 2020, 700 patients were recruited (mean age: PCI+OMT=70 years, OMT=68 years; male (%): PCI+OMT=87, OMT=88); median follow-up was 3.4 years. Over all follow-ups, patients undergoing PCI yielded similar health benefits at higher costs compared with OMT alone (PCI+OMT: 4.14 QALYs, £22 352; OMT alone: 4.16 QALYs, £15 569; difference: −0.015, £6782). For both groups, most health resource consumption occurred in the first 2 years post-randomization. Probabilistic results showed that the probability of PCI being cost-effective was 0. CONCLUSIONS: A minimal difference in total QALYs was identified between arms, and PCI+OMT was not cost-effective compared with OMT, given its additional cost. A strategy of routine PCI to treat ischemic left ventricular systolic dysfunction does not seem to be a justifiable use of health care resources in the United Kingdom

Arrhythmia and death following percutaneous revascularization in ischemic left ventricular dysfunction: Prespecified analyses from the REVIVED-BCIS2 trial

BACKGROUND: Ventricular arrhythmia is an important cause of mortality in patients with ischemic left ventricular dysfunction. Revascularization with coronary artery bypass graft or percutaneous coronary intervention is often recommended for these patients before implantation of a cardiac defibrillator because it is assumed that this may reduce the incidence of fatal and potentially fatal ventricular arrhythmias, although this premise has not been evaluated in a randomized trial to date. METHODS: Patients with severe left ventricular dysfunction, extensive coronary disease, and viable myocardium were randomly assigned to receive either percutaneous coronary intervention (PCI) plus optimal medical and device therapy (OMT) or OMT alone. The composite primary outcome was all-cause death or aborted sudden death (defined as an appropriate implantable cardioverter defibrillator therapy or a resuscitated cardiac arrest) at a minimum of 24 months, analyzed as time to first event on an intention-to-treat basis. Secondary outcomes included cardiovascular death or aborted sudden death, appropriate implantable cardioverter defibrillator (ICD) therapy or sustained ventricular arrhythmia, and number of appropriate ICD therapies. RESULTS: Between August 28, 2013, and March 19, 2020, 700 patients were enrolled across 40 centers in the United Kingdom. A total of 347 patients were assigned to the PCI+OMT group and 353 to the OMT alone group. The mean age of participants was 69 years; 88% were male; 56% had hypertension; 41% had diabetes; and 53% had a clinical history of myocardial infarction. The median left ventricular ejection fraction was 28%; 53.1% had an implantable defibrillator inserted before randomization or during follow-up. All-cause death or aborted sudden death occurred in 144 patients (41.6%) in the PCI group and 142 patients (40.2%) in the OMT group (hazard ratio, 1.03 [95% CI, 0.82–1.30]; P =0.80). There was no between-group difference in the occurrence of any of the secondary outcomes. CONCLUSIONS: PCI was not associated with a reduction in all-cause mortality or aborted sudden death. In patients with ischemic cardiomyopathy, PCI is not beneficial solely for the purpose of reducing potentially fatal ventricular arrhythmias. REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01920048

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste

INJURY PROFILE IN WOMEN SHOTOKAN KARATE CHAMPIONSHIPS IN IRAN(2004-2005)

The aims of this paper were to record injury rates among Iranian women competitive Shotokan karate athletes and propose possible predisposing factors. A prospective recording of the injuries resulting from all matches in 6 consecutive women national Shotokan Karate Championships in all age groups in Iran (season 2004-2005) was performed. Data recorded included demographic characteristics (Age and Weight), athletic background (rank, years of experience, time spent training and previous injuries), type, location and reason for the injury, and the result of the match. Results indicate 186 recorded injuries from a total of 1139 bouts involving 1019 athletes, therefore there were 0.163 injury per bout [C.I. 95%: 0.142-0.184] and 183 injuries per 1000 athletes [C.I. 95%: 159-205]. Injuries were most commonly located in the head and neck (55.4%) followed by the lower limb (21%), upper limb (12.9%) and trunk (10.8%). Punches (48. 4%) were associated with more injuries than kicks (33.3%). The injuries consisted of muscle strain and contusion (81, 43.6%), hematoma and epistaxis (49, 26.3%), lacerations and abrasions (28, 15. 1%), concussion (13, 7%), tooth avulsion or subluxation (3, 1.6%), joint dislocation (3, 1.6%) and fractures (3, 1.6%). In conclusion, as the majority of injuries are minor, and severe or longstanding injuries are uncommon, it can be argued that shotokan karate is a relatively safe for females, despite its image as a combat sport, where ostensibly the aim appears to injure your opponent. Further research is needed to evaluate the effective strategies to minimize the risk of injurie