Monocyte migration profiles define disease severity in acute COVID-19 and unique features of long COVID.

BACKGROUND: COVID-19 is associated with a dysregulated immune response but it is unclear how immune dysfunction contributes to the chronic morbidity persisting in many COVID-19 patients during convalescence (long COVID). METHODS: We assessed phenotypical and functional changes of monocytes in COVID-19 patients during hospitalisation and up to 9 months of convalescence following COVID-19, respiratory syncytial virus or influenza A. Patients with progressive fibrosing interstitial lung disease were included as a positive control for severe, ongoing lung injury. RESULTS: Monocyte alterations in acute COVID-19 patients included aberrant expression of leukocyte migration molecules, continuing into convalescence (n=142) and corresponding with specific symptoms of long COVID. Long COVID patients with unresolved lung injury, indicated by sustained shortness of breath and abnormal chest radiology, were defined by high monocyte expression of C-X-C motif chemokine receptor 6 (CXCR6) (p<0.0001) and adhesion molecule P-selectin glycoprotein ligand 1 (p<0.01), alongside preferential migration of monocytes towards the CXCR6 ligand C-X-C motif chemokine ligand 16 (CXCL16) (p<0.05), which is abundantly expressed in the lung. Monocyte CXCR6 and lung CXCL16 were heightened in patients with progressive fibrosing interstitial lung disease (p<0.001), confirming a role for the CXCR6-CXCL16 axis in ongoing lung injury. Conversely, monocytes from long COVID patients with ongoing fatigue exhibited a sustained reduction of the prostaglandin-generating enzyme cyclooxygenase 2 (p<0.01) and CXCR2 expression (p<0.05). These monocyte changes were not present in respiratory syncytial virus or influenza A convalescence. CONCLUSIONS: Our data define unique monocyte signatures that define subgroups of long COVID patients, indicating a key role for monocyte migration in COVID-19 pathophysiology. Targeting these pathways may provide novel therapeutic opportunities in COVID-19 patients with persistent morbidity.

Composition-Dependent Charge Transport in Boron Carbides Alloyed with Aromatics: Plasma Enhanced Chemical Vapor Deposition Aniline/Orthocarborane Films.

Boron carbide films, alloyed with aniline moieties, were deposited by plasma enhanced chemical vapor deposition (PECVD) from aniline and orthocarborane precursors and were found to exhibit composition-dependent drift carrier lifetimes as derived from I( V) and C( V)) measurements. For a film with an aniline/carborane ratio of 5:1, the effective drift carrier lifetimes are ∼80 µs at low bias voltage but quickly drop to a few microseconds with increasing bias. A film with a 10:1 aniline/carborane ratio, however, exhibited lifetimes of ∼6 µs, or less, at 1 kHz, and much smaller values at 10 kHz. These lifetimes are orders of magnitude longer than those in polyaniline films and comparable to those in PECVD carborane films without aromatic content. X-ray photoelectron spectroscopy (XPS), FTIR, and ellipsometry, combined with density functional theory (DFT)-based cluster calculations, indicate that aniline and orthocarborane moieties are largely intact within the films. Bonding occurs primarily between aniline C sites and carborane B sites, and the aniline coordination number per carborane icosahedron is ∼2 as the aniline/carborane ratio is increased from 3:1 to 10:1. This aniline/carborane coordination ratio independent of aniline/orthocarborane stoichiometry is consistent with the dependence of charge transport properties on aniline film content at high bias voltage.

The Role of Optical Phonon Confinement in the Infrared Dielectric Response of III-V Superlattices.

Polar dielectrics are key materials of interest for infrared (IR) nanophotonic applications due to their ability to host phonon-polaritons that allow for low-loss, subdiffractional control of light. The properties of phonon-polaritons are limited by the characteristics of optical phonons, which are nominally fixed for most "bulk" materials. Superlattices composed of alternating atomically thin materials offer control over crystal anisotropy through changes in composition, optical phonon confinement, and the emergence of new modes. In particular, the modified optical phonons in superlattices offer the potential for so-called crystalline hybrids whose IR properties cannot be described as a simple mixture of the bulk constituents. To date, however, studies have primarily focused on identifying the presence of new or modified optical phonon modes rather than assessing their impact on the IR response. This study focuses on assessing the impact of confined optical phonon modes on the hybrid IR dielectric function in superlattices of GaSb and AlSb. Using a combination of first principles theory, Raman, FTIR, and spectroscopic ellipsometry, the hybrid dielectric function is found to track the confinement of optical phonons, leading to optical phonon spectral shifts of up to 20 cm-1 . These results provide an alternative pathway toward designer IR optical materials.

Safer, silencing-resistant lentiviral vectors: optimization of the ubiquitous chromatin-opening element through elimination of aberrant splicing.

Gammaretroviral and lentiviral vectors have been used successfully in several clinical gene therapy trials, although powerful enhancer elements have caused insertional mutagenesis and clonal dysregulation. Self-inactivating vectors with internal heterologous regulatory elements have been developed as potentially safer and more effective alternatives. Lentiviral vectors containing a ubiquitous chromatin opening element from the human HNRPA2B1-CBX3 locus (A2UCOE), which allows position-independent, long-term transgene expression, are particularly promising. In a recently described assay, aberrantly spliced mRNA transcripts initiated in the vector A2UCOE sequence were found to lead to upregulation of growth hormone receptor gene (Ghr) expression in transduced murine Bcl-15 cells. Aberrant hybrid mRNA species formed between A2UCOE and a number of other cellular genes were also detected in transduced human PLB-985 myelomonocytic cells. Modification of the A2UCOE by mutation or deletion of recognized and potential cryptic splice donor sites was able to abrogate these splicing events and hybrid mRNA formation in Bcl-15 cells. This modification did not compromise A2UCOE regulatory activity in terms of resistance to CpG methylation and gene silencing in murine P19 embryonic carcinoma cells. These refined A2UCOE regulatory elements are likely to improve intrinsic biosafety and may be particularly useful for a number of clinical applications where robust gene expression is desirable.

The ß-globin locus control region in combination with the EF1α short promoter allows enhanced lentiviral vector-mediated erythroid gene expression with conserved multilineage activity.

Some gene therapy strategies are compromised by the levels of gene expression required for therapeutic benefit, and also by the breadth of cell types that require correction. We designed a lentiviral vector system in which a transgene is under the transcriptional control of the short form of constitutively acting elongation factor 1α promoter (EFS) combined with essential elements of the locus control region of the ß-globin gene (ß-LCR). We show that the ß-LCR can upregulate EFS activity specifically in erythroid cells but does not alter EFS activity in myeloid or lymphoid cells. Experiments using the green fluorescent protein (GFP) reporter or the human adenosine deaminase (ADA) gene demonstrate 3-7 times upregulation in vitro but >20 times erythroid-specific upregulation in vivo, the effects of which were sustained for 1 year. The addition of the ß-LCR did not alter the mutagenic potential of the vector in in vitro mutagenesis (IM) assays although microarray analysis showed that the ß-LCR upregulates ~9% of neighboring genes. This vector design therefore combines the benefits of multilineage gene expression with high-level erythroid expression, and has considerable potential for correction of multisystem diseases including certain lysosomal storage diseases through a hematopoietic stem cell (HSC) gene therapy approach.

Autoantibodies Neutralizing Type III Interferons Are Uncommon in Patients with Severe Coronavirus Disease 2019 Pneumonia.

Autoantibodies (AABs) neutralizing type I interferons (IFN) underlie about 15% of cases of critical coronavirus disease 2019 (COVID-19) pneumonia. The impact of autoimmunity toward type III IFNs remains unexplored. We included samples from 1,002 patients with COVID-19 (50% with severe disease) and 1,489 SARS-CoV-2-naive individuals. We studied the prevalence and neutralizing capacity of AABs toward IFNλ and IFNα. Luciferase-based immunoprecipitation method was applied using pooled IFNα (subtypes 1, 2, 8, and 21) or pooled IFNλ1-IFNλ3 as antigens, followed by reporter cell-based neutralization assay. In the SARS-CoV-2-naive cohort, IFNλ AABs were more common (8.5%) than those targeting IFNα2 (2.9%) and were related with older age. In the COVID-19 cohort the presence of autoreactivity to IFNλ did not associate with severe disease [odds ratio (OR) 0.84; 95% confidence interval (CI) 0.40-1.73], unlike to IFNα (OR 4.88; 95% CI 2.40-11.06; P < 0.001). Most IFNλ AAB-positive COVID-19 samples (67%) did not neutralize any of the 3 IFNλ subtypes. Pan-IFNλ neutralization occurred in 5 patients (0.50%), who all suffered from severe COVID-19 pneumonia, and 4 of them neutralized IFNα2 in addition to IFNλ. Overall, AABs to type III IFNs are rarely neutralizing, and do not seem to predispose to severe COVID-19 pneumonia on their own.

Large scale clinical trials: lessons from the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic has presented substantial new challenges to clinical and research teams. Our objective was to analyse the experience of investigators and research delivery staff regarding the research response to COVID-19 in order to identify these challenges as well as solutions for future pandemic planning. METHODS: We conducted a survey of diverse research staff involved in delivery of COVID-19 clinical trials across the UK. This was delivered online across centres linked to the NIHR Respiratory Translational Research Collaboration. Responses were analysed using a formal thematic analysis approach to identify common themes and recommendations. RESULTS: 83 survey participants from ten teaching hospitals provided 922 individual question responses. Respondents were involved in a range of research delivery roles but the largest cohort (60%) was study investigators. A wide range of research experiences were captured, including early and late phase trials. Responses were coded into overarching themes. Among common observations, complex protocols without adaptation to a pandemic were noted to have hampered recruitment. Recommendations included the need to develop and test pandemic-specific protocols, and make use of innovations in information technology. Research competition needs to be avoided and drug selection processes should be explicitly transparent. CONCLUSIONS: Delivery of clinical trials, particularly earlier phase trials, in a pandemic clinical environment is highly challenging, and was reactive rather than anticipatory. Future pandemic studies should be designed and tested in advance, making use of pragmatic study designs as far as possible and planning for integration between early and later phase trials and regulatory frameworks.

Integrated miRNA/cytokine/chemokine profiling reveals severity-associated step changes and principal correlates of fatality in COVID-19.

Inflammatory cytokines and chemokines (CC) drive COVID-19 pathology. Yet, patients with similar circulating CC levels present with different disease severity. Here, we determined 171 microRNAomes from 58 hospitalized COVID-19 patients (Cohort 1) and levels of 25 cytokines and chemokines (CC) in the same samples. Combining microRNA (miRNA) and CC measurements allowed for discrimination of severe cases with greater accuracy than using miRNA or CC levels alone. Severity group-specific associations between miRNAs and COVID-19-associated CC (e.g., IL6, CCL20) or clinical hallmarks of COVID-19 (e.g., neutrophilia, hypoalbuminemia) separated patients with similar CC levels but different disease severity. Analysis of an independent cohort of 108 patients from a different center (Cohort 2) demonstrated feasibility of CC/miRNA profiling in leftover hospital blood samples with similar severe disease CC and miRNA profiles, and revealed CCL20, IL6, IL10, and miR-451a as key correlates of fatal COVID-19. These findings highlight that systemic miRNA/CC networks underpin severe COVID-19.

Effect of the internal promoter on insertional gene activation by lentiviral vectors with an intact HIV long terminal repeat.

Insertional mutagenesis by viral vectors is a problem in gene therapy. We recently reported that lentiviral vectors with an intact HIV long terminal repeat (LTR) caused insertional gene activation by transcripts from the 5' LTR splicing to an adjacent gene. Here we demonstrate that the level of transcription from the 5' LTR, and also insertional gene activation, is dependent on the internal promoter in the vector. We also show that there are more transcripts originating from the 5' LTR than from, or reading through, the 3' LTR. This study will allow the design of safer lentiviral vectors for applications in which an intact HIV LTR is required.

Alterations in T and B cell function persist in convalescent COVID-19 patients.

BACKGROUND: Emerging studies indicate that some coronavirus disease 2019 (COVID-19) patients suffer from persistent symptoms, including breathlessness and chronic fatigue; however, the long-term immune response in these patients presently remains ill-defined. METHODS: Here, we describe the phenotypic and functional characteristics of B and T cells in hospitalized COVID-19 patients during acute disease and at 3-6 months of convalescence. FINDINGS: We report that the alterations in B cell subsets observed in acute COVID-19 patients were largely recovered in convalescent patients. In contrast, T cells from convalescent patients displayed continued alterations with persistence of a cytotoxic program evident in CD8+ T cells as well as elevated production of type 1 cytokines and interleukin-17 (IL-17). Interestingly, B cells from patients with acute COVID-19 displayed an IL-6/IL-10 cytokine imbalance in response to Toll-like receptor activation, skewed toward a pro-inflammatory phenotype. Whereas the frequency of IL-6+ B cells was restored in convalescent patients irrespective of clinical outcome, the recovery of IL-10+ B cells was associated with the resolution of lung pathology. CONCLUSIONS: Our data detail lymphocyte alterations in previously hospitalized COVID-19 patients up to 6 months following hospital discharge and identify 3 subgroups of convalescent patients based on distinct lymphocyte phenotypes, with 1 subgroup associated with poorer clinical outcome. We propose that alterations in B and T cell function following hospitalization with COVID-19 could affect longer-term immunity and contribute to some persistent symptoms observed in convalescent COVID-19 patients. FUNDING: Provided by UKRI, Lister Institute of Preventative Medicine, the Wellcome Trust, The Kennedy Trust for Rheumatology Research, and 3M Global Giving.

Insertional gene activation by lentiviral and gammaretroviral vectors.

Gammaretroviral and lentiviral vectors are promising tools for gene therapy, but they can be oncogenic. The development of safer vectors depends on a quantitative assay for insertional mutagenesis. Here we report a rapid, inexpensive, and reproducible assay which uses a murine cell line to measure the frequency of interleukin-3 (IL-3)-independent mutants. Lentiviral and gammaretroviral vectors cause insertional mutagenesis at similar frequencies; however, they use different mechanisms. Human immunodeficiency virus (HIV)-based vectors generate mutants by insertion only into the growth hormone receptor (Ghr) locus. The HIV enhancer/promoter is active in the absence of the HIV Tat protein in this locus, and an HIV/Ghr spliced transcript expresses GHR and cells respond to GH. Deletion of the enhancer/promoter in a self-inactivating HIV-based vector prevents this mechanism of insertional mutagenesis. In contrast, gammaretroviral vectors insert into other loci, including IL-3 and genes identified as common insertion sites in the Retroviral Tagged Cancer Gene Database (RTCGD).

Tunable cavity-enhanced terahertz frequency-domain optical Hall effect.

Presented here is the development and demonstration of a tunable cavity-enhanced terahertz (THz) frequency-domain optical Hall effect (OHE) technique. The cavity consists of at least one fixed and one tunable Fabry-Pérot resonator. The approach is suitable for the enhancement of the optical signatures produced by the OHE in semi-transparent conductive layer structures with plane parallel interfaces. Tuning one of the cavity parameters, such as the external cavity thickness, permits shifting of the frequencies of the constructive interference and provides substantial enhancement of the optical signatures produced by the OHE. A cavity-tuning optical stage and gas flow cell are used as examples of instruments that exploit tuning an external cavity to enhance polarization changes in a reflected THz beam. Permanent magnets are used to provide the necessary external magnetic field. Conveniently, the highly reflective surface of a permanent magnet can be used to create the tunable external cavity. The signal enhancement allows the extraction of the free charge carrier properties of thin films and can eliminate the need for expensive superconducting magnets. Furthermore, the thickness of the external cavity establishes an additional independent measurement condition, similar to, for example, the magnetic field strength, THz frequency, and angle of incidence. A high electron mobility transistor (HEMT) structure and epitaxial graphene are studied as examples. The tunable cavity-enhancement effect provides a maximum increase of more than one order of magnitude in the change of certain polarization components for both the HEMT structure and epitaxial graphene at particular frequencies and external cavity sizes.

Outcomes Evaluated in Controlled Clinical Trials on the Management of COVID-19: A Methodological Systematic Review.

It is crucial that randomized controlled trials (RCTs) on the management of coronavirus disease 2019 (COVID-19) evaluate the outcomes that are critical to patients and clinicians, to facilitate relevance, interpretability, and comparability. This methodological systematic review describes the outcomes evaluated in 415 RCTs on the management of COVID-19, that were registered with ClinicalTrials.gov, by 5 May 2020, and the instruments used to measure these outcomes. Significant heterogeneity was observed in the selection of outcomes and instruments. Mortality, adverse events and treatment success or failure are only evaluated in 64.4%, 48.4% and 43% of the included studies, respectively, while other outcomes are selected less often. Studies focusing on more severe presentations (hospitalized patients or requiring intensive care) most frequently evaluate mortality (72.5%) and adverse events (55.6%), while hospital admission (50.8%) and viral detection/load (55.6%) are most frequently assessed in the community setting. Outcome measurement instruments are poorly reported and heterogeneous. Follow-up does not exceed one month in 64.3% of these earlier trials, and long-term COVID-19 burden is rarely assessed. The methodological issues identified could delay the introduction of potentially life-saving treatments in clinical practice. Our findings demonstrate the need for greater consistency, to enable decision makers to compare and contrast studies.

Longitudinal immune profiling reveals key myeloid signatures associated with COVID-19.

COVID-19 pathogenesis is associated with an exaggerated immune response. However, the specific cellular mediators and inflammatory components driving diverse clinical disease outcomes remain poorly understood. We undertook longitudinal immune profiling on both whole blood and peripheral blood mononuclear cells (PBMCs) of hospitalized patients during the peak of the COVID-19 pandemic in the UK. Here, we report key immune signatures present shortly after hospital admission that were associated with the severity of COVID-19. Immune signatures were related to shifts in neutrophil to T cell ratio, elevated serum IL-6, MCP-1 and IP-10, and most strikingly, modulation of CD14+ monocyte phenotype and function. Modified features of CD14+ monocytes included poor induction of the prostaglandin-producing enzyme, COX-2, as well as enhanced expression of the cell cycle marker Ki-67. Longitudinal analysis revealed reversion of some immune features back to the healthy median level in patients with a good eventual outcome. These findings identify previously unappreciated alterations in the innate immune compartment of COVID-19 patients and lend support to the idea that therapeutic strategies targeting release of myeloid cells from bone marrow should be considered in this disease. Moreover, they demonstrate that features of an exaggerated immune response are present early after hospital admission suggesting immune-modulating therapies would be most beneficial at early timepoints.

Attending community-based lung cancer screening influences smoking behaviour in deprived populations.

OBJECTIVES: The impact of lung cancer screening on smoking is unclear, especially in deprived populations who are underrepresented in screening trials. The aim of this observational cohort study was to investigate whether a community-based lung cancer screening programme influenced smoking behaviour and smoking attitude in socio-economically deprived populations. MATERIAL AND METHODS: Ever-smokers, age 55-74, registered at participating General Practices were invited to a community-based Lung Health Check (LHC). This included an assessment of respiratory symptoms, lung cancer risk (PLCOm2012), spirometry and signposting to stop smoking services. Those at high risk (PLCOM2012≥1.51%) were offered annual low-dose CT screening over two rounds. Self-reported smoking status and behaviour were recorded at the LHC and again 12 months later, when attitudes to smoking were also assessed. RESULTS: 919 participants (51% women) were included in the analysis (77% of attendees); median deprivation rank in the lowest decile for England. At baseline 50.3% were current smokers. One-year quit rate was 10.2%, quitting was associated with increased baseline symptoms (adjOR 2.62, 95% CI 1.07-6.41; p = 0.035) but not demographics or screening results. 55% attributed quitting to the LHC. In current smokers, 44% reported the LHC had made them consider stopping, 29% it made them try to stop and 25% made them smoke less whilst only 1.7% and 0.7% said it made them worry less about smoking or think it acceptable to smoke. CONCLUSIONS: Our data suggest a community-based lung cancer screening programme in deprived areas positively impacts smoking behaviour, with no evidence of a 'licence to smoke' in those screened.

Electromagnon excitation in cupric oxide measured by Fabry-Pérot enhanced terahertz Mueller matrix ellipsometry.

Here we present the use of Fabry-Pérot enhanced terahertz (THz) Mueller matrix ellipsometry to measure an electromagnon excitation in monoclinic cupric oxide (CuO). As a magnetically induced ferroelectric multiferroic, CuO exhibits coupling between electric and magnetic order. This gives rise to special quasiparticle excitations at THz frequencies called electromagnons. In order to measure the electromagnons in CuO, we exploit single-crystal CuO as a THz Fabry-Pérot cavity to resonantly enhance the excitation's signature. This enhancement technique enables the complex index of refraction to be extracted. We observe a peak in the absorption coefficient near 0.705 THz and 215 K, which corresponds to the electromagnon excitation. This absorption peak is observed along only one major polarizability axis in the monoclinic a-c plane. We show the excitation can be represented using the Lorentz oscillator model, and discuss how these Lorentz parameters evolve with temperature. Our findings are in excellent agreement with previous characterizations by THz time-domain spectroscopy (THz-TDS), which demonstrates the validity of this enhancement technique.

Analysis of Robotic Procedural Times Using Colpassist Versus End-to-End Anastomosis Sizer for Robotic-Assisted Sacrocolpopexy: A Randomized Controlled Trial.

OBJECTIVE: The impact of vaginal instrumentation on operative time for robotic-assisted sacrocolpopexy (RSC) is not well understood. Colpassist (Boston Scientific, Quincy, Mass) is a vaginal positioning device designed to improve exposure with a curved double-sided handle that can be adapted to patient anatomy with 2 different sized functional ends. We sought to compare operative time for RSC using a Colpassist compared with a traditional end-to-end anastomosis sizer (EEAS). METHODS: This was a single-center randomized controlled trial of consecutive women undergoing RSC for apical pelvic organ prolapse. Participants were randomized to undergo RSC using either Colpassist or EEAS. The primary outcome was total operative time for the steps of RSC requiring use of a vaginal positioning device. Secondary outcomes included injury rates and satisfaction of the participants. RESULTS: Fifty-two women were enrolled (25 Colpassist, 27 EEAS). For the primary outcome, there was no significant difference in total operative time between procedures performed with Colpassist versus EEAS (P = 0.15). However, 16 (64%) of the 25 Colpassist arm versus 0 of EEAS (P < 0.01) required use of an alternative vaginal positioning device intraoperatively. Comparing secondary outcomes, there were no differences in rate of intraoperative injury between groups. Surgeon and fellow satisfaction scores were lower with Colpassist (P < 0.01). Surgical vaginal assistants were equally satisfied with both vaginal positioning devices (P > 0.05). CONCLUSIONS: Colpassist is a potential alternative to EEAS with no significant difference in operative time or complication rate. However, case completion with Colpassist was significantly lower than EEAS and was associated with lower surgeon and fellow satisfaction.

Progress and prospects of early detection in lung cancer.

Lung cancer is the leading cause of cancer-related death in the world. It is broadly divided into small cell (SCLC, approx. 15% cases) and non-small cell lung cancer (NSCLC, approx. 85% cases). The main histological subtypes of NSCLC are adenocarcinoma and squamous cell carcinoma, with the presence of specific DNA mutations allowing further molecular stratification. If identified at an early stage, surgical resection of NSCLC offers a favourable prognosis, with published case series reporting 5-year survival rates of up to 70% for small, localized tumours (stage I). However, most patients (approx. 75%) have advanced disease at the time of diagnosis (stage III/IV) and despite significant developments in the oncological management of late stage lung cancer over recent years, survival remains poor. In 2014, the UK Office for National Statistics reported that patients diagnosed with distant metastatic disease (stage IV) had a 1-year survival rate of just 15-19% compared with 81-85% for stage I.