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Background Intra-operative aspiration of oropharyngeal secretions is associated with post-operative pneumonia. The use of endotracheal tubes ( ETTs) with a modified cuff shape could be one preventive action. In this clinical, prospective, randomised controlled trial, we hypothesised that altering the cuff shape to a tapered shape could reduce the aspiration incidence. The primary outcome was aspiration of dye solution into the trachea. Methods Patients scheduled for lumbar surgery were intubated with either an ETT with a barrel-shaped polyvinylchloride cuff (control group, n = 30) or tapered-shaped polyvinylchloride cuff (intervention group, n = 30). Subsequently, instillation with methylthioninium chloride was performed. At 10, 30, 60, 90, and 120 min after intubation, bronchoscopy was performed assessing the degree of dye descent along the cuff and digitally stored. Single blind review of the videoclips provided data on incidence of dye aspiration and depth of penetration along the cuff. Results The traditional cuff showed descent of dye into the trachea in 20% of the patients. Although a tapered-shaped polyvinylchloride cuff leaked up to the second third of the cuff, no dye leakage into the trachea was observed. The use of a tapered-shaped cuff had a protective role against aspiration ( T30: OR 3.0, CI 1.57-5.75; P = 0.001). Conclusions Short-term use of tapered-shaped polyvinylchloride cuffs in surgical patients results in more effective sealing of the tracheal lumen in comparison with a traditional barrel-shaped polyvinylchloride cuffs. Further evaluation is needed to determine whether a reduction in post-operative pneumonia can be demonstrated when these cuffs are used. [ABSTRACT FROM AUTHOR]

Immunomodulatory therapies have fueled interest in targeting microglial cells as part of the innate immune response after infection or injury. In this context, the colony-stimulating factor 1 (CSF-1) and its receptor (CSF-1R) have gained attention in various neurological conditions to deplete and reprogram the microglia/macrophages compartment. Published data in physiological conditions support the use of small-molecule inhibitors to study microglia/macrophages dynamics under inflammatory conditions and as a therapeutic strategy in pathologies where those cells support disease progression. However, preclinical and clinical data highlighted that the complexity of the spatiotemporal inflammatory response could limit their efficiency due to compensatory mechanisms, ultimately leading to therapy resistance. We review the current state-of-art in the field of CSF-1R inhibition in glioma and stroke and provide an overview of the fundamentals, ongoing research, potential developments of this promising therapeutic strategy and further application toward molecular imaging., Competing Interests: The author BZ is currently employed by F. Hoffman-La Roche Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Barca, Foray, Hermann, Herrlinger, Remory, Laoui, Schäfers, Grauer, Zinnhardt and Jacobs.)

Broadly neutralizing antibodies are an important treatment for individuals with coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antibody-based therapeutics are also essential for pandemic preparedness against future Sarbecovirus outbreaks. Camelid-derived single domain antibodies (VHHs) exhibit potent antimicrobial activity and are being developed as SARS-CoV-2–neutralizing antibody-like therapeutics. Here, we identified VHHs that neutralize both SARS-CoV-1 and SARS-CoV-2, including now circulating variants. We observed that the VHHs bound to a highly conserved epitope in the receptor binding domain of the viral spike protein that is difficult to access for human antibodies. Structure-guided molecular modeling, combined with rapid yeast-based prototyping, resulted in an affinity enhanced VHH-human immunoglobulin G1 Fc fusion molecule with subnanomolar neutralizing activity. This VHH-Fc fusion protein, produced in and purified from cultured Chinese hamster ovary cells, controlled SARS-CoV-2 replication in prophylactic and therapeutic settings in mice expressing human angiotensin converting enzyme 2 and in hamsters infected with SARS-CoV-2. These data led to affinity-enhanced selection of the VHH, XVR011, a stable anti–COVID-19 biologic that is now being evaluated in the clinic.

Radiolabeling of nanobodies with radiometals by chelation has the advantage of being simple, fast and easy to implement in clinical routine. In this study, we validated 68Ga/111In-labeled anti-VCAM-1 nanobodies as potential radiometal-based tracers for molecular imaging of atherosclerosis. Both showed specific targeting of atherosclerotic lesions in ApoE-/- mice. Nevertheless, uptake in lesions and constitutively VCAM-1 expressing organs was lower than previously reported for the 99mTc-labeled analog. We further investigated the impact of different radiolabeling strategies on the in vivo biodistribution of nanobody-based tracers. Comparison of the pharmacokinetics between 68Ga-, 18F-, 111In- and 99mTc-labeled anti-VCAM-1 nanobodies showed highest specific uptake for 99mTc-nanobody at all time-points, followed by the 68Ga-, 111In- and 18F-labeled tracer. No correlation was found with the estimated number of radioisotopes per nanobody, and mimicking specific activity of other radiolabeling methods did not result in an analogous biodistribution. We also demonstrated specificity of the tracer using mice with a VCAM-1 knocked-down phenotype, while showing for the first time the in vivo visualization of a protein knock-down using intrabodies. Conclusively, the chosen radiochemistry does have an important impact on the biodistribution of nanobodies, in particular on the specific targeting, but differences are not purely due to the tracer's specific activity.

Purpose: Macrophage accumulation characterizes the development of atherosclerotic plaques, and the presence of certain macrophage subsets might be an indicator of plaque phenotype and (in)stability. The macrophage mannose receptor (MMR) is expressed on alternatively activated macrophages and found at sites of intraplaque hemorrhage and neovascularization. It has been proposed as target to identify vulnerable plaques. Therefore, we aimed to assess the feasibility of using anti-MMR nanobodies (Nbs) as molecular tracers for nuclear imaging in an animal model of atherosclerosis., Procedure: Anti-MMR and control Nb, radiolabeled with Tc-99m, were injected in ApoE -/- and/or C57Bl/6 mice (n = 6). In vivo competition studies involving pre-injection of excess of unlabeled anti-MMR Nb (n = 3) and injection of anti-MMR Nb in MMR -/- mice (n = 3) were performed to demonstrate specificity. At 3 h p.i. radioactive uptake in organs, tissues and aorta segments were evaluated. Autoradiography and immunofluorescence were performed on aortic sections., Results: Significantly higher uptake was observed in all aortic segments of ApoE -/- mice injected with anti-MMR Nb compared to control Nb (1.36 ± 0.67 vs 0.38 ± 0.13 percent of injected dose per gram (%ID/g), p ≤ 0.001). Surprisingly, high aortic uptake was also observed in C57Bl/6 mice (1.50 ± 0.43%ID/g, p ≥ 0.05 compared to ApoE -/- ), while aortic uptake was reduced to background levels in the case of competition and in MMR -/- mice (0.46 ± 0.10 and 0.22 ± 0.06%ID/g, respectively; p ≤ 0.001). Therefore, expression of MMR along healthy aortas was suggested. Autoradiography showed no specific radioactive signal within atherosclerotic plaques, but rather localization of the signal along the aorta, correlating with MMR expression in perivascular tissue as demonstrated by immunofluorescence., Conclusions: No significant uptake of MMR-specific Nb could be observed in atherosclerotic lesions of ApoE -/- mice in this study. A specific perivascular signal causing a non-negligible background level was demonstrated. This observation should be considered when using MMR as a target in molecular imaging of atherosclerosis, as well as use of translational animal models with vulnerable plaques.

Objective: Calcific aortic valve disease (CAVD) is currently the most common heart valve disease worldwide and is known to be an active process. Both renal failure and dyslipidaemia are considered to be promoting factors for the development of valvular calcifications. The aim of this study is to prospectively evaluate the respective contribution and interaction of renal failure and dyslipidaemia on CAVD in a rat model, using echocardiography and compared with histology., Methods and Results: Sixty-eight male Wistar rats were prospectively divided in eight groups, each fed a different diet to induce renal failure alone and combined with hyperlipidaemia or hypercholesterolemia. CAVD was detected and quantified by calibrated integrated backscatter of ultrasound (cIB) and compared with the histological calcium score. The study follow-up was 20 weeks. At the end of the study, the cIB value and the calcium score of the aortic valve were significantly increased in the group with isolated renal failure but not with dyslipidaemia. The combination of renal failure with high cholesterol or high-fat diet did not significantly increase calcifications further., Conclusions: Renal failure alone does induce aortic valve calcifications in a rat model of CAVD, whereas dyslipidaemia alone does not. The combination of renal failure with dyslipidaemia does not increase calcification further. These findings suggest that a combination of atherosclerotic and calcifying factors is not required to induce aortic valve calcifications in this model.

Purpose: Calcification is an important prognostic factor in aortic valve stenosis. However, there is no ultrasound (US) method available to accurately quantify calcification in this setting to date. We aimed to validate a new US method for measuring the amount of calcium in an in vitro model, and compare it to computed tomography (CT), the current imaging gold standard., Materials and Methods: An agar phantom (2% agar) was made, containing 9 different amounts of calcium-hydroxyapatite Ca5(PO4)3OH (2 to 50 mg). The phantoms were imaged with micro-CT and US (10 MHz probe). The calcium area (areacalcium) and its maximum pixel value (PVmax) were obtained. These values were summed to calculate CT and US calcium scores (∑(areacalcium × PVmax)) and volumes (∑areacalcium). Both US- and CT-calcium scores were compared with the calcium amounts, and with each other., Results: Both calcium scores correlated significantly with the calcium amount (R2 = 0.9788, p<0.0001 and R2 = 0.8154, p<0.0001 for CT and US respectively). Furthermore, there was a significant correlation between US and CT for calcium volumes (R2 = 0.7392, p<0.0001) and scores (R2 = 0.7391, p<0.0001)., Conclusion: We developed a new US method that accurately quantifies the amount of calcium in an in vitro model. Moreover it is strongly correlated with CT.

Aims: Calcification is an independent predictor of mortality in aortic valve (AV) stenosis. Echocardiographic calibrated integrated backscatter (cIB) is a promising parameter for quantifying AV calcification. However, the ability of cIB to differentiate between calcification and valvular thickening has been questioned. Therefore, we aimed to use cIB to study AV calcification compared with non-calcified AV thickening in rats, with histology as reference., Methods and Results: Twenty male Wistar rats were studied. Group 1 (N = 6) received subcutaneous (SC) serotonin injections (60 mg/kg/day) for 12 weeks to induce myxoid non-calcified AV thickening. Group 2 (N = 7) received vitamin D3 (25,000 UI/kg/day) SC to induce AV calcification, and Group 3 (N = 7) received only vehicle SC for 10 weeks. cIB of the AV was calculated at the end of the study, followed by measurement of the percentage of the histological AV calcification. At the end of the study, cIB values and calcification percentages were significantly higher in vitamin D3-injected rats compared with serotonin-injected rats and controls. There was no significant difference in cIB values between serotonin-injected rats and controls (vitamin D3: 21.5 ± 3.0 dB*; serotonin: 11.8 ± 3.1 dB; control: 10.3 ± 3.4 dB; *P < 0.05). The percentage of histological calcification was significantly higher in the vitamin D3 group compared with the other groups. Serotonin-injected rats developed significant AV thickening., Conclusion: Increased cIB values of the AV are related to increased calcification at histology and not to myxoid non-calcified valvular thickening. Therefore, cIB may be considered as a sensitive technique to quantify calcification of AV rather than for detecting non-calcified valvular thickening., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Background: Calcification is an independent predictor of mortality in calcific aortic valve disease (CAVD). The aim of this study was to evaluate the use of non-invasive, non-ionizing echocardiographic calibrated integrated backscatter (cIB) for monitoring progression and subsequent regression of aortic valvular calcifications in a rat model of reversible renal failure with CAVD, compared to histology., Methods: 28 male Wistar rats were prospectively followed during 21 weeks. Group 1 (N=14) was fed with a 0.5% adenine diet for 9 weeks to induce renal failure and CAVD. Group 2 (N=14) received a standard diet. At week 9, six animals of each group were killed. The remaining animals of group 1 (N=8) and group 2 (N=8) were kept on a standard diet for an additional 12 weeks. cIB of the aortic valve was calculated at baseline, 9 and 21 weeks, followed by measurement of the calcified area (Ca Area) on histology., Results: At week 9, cIB values and Ca Area of the aortic valve were significantly increased in the adenine-fed rats compared to baseline and controls. After 12 weeks of adenine diet cessation, cIB values and Ca Area of group 1 decreased compared to week 9, while there was no longer a significant difference compared to age-matched controls of group 2., Conclusions: cIB is a non-invasive tool allowing quantitative monitoring of CAVD progression and regression in a rat model of reversible renal failure, as validated by comparison with histology. This technique might become useful for assessing CAVD during targeted therapy.

Unlabelled: Bioluminescence imaging is routinely performed in anesthetized mice. Often isoflurane anesthesia is used because of its ease of use and fast induction/recovery. However, general anesthetics have been described as important inhibitors of the luciferase enzyme reaction., Aim: To investigate frequently used mouse anesthetics for their direct effect on the luciferase reaction, both in vitro and in vivo., Materials and Methods: isoflurane, sevoflurane, desflurane, ketamine, xylazine, medetomidine, pentobarbital and avertin were tested in vitro on luciferase-expressing intact cells, and for non-volatile anesthetics on intact cells and cell lysates. In vivo, isoflurane was compared to unanesthetized animals and different anesthetics. Differences in maximal photon emission and time-to-peak photon emission were analyzed., Results: All volatile anesthetics showed a clear inhibitory effect on the luciferase activity of 50% at physiological concentrations. Avertin had a stronger inhibitory effect of 80%. For ketamine and xylazine, increased photon emission was observed in intact cells, but this was not present in cell lysate assays, and was most likely due to cell toxicity and increased cell membrane permeability. In vivo, the highest signal intensities were measured in unanesthetized mice and pentobarbital anesthetized mice, followed by avertin. Isoflurane and ketamine/medetomidine anesthetized mice showed the lowest photon emission (40% of unanesthetized), with significantly longer time-to-peak than unanesthetized, pentobarbital or avertin-anesthetized mice. We conclude that, although strong inhibitory effects of anesthetics are present in vitro, their effect on in vivo BLI quantification is mainly due to their hemodynamic effects on mice and only to a lesser extent due to the direct inhibitory effect.