Determination of Fibrin Fiber Diameter Using Scanning Electron Microscopy and Image Processing Software

Injectable, biodegradable scaffolds that mimic local tissue properties have great potential for aiding and accelerating the natural wound healing process. Ideally, scaffolds will have physical properties e.g., stiffness and microstructures, that are comparable to the tissue in which they will be applied. Fibrin is an insoluble protein that is formed in vivo during hemostasis via action of the enzyme thrombin on the soluble protein fibrinogen. It acts as a glue that holds together a loose platelet plug - a blood clot - that is degraded as wound healing progresses. The stiffness of this material can be easily tuned by adjusting its composition, as has been previously shown. These combined factors make fibrin a suitable scaffolding candidate for promoting cell delivery to wound sites, cell growth, and proliferation which are important parameters influencing the healing process. Scanning electron microscopy as well as other techniques are currently being leveraged in order to better understand fibrin's microstructure. Fibrin hydrogels are prepared in vitro by mixing fibrinogen, thrombin, and CaCl2 at various compositions. After additional processing, samples are then dried using either critical point drying or freeze-drying techniques to retain structure, and are subsequently sputter coated with gold/palladium, and imaged using a Hitachi SU7000. A MATLAB script was created that allows for the random selection and analysis of fibers. The resulting image is transferred into ImageJ where fiber sizes are measured. Average fiber diameter for fibrin gels prepared using fibrinogen at 6mg/ml and thrombin at 1U/ml is estimated to be 77nm

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2,3,4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

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

*WINNER* Rheological and Turbidimetric Characterization of Early-phase Wound Gels

Treatment of dermal wounds and development of new techniques by which dermal wound healing can be accelerated with minimal scarring are of global interest. During the early stages of the wound healing process, a loose platelet plug is stabilized by the formation of a fibrin gel matrix. A fibrin gel is formed when the protein fibrinogen is enzymatically cleaved by thrombin and crosslinked by Factor XIII. These fibrin matrices help to halt blood flow from the wounded site and serve as a scaffold by which cell transport and adhesion may occur. Various medical disorders, deficiencies, and diseases can result in abnormal wound healing, i.e. scarring or inability to form stable, lasting clots. The study of these bio-gels is expected to result in advancements in wound healing techniques and a better understanding of the behavior of dermal wounds, transport of cellular and other items through such wounds, and resultant scarring control. Towards this end, rheological techniques were explored to characterize structural properties of such early-phase wound media during gel formation. Fibrin gels were prepared using 1, 3, 6, and 12 mg/ml fibrinogen, 1 U/ml thrombin, and 5mM CaCl2, final concentrations. Rheology and turbidity data indicate that gels formed in the presence of higher fibrinogen concentrations develop more rigid structures sooner than those formed at lower fibrinogen concentrations. Such results provide a foundation for future studies to explore the effects of mixing and the influence of modified versions of fibrinogen on gel properties and species transport through such gels

Simulation of Microfluidics in a Meandering Channel and Viability of a Manufactured Microfluidic Device for Medical Applications

A study was conducted on microfluidic mixers for the medical applications of screening alpha-1 antitrypsin deficiency and wound transport phenomena. A micromixer utilizes a microfluidic channel to mix feed streams, such as elastase and substrate or fibrinogen and thrombin. There are several micromixer designs, the simplest being a zig-zag channel. The research focuses on exploring how the design of microfluidic channel affects the mixing, using experimental and simulation approaches. A microfluidic mixer must be constructed to conduct microfluidics experiments, and a simulation is built using software as a precursory step to experimentation. The simulations were constructed and calculated using ComSol® . After building the initial mixer channel, the particular physics and conditions were applied in order to produce a concentration profile. The steps to manufacture a micromixer include: designing the zig-zag template, preparing a PDMS gel, submerging the template in PDMS, curing PDMS, dissolving the template, then running microfluidic experiments. Inconsistencies were found in the produced PDMS gels for each trial. Some gels were solid and firm, while others remained viscous. Also, the ABS templates were not easily dissolved with acetone. The simulations yielded results about the relationship between geometry, diffusion coefficient, degree of mixing, and residence time. To further the research, the procedure must be refined to produce gels that are consistent for each trial. Some recommendations include: finding the ideal ratio of elastomer to curing agent, finding the ideal oven temperature and heating time, finalizing a micromixer geometry, and finding the solubility of ABS in concentrated acetone