Aptamer BT200 blocks interaction of K1405-1408 in the VWF-A1 domain with macrophage LRP1.

Rondaptivan pegol (previously BT200) is a PEGylated RNA aptamer that binds to the A1 domain of VWF. Recent clinical trials demonstrated that BT200 significantly increased plasma VWF-FVIII levels by attenuating VWF clearance. The biological mechanism(s) through which BT200 attenuates in vivo clearance of VWF have not been defined. We hypothesized that BT200 interaction with the VWF-A1 domain may increase plasma VWF levels by attenuating macrophage-mediated clearance. We observed that full length- and VWF-A1A2A3 binding to macrophages, and VWF-A1 domain binding to LRP1 cluster II and cluster IV, were concentration-dependently inhibited by BT200. Additionally, full length VWF binding to LRP1 expressed on HEK293T (HEK-LRP1) cells was also inhibited by BT200. Importantly, BT200 interacts with the VWF-A1 domain in proximity to a conserved cluster of four lysine residues (K1405, K1406, K1407 and K1408). Alanine mutagenesis of this K1405-K1408 cluster (VWF-4A) significantly (p<0.001) attenuated binding of VWF to both LRP1 clusters II and IV. Furthermore, in vivo clearance of VWF-4A was significantly (p<0.001) reduced compared to wild type VWF. BT200 did not significantly inhibit binding of VWF-4A to LRP1 cluster IV or HEK-LRP1 cells. Finally, BT200 interaction with the VWF-A1 domain also inhibited binding to macrophage galactose lectin (MGL) and the SR-AI scavenger receptor. Collectively, our findings demonstrate that BT200 prolongs VWF half-life by attenuating macrophage-mediated clearance and specifically the interaction of K1405-1408 in the VWF-A1 domain with macrophage LRP1. These data support the concept that targeted inhibition of VWF clearance pathways represent a novel therapeutic approach for VWD and hemophilia A.

Macrophage Galactose Lectin Contributes to the Regulation of FVIII (Factor VIII) Clearance in Mice-Brief Report.

BACKGROUND: Although most plasma FVIII (Factor VIII) circulates in complex with VWF (von Willebrand factor), a minority (3%-5%) circulates as free-FVIII, which is rapidly cleared. Consequently, 20% of total FVIII may be cleared as free-FVIII. Critically, the mechanisms of free-FVIII clearance remain poorly understood. However, recent studies have implicated the MGL (macrophage galactose lectin) in modulating VWF clearance. METHODS: Since VWF and FVIII share similar glycosylation, we investigated the role of MGL in FVIII clearance. FVIII binding to MGL was assessed in immunosorbent and cell-based assays. In vivo, FVIII clearance was assessed in MGL1-/- and VWF-/-/FVIII-/- mice. RESULTS: In vitro-binding studies identified MGL as a novel macrophage receptor that binds free-FVIII in a glycan-dependent manner. MGL1-/- and MGL1-/- mice who received an anti-MGL1/2 blocking antibody both showed significantly increased endogenous FVIII activity compared with wild-type mice (P=0.036 and P<0.0001, respectively). MGL inhibition also prolonged the half-life of infused FVIII in FVIII-/- mice. To assess whether MGL plays a role in the clearance of free FVIII in a VWF-independent manner, in vivo clearance experiments were repeated in dual VWF-/-/FVIII-/- mice. Importantly, the rapid clearance of free FVIII in VWF-/-/FVIII-/- mice was significantly (P=0.012) prolonged in the presence of anti-MGL1/2 antibodies. Finally, endogenous plasma FVIII levels in VWF-/- mice were significantly increased following MGL inhibition (P=0.016). CONCLUSIONS: Cumulatively, these findings demonstrate that MGL plays an important role in regulating macrophage-mediated clearance of both VWF-bound FVIII and free-FVIII in vivo. We propose that this novel FVIII clearance pathway may be of particular clinical importance in patients with type 2N or type 3 Von Willebrand disease.

Comparing code-free and bespoke deep learning approaches in ophthalmology.

AIM: Code-free deep learning (CFDL) allows clinicians without coding expertise to build high-quality artificial intelligence (AI) models without writing code. In this review, we comprehensively review the advantages that CFDL offers over bespoke expert-designed deep learning (DL). As exemplars, we use the following tasks: (1) diabetic retinopathy screening, (2) retinal multi-disease classification, (3) surgical video classification, (4) oculomics and (5) resource management. METHODS: We performed a search for studies reporting CFDL applications in ophthalmology in MEDLINE (through PubMed) from inception to June 25, 2023, using the keywords 'autoML' AND 'ophthalmology'. After identifying 5 CFDL studies looking at our target tasks, we performed a subsequent search to find corresponding bespoke DL studies focused on the same tasks. Only English-written articles with full text available were included. Reviews, editorials, protocols and case reports or case series were excluded. We identified ten relevant studies for this review. RESULTS: Overall, studies were optimistic towards CFDL's advantages over bespoke DL in the five ophthalmological tasks. However, much of such discussions were identified to be mono-dimensional and had wide applicability gaps. High-quality assessment of better CFDL applicability over bespoke DL warrants a context-specific, weighted assessment of clinician intent, patient acceptance and cost-effectiveness. We conclude that CFDL and bespoke DL are unique in their own assets and are irreplaceable with each other. Their benefits are differentially valued on a case-to-case basis. Future studies are warranted to perform a multidimensional analysis of both techniques and to improve limitations of suboptimal dataset quality, poor applicability implications and non-regulated study designs. CONCLUSION: For clinicians without DL expertise and easy access to AI experts, CFDL allows the prototyping of novel clinical AI systems. CFDL models concert with bespoke models, depending on the task at hand. A multidimensional, weighted evaluation of the factors involved in the implementation of those models for a designated task is warranted.

Evaluating an automated machine learning model that predicts visual acuity outcomes in patients with neovascular age-related macular degeneration.

PURPOSE: Neovascular age-related macular degeneration (nAMD) is a major global cause of blindness. Whilst anti-vascular endothelial growth factor (anti-VEGF) treatment is effective, response varies considerably between individuals. Thus, patients face substantial uncertainty regarding their future ability to perform daily tasks. In this study, we evaluate the performance of an automated machine learning (AutoML) model which predicts visual acuity (VA) outcomes in patients receiving treatment for nAMD, in comparison to a manually coded model built using the same dataset. Furthermore, we evaluate model performance across ethnic groups and analyse how the models reach their predictions. METHODS: Binary classification models were trained to predict whether patients' VA would be 'Above' or 'Below' a score of 70 one year after initiating treatment, measured using the Early Treatment Diabetic Retinopathy Study (ETDRS) chart. The AutoML model was built using the Google Cloud Platform, whilst the bespoke model was trained using an XGBoost framework. Models were compared and analysed using the What-if Tool (WIT), a novel model-agnostic interpretability tool. RESULTS: Our study included 1631 eyes from patients attending Moorfields Eye Hospital. The AutoML model (area under the curve [AUC], 0.849) achieved a highly similar performance to the XGBoost model (AUC, 0.847). Using the WIT, we found that the models over-predicted negative outcomes in Asian patients and performed worse in those with an ethnic category of Other. Baseline VA, age and ethnicity were the most important determinants of model predictions. Partial dependence plot analysis revealed a sigmoidal relationship between baseline VA and the probability of an outcome of 'Above'. CONCLUSION: We have described and validated an AutoML-WIT pipeline which enables clinicians with minimal coding skills to match the performance of a state-of-the-art algorithm and obtain explainable predictions.

Automated deep learning in ophthalmology: AI that can build AI.

PURPOSE OF REVIEW: The purpose of this review is to describe the current status of automated deep learning in healthcare and to explore and detail the development of these models using commercially available platforms. We highlight key studies demonstrating the effectiveness of this technique and discuss current challenges and future directions of automated deep learning. RECENT FINDINGS: There are several commercially available automated deep learning platforms. Although specific features differ between platforms, they utilise the common approach of supervised learning. Ophthalmology is an exemplar speciality in the area, with a number of recent proof-of-concept studies exploring classification of retinal fundus photographs, optical coherence tomography images and indocyanine green angiography images. Automated deep learning has also demonstrated impressive results in other specialities such as dermatology, radiology and histopathology. SUMMARY: Automated deep learning allows users without coding expertise to develop deep learning algorithms. It is rapidly establishing itself as a valuable tool for those with limited technical experience. Despite residual challenges, it offers considerable potential in the future of patient management, clinical research and medical education. VIDEO ABSTRACT: http://links.lww.com/COOP/A44.

Green Solvents as an Alternative to DMF in ZIF-90 Synthesis.

The use of green solvents as an alternative to dimethylformamide (DMF) in the synthesis of zeolitic imidazolate framework-90 (ZIF-90) was investigated. Two biobased aprotic dipolar solvents CyreneTM and γ-valerolactone (GVL) proved to successfully replace DMF in the synthesis at room temperature with a high product yield. While the CyreneTM-based product shows reduced porosity after activation, the use of GVL resulted in materials with preserved crystallinity and porosity after activation, without prior solvent exchange and a short treatment at 200 °C. The primary particles of 30 nm to 60 nm in all products further form agglomerates of different size and interparticle mesoporosity, depending on the type and molar ratios of solvents used.

Dysregulated skin barrier function in Tmem79 mutant mice promotes IL-17A-dependent spontaneous skin and lung inflammation.

BACKGROUND: Atopic dermatitis (AD) is associated with a dysregulation of the skin barrier and may predispose to the development of secondary allergic conditions, such as asthma. Tmem79ma/ma mice harbor a mutation in the gene encoding Transmembrane Protein 79 (or Mattrin), which has previously been associated with AD. As a result of the Tmem79 gene mutation, these mice have a defective skin barrier and develop spontaneous skin inflammation. In this study, Tmem79ma/ma mice were assessed for the underlying immunological response in the development of spontaneous skin and lung inflammation. METHODS: Development of spontaneous skin and lung inflammation in Tmem79ma/ma mice was analyzed. We further investigated susceptibility to cutaneous Staphylococcus aureus infection. Tmem79ma/ma were crossed to IL-17A-deficient mice to address the contribution of IL-17A to spontaneous skin and lung disease. RESULTS: Tmem79ma/ma mice developed IL-17A-dependent spontaneous AD-like inflammation and were refractory to S aureus infection. Mutant mice progressed to airway inflammation subsequent to the occurrence of dermatitis. The progression from skin to lung disease is dependent on adaptive immunity and is facilitated by cutaneous expansion of Th17 and TCRγδ T cells. CONCLUSION: Mice lacking Tmem79/Mattrin expression have a defective skin barrier. In adulthood, these mice develop dermatitis with secondary progression to lung inflammation. The development of skin and lung inflammation is IL-17A-dependent and mediated by TCRγδ T cells.

Therapeutic Intervention in Musculoskeletal Radiology: Current Practice and Future Directions.

Over the last several decades, the volume and range of therapeutic musculoskeletal (MSK) interventions that radiologists can offer their patients has dramatically increased. With new materials and improving imaging modalities, as well as significant investment in research, the field of MSK interventional radiologic intervention will likely continue to expand. In this article, we summarize the range of interventions currently available to the MSK radiologist. We also seek to explore new and emerging techniques that may become commonplace in the near future while considering the challenges that may lie ahead in the field of MSK radiology.

R1205H (Vicenza) causes conformational changes in the VWF-D'D3 domains and enhances VWF binding to clearance receptors LRP1 and SR-A1.

INTRODUCTION: Von Willebrand factor (VWF)-R1205H variant (Vicenza) results in markedly enhanced VWF clearance in humans that has been shown to be largely macrophage-mediated. However, the biological mechanisms underlying this enhanced clearance remain poorly understood. This study aimed to investigate the roles of (i) specific VWF domains and (ii) different macrophage receptors in regulating enhanced VWF-R1205H clearance. METHODS: In vivo clearance of full-length and truncated wild-type (WT)-VWF and VWF with R1205 substitutions was investigated in VWF-/- mice. Plate-binding assays were employed to characterize VWF binding to purified scavenger receptor class A member 1 (SR-A1), low-density lipoprotein receptor-related protein-1 (LRP1) cluster II or cluster IV receptors, and macrophage galactose-type lectin (MGL). RESULTS: In full-length VWF missing the A1 domain (VWF-ΔA1), introduction of R1205H led to significantly enhanced clearance in VWF-/- mice compared to WT-VWF-ΔA1. Importantly, R1205H in a truncated VWF-D'D3 fragment also triggered increased clearance compared to WT-VWF-D'D3. Additional in vivo studies demonstrated that VWF-R1205K (which preserves the positive charge at 1205) exhibited normal clearance, whereas VWF-R1205E (which results in loss of the positive charge) caused significantly enhanced clearance, pinpointing the importance of the positive charge at VWF-R1205. In vitro plate-binding studies confirmed increased VWF-R1205H interaction with SR-A1 compared to WT-VWF. Furthermore, significantly enhanced VWF-R1205H binding to LRP1 cluster IV (p<0.001) and less marked enhanced binding to LRP1 cluster II (p=0.034) was observed. In contrast, VWF-R1205H and WT-VWF demonstrated no difference in binding affinity to MGL. CONCLUSION: Disruption of the positive charge at amino acid 1205 causes conformational changes in the VWF-D'D3 domains, and triggers enhanced LRP1 and SR-A1 mediated clearance.

VWF-ADAMTS13 axis dysfunction in children with sickle cell disease treated with hydroxycarbamide vs blood transfusion.

Previous studies have reported elevated von Willebrand factor (VWF) levels in patients with sickle cell disease (SCD) and demonstrated a key role for the VWF-ADAMTS13 axis in the pathobiology of SCD vaso-occlusion. Although blood transfusion is the gold standard for stroke prevention in SCD, the biological mechanisms underpinning its improved efficacy compared with hydroxycarbamide are not fully understood. We hypothesized that the improved efficacy of blood transfusion might relate to differences in VWF-ADAMTS13 axis dysfunction. In total, 180 children with a confirmed diagnosis of SCD (hemoglobin SS) on hydroxycarbamide (n = 96) or blood transfusion (n = 84) were included. Despite disease-modifying treatment, plasma VWF and VWF propeptide were elevated in a significant proportion of children with SCD (33% and 47%, respectively). Crucially, all VWF parameters were significantly higher in the hydroxycarbamide compared with the blood transfusion cohort (P < .05). Additionally, increased levels of other Weibel-Palade body-stored proteins, including factor VIII (FVIII), angiopoietin-2, and osteoprotegerin were observed, indicated ongoing endothelial cell activation. Children treated with hydroxycarbamide also had higher FVIII activity and enhanced thrombin generation compared with those in the blood transfusion cohort (P < .001). Finally, hemolysis markers strongly correlated with VWF levels (P < .001) and were significantly reduced in the blood transfusion cohort (P < .001). Cumulatively, to our knowledge, our findings demonstrate for the first time that despite treatment, ongoing dysfunction of the VWF-ADAMTS13 axis is present in a significant subgroup of pediatric patients with SCD, especially those treated with hydroxycarbamide.

Omega-3 fatty acids in the management of dry eye disease-An updated systematic review and meta-analysis.

PURPOSE: The purpose of this study was to examine the effectiveness of omega-3 fatty acids in comparison to a placebo in the management of dry eye disease. METHODS: A systematic literature search was performed including randomised controlled trials (RCTs) comparing omega-3 versus placebo in the management of dry eye disease in human subjects. There were no language or time restrictions. Eligible trials were assessed for bias and assigned a risk-of-bias score. Data extraction was carried out using a standardised data extraction form, and meta-analysis was performed using a random effects model for continuous data. The outcome measures were Ocular Surface Disease Index (OSDI) scores, tear breakup time (TBUT) measurements, corneal staining and Schirmer's score. Statistical heterogeneity was defined as substantial if the I2 test achieved a value >60%. RESULTS: Eight parallel RCTs including 1107 subjects met eligibility criteria. None of the included studies achieved low risk of bias. Data synthesis demonstrated an improvement in the mean change in OSDI score from baseline to final assessment. Omega-3 supplementation conferred no evident improvement in corneal staining, TBUT or Schirmer's score. There was considerable statistical heterogeneity in all four outcome measures. CONCLUSIONS: This updated systematic review and meta-analysis indicates that omega-3 supplementation improves subjective symptoms in patients with dry eye disease.

Knowledge of face masks and attitudes to re-sterilisation among healthcare workers.

INTRODUCTION: COVID-19 was declared a pandemic in March 2020. With the sudden surge in demand for personal protective equipment (PPE), significant concerns regarding the ongoing availability emerged. One solution proposed is re-sterilisation of respirator masks and this has been commenced in some parts of the world. On review of the literature, very little is documented regarding the knowledge of masks and the attitudes of healthcare workers towards using re-sterilised masks. METHODS: A comprehensive questionnaire was used to assess general knowledge and attitudes around facemasks and respirators. RESULTS: There were 190 respondents. There were significant gaps in knowledge and understanding of when particular face masks should be worn. One-third had significant concerns about ongoing availability. One-third had concerns about the quality of the masks as the pandemic continued. Only 10% of respondents underwent formal face-fitting. Eighty percent of respondents stated they would wear a re-sterilised mask. A further 15% would use a re-sterilised mask but required certain reassurances. Five percent of our respondents would not use a re-sterilised mask under any circumstances. DISCUSSION: Ensuring an adequate understanding of face masks is crucial among healthcare workers (HCWs) and this study highlights a need for further education. It also demonstrates a general acceptability among HCWs towards the use of re-sterilised face masks.

Breast cancer cells mediate endothelial cell activation, promoting von Willebrand factor release, tumor adhesion, and transendothelial migration.

BACKGROUND: Breast cancer results in a three- to four-fold increased risk of venous thromboembolism (VTE), which is associated with reduced patient survival. Despite this, the mechanisms underpinning breast cancer-associated thrombosis remain poorly defined. Tumor cells can trigger endothelial cell (EC) activation resulting in increased von Willebrand factor (VWF) secretion. Importantly, elevated plasma VWF levels constitute an independent biomarker for VTE risk. Moreover, in a model of melanoma, treatment with low molecular weight heparin (LMWH) negatively regulated VWF secretion and attenuated tumor metastasis. OBJECTIVE: To investigate the role of VWF in breast cancer metastasis and examine the effect of LMWH in modulating EC activation and breast tumor transmigration. METHODS: von Willebrand factor levels were measured by ELISA. Primary ECs were used to assess tumor-induced activation, angiogenesis, tumor adhesion, and transendothelial migration. RESULTS AND CONCLUSION: Patients with metastatic breast cancer have markedly elevated plasma VWF:Ag levels that also correlate with poorer survival. MDA-MB-231 and MCF-7 breast cancer cells induce secretion of VWF, angiopoietin-2, and osteoprotegerin from ECs, which is further enhanced by the presence of platelets. Vascular endothelial growth factor-A (VEGF-A) plays an important role in modulating breast cancer-induced VWF release. Moreover, VEGF-A from breast tumor cells also contributes to a pro-angiogenic effect on ECs. VWF multimers secreted from ECs, in response to tumor-VEGF-A, mediate adhesion of breast tumor cells along the endothelium. LMWH inhibits VWF-breast tumor adhesion and transendothelial migration. Our findings highlight the significant crosstalk between tumor cells and the endothelium including increased VWF secretion which may contribute to tumor metastasis.

'It's just a way of approaching things now': staff perspectives of an anticipatory care programme in Edinburgh.

BACKGROUND: Keep Well, an anticipatory care programme aimed at reducing health inequalities in Scotland, commenced in 2006. There is currently little evidence exploring how frontline staff respond to the delivery of anticipatory care. AIM: To understand how staff view changes in practice resulting from the implementation of Keep Well. METHODS: Qualitative interviews were conducted with a purposively selected sample of 12 Keep Well staff. Data were analysed using a thematic analysis approach. RESULTS: Keep Well was viewed positively, especially in terms of increased knowledge and skills for staff and the benefits of a holistic health check to patients. Staff believed that Keep Well has improved their practice beyond the project. Concerns were raised, relating to potential inconveniences for patients, staff turnover, increased workload and procedures. There was a view that anticipatory care should continue to be delivered to deprived communities, provided that there are adequate resources, such as staff and funding, to do so. CONCLUSION: Keep Well staff believe that the project should continue to be offered as a 40-minute holistic health check targeted at patients in deprived areas.