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Since the precursor frequency of naive T cells is extremely low, investigating the early steps of antigen-specific T cell activation is challenging. To overcome this detection problem, adoptive transfer of a cohort of T cells purified from T cell receptor (TCR) transgenic donors has been extensively used but is not readily available for emerging pathogens. Constructing TCR transgenic mice from T cell hybridomas is a labor-intensive and sometimes erratic process, since the best clones are selected based on antigen-induced CD69 upregulation or IL-2 production in vitro, and TCR chains are polymerase chain reaction (PCR)-cloned into expression vectors. Here, we exploited the rapid advances in single-cell sequencing and TCR repertoire analysis to select the best clones without hybridoma selection, and generated CORSET8 mice ( COR ona S pike E pitope specific CD8 T cell), carrying a TCR specific for the Spike protein of SARS-CoV-2. Implementing newly created DALI software for TCR repertoire analysis in single-cell analysis enabled the rapid selection of the ideal responder CD8 T cell clone, based on antigen reactivity, proliferation, and immunophenotype in vivo. Identified TCR sequences were inserted as synthetic DNA into an expression vector and transgenic CORSET8 donor mice were created. After immunization with Spike/CpG-motifs, mRNA vaccination or SARS-CoV-2 infection, CORSET8 T cells strongly proliferated and showed signs of T cell activation. Thus, a combination of TCR repertoire analysis and scRNA immunophenotyping allowed rapid selection of antigen-specific TCR sequences that can be used to generate TCR transgenic mice., Competing Interests: ND, SL, MV, JD, CD, WT, RV, KV, HH, SV, BL No competing interests declared, (© 2024, Debeuf, Lameire et al.)

Background: Asthma is often accompanied by type 2 immunity rich in IL-4, IL-5, and IL-13 cytokines produced by T H 2 lymphocytes or type 2 innate lymphoid cells (ILC2s). IL-2 family cytokines play a key role in the differentiation, homeostasis, and effector function of innate and adaptive lymphocytes., Objective: IL-9 and IL-21 boost activation and proliferation of T H 2 and ILC2s, but the relative importance and potential synergism between these γ common chain cytokines are currently unknown., Methods: Using newly generated antibodies, we inhibited IL-9 and IL-21 alone or in combination in various murine models of asthma. In a translational approach using segmental allergen challenge, we recently described elevated IL-9 levels in human subjects with allergic asthma compared with nonasthmatic controls. Here, we also measured IL-21 in both groups., Results: IL-9 played a central role in controlling innate IL-33-induced lung inflammation by promoting proliferation and activation of ILC2s in an IL-21-independent manner. Conversely, chronic house dust mite-induced airway inflammation, mainly driven by adaptive immunity, was solely dependent on IL-21, which controlled T H 2 activation, eosinophilia, total serum IgE, and formation of tertiary lymphoid structures. In a model of innate on adaptive immunity driven by papain allergen, a clear synergy was found between both pathways, as combined anti-IL-9 or anti-IL-21 blockade was superior in reducing key asthma features. In human bronchoalveolar lavage samples we measured elevated IL-21 protein within the allergic asthmatic group compared with the allergic control group. We also found increased IL21R transcripts and predicted IL-21 ligand activity in various disease-associated cell subsets., Conclusions: IL-9 and IL-21 play important and nonredundant roles in allergic asthma by boosting ILC2s and T H 2 cells, revealing a dual IL-9 and IL-21 targeting strategy as a new and testable approach., Competing Interests: Disclosure statement This work was funded by a Baekeland Mandate of Flanders Innovation & Entrepreneurship (VLAIO) (HBC.2019.2598) to F.B. B.N.L. acknowledges support from a European Research Council (ERC) advanced grant (789384 ASTHMA CRYSTAL CLEAR), a concerted research initiative grant from Ghent University (GOA, 01G010C9), a Fonds Wetenschappelijk Onderzoek (FWO) Methusalem grant (01M01521) and an FWO Excellence of Science (EOS) Consortium research grant (3G0H1222), and the Flanders Institute of Biotechnology (VIB). H.H. is supported by a concerted research initiative grant from Ghent University (GOA, 01G010C9) and FWO EOS Consortium research grant (3G0H1222). M.J.S. acknowledges support from FWO Vlaanderen (12Y5322N), Fund Suzanne Duchesne (managed by the King Baudouin Foundation), and Fondation ACTERIA. Disclosure of potential conflict of interest: N. P. Smith is a consultant for Hera Biotech. A.-C. Villani has financial interest in 10X Genomics. B. D. Medoff serves on advisory boards for Sanofi, Verona Pharma, and Apogee Therapeutics and has sponsored research agreements from Sanofi and Regeneron. R. Bigirimana is an employee of argenx. C. Blanchetot is a consultant and shareholder of argenx. B. N. Lambrecht receives consultancy fees from GSK Biologics, Novartis, Sanofi, AstraZeneca, ALK, OncoArendi, and argenx and has research grants from ALK, argenx, AstraZeneca, GSK, GSK Biologics, and Johnson & Johnson. The rest of the authors declare that they have no relevant conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The sensitive, rapid and accurate diagnosis of Mycobacterium tuberculosis ( Mtb ) infection is a central challenge in controlling the global tuberculosis (TB) pandemic. Yet the detection of mycobacteria is often made difficult by the low sensitivity of current diagnostic tools, with over 3.6 million TB cases missed each year. To overcome these limitations there is an urgent need for next-generation TB diagnostic technologies. Here we report the use of a discrete panel of native 19 F-trehalose (F-Tre) analogues to label and directly visualise Mtb by exploiting the uptake of fluorine-modified trehalose analogues via the mycobacterial trehalose LpqY-SugABC ATP-binding cassette (ABC) importer. We discovered the extent of modified F-Tre uptake correlates with LpqY substrate recognition and characterisation of the interacting sites by saturation transfer difference NMR coupled with molecular dynamics provides a unique glimpse into the molecular basis of fluorine-modified trehalose import in Mtb . Lipid profiling demonstrated that F-Tre analogues modified at positions 2, 3 and 6 are incorporated into mycobacterial cell-surface trehalose-containing glycolipids. This rapid one-step labelling approach facilitates the direct visualisation of F-Tre-labelled Mtb by Focused Ion Beam (FIB) Secondary Ion Mass Spectrometry (SIMS), enabling detection of the Mtb pathogen. Collectively, our findings highlight that F-Tre analogues have potential as tools to probe and unravel Mtb biology and can be exploited to detect and image TB., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The intersection of digital transformation and circular construction practices presents significant potential to mitigate environmental impacts through optimised material reuse. We propose a five-step (D5) digital circular workflow that integrates these digital innovations towards reuse, validated through real-world case studies. We assessed a variety of digital tools for enhancing the reuse of construction materials, including digital product passports, material classification assisted by artificial intelligence (AI), reality capture, computational design, design inspired by generative AI, digital fabrication techniques, extended reality, and blockchain technology. Using action research through a multiple case study approach, we disassembled several buildings that were set for demolition and subsequently designed and executed construction projects using the salvaged materials. Our findings indicate that digital transformation for detection, disassembly, distribution, design, and finally deployment significantly support the application of circular economy principles. We demonstrate the potential of the proposed workflow for industry implementation and scalability., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2024.)

Efforts to prevent human-to-human transmission of variant Creutzfeldt-Jakob disease (vCJD) by contaminated blood would be aided by the development of a sensitive diagnostic test that could be routinely used to screen blood donations. As blood samples from vCJD patients are extremely rare, here we describe the optimisation of real-time quaking-induced conversion (RT-QuIC) for detection of PrPSc (misfolded prion protein, a marker of prion infection) in blood samples from an established large animal model of vCJD, sheep experimentally infected with bovine spongiform encephalopathy (BSE). Comparative endpoint titration experiments with RT-QuIC, miniaturized bead protein misfolding cyclic amplification (mb-PMCA) and intracerebral inoculation of a transgenic mouse line expressing sheep PrP (tgOvARQ), demonstrated highly sensitive detection of PrPSc by RT-QuIC in a reference sheep brain homogenate. Upon addition of a capture step with iron oxide beads, the RT-QuIC assay was able to detect PrPSc in whole blood samples from BSE-infected sheep up to two years before disease onset. Both RT-QuIC and mb-PMCA also demonstrated sensitive detection of PrPSc in a reference vCJD-infected human brain homogenate, suggesting that either assay may be suitable for application to human blood samples. Our results support the further development and evaluation of RT-QuIC as a diagnostic or screening test for vCJD., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Thomas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Healthy adipose tissue (AT) contains ST2+ Tregs, ILC2s, and alternatively activated macrophages that are lost in mice or humans on high caloric diet. Understanding how this form of type 2 immunity is regulated could improve treatment of obesity. The STE20 kinase Thousand And One amino acid Kinase-3 (TAOK3) has been linked to obesity in mice and humans, but its precise function is unknown. We found that ST2+ Tregs are upregulated in visceral epididymal white AT (eWAT) of Taok3-/- mice, dependent on IL-33 and the kinase activity of TAOK3. Upon high fat diet feeding, metabolic dysfunction was attenuated in Taok3-/- mice. ST2+ Tregs disappeared from eWAT in obese wild-type mice, but this was not the case in Taok3-/- mice. Mechanistically, AT Taok3-/- Tregs were intrinsically more responsive to IL-33, through higher expression of ST2, and expressed more PPARγ and type 2 cytokines. Thus, TAOK3 inhibits adipose tissue Tregs and regulates immunometabolism under excessive caloric intake., (© 2023 Maes et al.)

Oxymel , a combination of honey and vinegar, has been used as a remedy for wounds and infections in historical and traditional medical settings. While honey is now clinically used to treat infected wounds, this use of a complex, raw natural product (NP) mixture is unusual in modern western medicine. Research into the antimicrobial activity of NPs more usually focuses on finding a single active compound. The acetic acid in vinegar is known to have antibacterial activity at low concentrations and is in clinical use to treat burn wound infections. Here, we investigated the potential for synergistic activity of different compounds present in a complex ingredient used in historical medicine (vinegar) and in an ingredient mixture ( oxymel ). We conducted a systematic review to investigate published evidence for antimicrobial effects of vinegars against human pathogenic bacteria and fungi. No published studies have explicitly compared the activity of vinegar with that of a comparable concentration of acetic acid. We then characterized selected vinegars by HPLC and assessed the antibacterial and antibiofilm activity of the vinegars and acetic acid, alone and in combination with medical-grade honeys, against Pseudomonas aeruginosa and Staphylococcus aureus . We found that some vinegars have antibacterial activity that exceeds that predicted by their acetic acid content alone, but that this depends on the bacterial species being investigated and the growth conditions (media type, planktonic vs. biofilm). Pomegranate vinegars may be particularly interesting candidates for further study. We also conclude that there is potential for acetic acid, and some vinegars, to show synergistic antibiofilm activity with manuka honey.

The application of the circular economy (CE) in the building industry is critical for achieving the carbon reduction goals defined in the Paris Agreement and is increasingly promoted through European policies. In recent years, CE strategies have been applied and tested in numerous building projects in practice. However, insights into their application and decarbonisation potential are limited. This study analysed and visualised 65 novel real-world cases of new build, renovation, and demolition projects in Europe compiled from academic and grey literature. Cases were analysed regarding the circular solution applied, level of application in buildings, and decarbonisation potential reported, making this study one of the first comprehensive studies on the application and decarbonisation potential of circular strategies in the building industry in practice. The identified challenges of using LCA for CE assessment in buildings are discussed and methodological approaches for future research are suggested., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (.)

Infectious prion diseases have very long incubation periods, and the role that subclinical infections play in transmission, persistence and re-emergence of these diseases is unclear. In this study, we used a well-established model of vCJD (sheep experimentally infected with bovine spongiform encephalopathy, BSE) to determine the prevalence of subclinical infection following exposure by blood transfusion from infected donors. Many recipient sheep survived for years post-transfusion with no clinical signs and no disease-associated PrP (PrP Sc ) found in post mortem tissue samples by conventional tests. Using a sensitive protein misfolding cyclic amplification assay (PMCA), we found that the majority of these sheep had detectable PrP Sc in lymph node samples, at levels approximately 10 5 -10 6 times lower than in equivalent samples from clinically positive sheep. Further testing revealed the presence of PrP Sc in other tissues, including brain, but not in blood samples. The results demonstrate that subclinical infection is a frequent outcome of low dose prion infection by a clinically relevant route for humans (blood transfusion). The long term persistence of low levels of infection has important implications for prion disease control and the risks of re-emergent infections in both humans and animals., (© 2022. The Author(s).)

Background: The most common endotype of asthma is type 2-high asthma, which is sometimes driven by adaptive allergen-specific T H 2 lymphocytes that react to allergens presented by dendritic cells (DCs), or sometimes by an innate immune response dominated by type 2 innate lymphocytes (ILC2s). Understanding the underlying pathophysiology of asthma is essential to improve patient-tailored therapy. The STE20 kinase thousand-and-one kinase 3 (TAOK3) controls key features in the biology of DCs and lymphocytes, but to our knowledge, its potential usefulness as a target for asthma therapy has not yet been addressed., Objective: We examined if and how loss of Taok3 affects the development of house dust mite (HDM)-driven allergic asthma in an in vivo mouse model., Methods: Wild-type Taok3 +/+ and gene-deficient Taok3 -/- mice were sensitized and challenged with HDM, and bronchoalveolar lavage fluid composition, mediastinal lymph node cytokine production, lung histology, and bronchial hyperreactivity measured. Conditional Taok3 fl/fl mice were crossed to tissue- and cell-specific specific deletor Cre mice to understand how Taok3 acted on asthma susceptibility. Kinase-dead (KD) Taok3 KD mice were generated to probe for the druggability of this pathway. Activation of HDM-specific T cells was measured in adoptively transferred HDM-specific T-cell receptor-transgenic CD4 + T cells. ILC2 biology was assessed by in vivo and in vitro IL-33 stimulation assays in Taok3 -/- and Taok3 +/+ , Taok3 KD , and Red5-Cre Taok3 fl/fl mice., Results: Taok3 -/- mice failed to mount salient features of asthma, including airway eosinophilia, T H 2 cytokine production, IgE secretion, airway goblet cell metaplasia, and bronchial hyperreactivity compared to controls. This was due to intrinsic loss of Taok3 in hematopoietic and not epithelial cells. Loss of Taok3 resulted in hampered HDM-induced lung DC migration to the draining lymph nodes and defective priming of HDM-specific T H 2 cells. Strikingly, HDM and IL-33-induced ILC2 proliferation and function were also severely affected in Taok3-deficient and Taok3 KD mice., Conclusions: Absence of Taok3 or loss of its kinase activity protects from HDM-driven allergic asthma as a result of defects in both adaptive DC-mediated T H 2 activation and innate ILC2 function. This identifies Taok3 as an interesting drug target, justifying further testing as a new treatment for type 2-high asthma., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

With the emergence of disease-modifying therapies for Parkinson's disease, reliable longitudinal markers are needed to quantify pathology and demonstrate disease progression. We developed the A53T-AAV rat model of synucleinopathy by combining longitudinal measures over 12 weeks. We first characterized the progression of the motor and dopaminergic deficits. Then, we monitored the disease progression using the [ 18 F]FMT Positron Emission Tomography (PET) radiotracer. The nigral injection of A53T-AAV led to an increase in phosphorylated α-synuclein on S129, a progressive accumulation of α-synuclein aggregates, and a decrease of dopaminergic function associated with a deterioration of motor activity. The longitudinal monitoring of A53T-AAV rats with [ 18 F]FMT PET showed a progressive reduction of the K c outcome parameter in the caudate putamen from the lesioned side. Interestingly, the progressive reduction in the [ 18 F]FMT PET signal correlated with defects in the stepping test. In conclusion, we established a progressive rat model of α-synuclein pathology which monitors the deficit longitudinally using both the [ 18 F]FMT PET tracer and behavioral parameters, 2 features that have strong relevance for translational approaches., (Copyright © 2021. Published by Elsevier Inc.)

We present the first clinical integration of a prototype device based on integrated auto-fluorescence imaging and Raman spectroscopy (Fast Raman device) for intra-operative assessment of surgical margins during Mohs micrographic surgery of basal cell carcinoma (BCC). Fresh skin specimens from 112 patients were used to optimise the tissue pre-processing and the Fast Raman algorithms to enable an analysis of complete Mohs layers within 30 minutes. The optimisation allowed >95% of the resection surface area to be investigated (including the deep and epidermal margins). The Fast Raman device was then used to analyse skin layers excised from the most relevant anatomical sites (nose, temple, eyelid, cheek, forehead, eyebrow and lip) and to detect the three main types of BCC (nodular, superficial and infiltrative). These results suggest that the Fast Raman technique is a promising tool to provide an objective diagnosis "tumour clear yes/no" during Mohs surgery of BCC. This clinical integration study is a key step towards a larger scale diagnosis test accuracy study to reliably determine the sensitivity and specificity in a clinical setting., Competing Interests: I Notingher, H Williams, S Varma, A. Koloydenko hold a patent related to the Fast Raman technology. The Fast Raman device was built by RiverD International., (Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.)

Variant Creutzfeldt-Jakob disease (vCJD) is a human prion disease resulting from zoonotic transmission of bovine spongiform encephalopathy (BSE). Documented cases of vCJD transmission by blood transfusion necessitate on-going risk reduction measures to protect blood supplies, such as leucodepletion (removal of white blood cells, WBCs). This study set out to determine the risks of prion transmission by transfusion of labile blood components (red blood cells, platelets, plasma) commonly used in human medicine, and the effectiveness of leucodepletion in preventing infection, using BSE-infected sheep as a model. All components were capable of transmitting prion disease when donors were in the preclinical phase of infection, with the highest rates of infection in recipients of whole blood and buffy coat, and the lowest in recipients of plasma. Leucodepletion of components (<106 WBCs/unit) resulted in significantly lower transmission rates, but did not completely prevent transmission by any component. Donor PRNP genotype at codon 141, which is associated with variation in incubation period, also had a significant effect on transfusion transmission rates. A sensitive protein misfolding cyclic amplification (PMCA) assay, applied to longitudinal series of blood samples, identified infected sheep from 4 months post infection. However, in donor sheep (orally infected), the onset of detection of PrPSc in blood was much more variable, and generally later, compared to recipients (intravenous infection). This shows that the route and method of infection may profoundly affect the period during which an individual is infectious, and the test sensitivity required for reliable preclinical diagnosis, both of which have important implications for disease control. Our results emphasize that blood transfusion can be a highly efficient route of transmission for prion diseases. Given current uncertainties over the prevalence of asymptomatic vCJD carriers, this argues for the maintenance and improvement of current measures to reduce the risk of transmission by blood products., Competing Interests: The authors have declared that no competing interests exist.

The Adjuvant System AS01 contains monophosphoryl lipid A (MPL) and the saponin QS-21 in a liposomal formulation. AS01 is included in recently developed vaccines against malaria and varicella zoster virus. Like for many other adjuvants, induction of adaptive immunity by AS01 is highly dependent on the ability to recruit and activate dendritic cells (DCs) that migrate to the draining lymph node for T and B cell stimulation. The objective of this study was to more precisely address the contribution of the different conventional (cDC) and monocyte-derived DC (MC) subsets in the orchestration of the adaptive immune response after immunization with AS01 adjuvanted vaccine. The combination of MPL and QS-21 in AS01 induced strong recruitment of CD26 + XCR1 + cDC1s, CD26 + CD172 + cDC2s and a recently defined CCR2-dependent CD64-expressing inflammatory cDC2 (inf-cDC2) subset to the draining lymph node compared to antigen alone, while CD26 - CD64 + CD88 + MCs were barely detectable. At 24 h post-vaccination, cDC2s and inf-cDC2s were superior amongst the different subsets in priming antigen-specific CD4 + T cells, while simultaneously presenting antigen to CD8 + T cells. Diphtheria toxin (DT) mediated depletion of all DCs prior to vaccination completely abolished adaptive immune responses, while depletion 24 h after vaccination mainly affected CD8 + T cell responses. Vaccinated mice lacking Flt3 or the chemokine receptor CCR2 showed a marked deficit in inf-cDC2 recruitment and failed to raise proper antibody and T cell responses. Thus, the adjuvant activity of AS01 is associated with the potent activation of subsets of cDC2s, including the newly described inf-cDC2s., Competing Interests: CC and AC are employees of the GSK group of companies. AD was an employee of GSK at the time of the study and owns GSK stocks. 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 Bosteels, Fierens, De Prijck, Van Moorleghem, Vanheerswynghels, De Wolf, Chalon, Collignon, Hammad, Didierlaurent and Lambrecht.)

Background: During healthcare guideline development, panel members often have implicit, different definitions of health outcomes that can lead to misunderstandings about how important these outcomes are and how to balance benefits and harms. McMaster GRADE Centre researchers developed 'health outcome descriptors' for standardizing descriptions of health outcomes and overcoming these problems to support the European Commission Initiative on Breast Cancer (ECIBC) Guideline Development Group (GDG). We aimed to determine which aspects of the development, content, and use of health outcome descriptors were valuable to guideline developers., Methods: We developed 24 health outcome descriptors related to breast cancer screening and diagnosis for the European Commission Breast Guideline Development Group (GDG). Eighteen GDG members provided feedback in written format or in interviews. We then evaluated the process and conducted two health utility rating surveys., Results: Feedback from GDG members revealed that health outcome descriptors are probably useful for developing recommendations and improving transparency of guideline methods. Time commitment, methodology training, and need for multidisciplinary expertise throughout development were considered important determinants of the process. Comparison of the two health utility surveys showed a decrease in standard deviation in the second survey across 21 (88%) of the outcomes., Conclusions: Health outcome descriptors are feasible and should be developed prior to the outcome prioritization step in the guideline development process. Guideline developers should involve a subgroup of multidisciplinary experts in all stages of development and ensure all guideline panel members are trained in guideline methodology that includes understanding the importance of defining and understanding the outcomes of interest.

This observational study with a pre-post design, conducted in two Dutch primary healthcare centres, aimed to evaluate the effect of a supervised walking programme in highly motivated individuals with or at risk for type 2 diabetes mellitus (T2DM). Those able and willing to walk at least 6 km, were invited for a 28-week walking programme (February to August 2017), in which participants walked in groups, once weekly under supervision of volunteer healthcare professionals. Changes in bodyweight, BMI, waist circumference, HbA1c, blood pressure, well-being, health status and patient activation were analysed using paired t -tests and the Wilcoxon signed-rank test. Fifty-six people were included (30 T2DM; 26 at risk), of whom 60.7% were female. Mean age was 60.6 years, median BMI 30.8 kg/m 2 and mean systolic blood pressure 146.9 mm Hg. Participants with T2DM had median HbA1c of 50.0 mmol/mol. Post-challenge, BMI had decreased to 29.7 kg/m 2 , and waist circumference decreased 3.4 cm (95% CI 2.1-4.8), both p  < 0.01. Systolic and diastolic blood pressure decreased significantly (mean difference 6.5 mm Hg (95% CI 1.6-11.3, p  = 0.01) and 3.5 mm Hg (95% CI 1.0-6.0, p  < 0.01), respectively). Participants with HbA1c >53 mmol/mol at baseline ( n  = 8), had median decrease in HbA1c of 6.5 mmol/mol ( p  = 0.03). Well-being, but not health status and patient activation, improved significantly. In conclusion, in highly motivated individuals with or at risk for T2DM, this simple to implement and low-cost, but intensive, volunteer-based supervised walking programme is favourable, and therefore, can be seen as an option for clinical programs to implement to support highly motivated patients.

Dendritic cells (DCs) are key connectors between the innate and adaptive immune system and have an important role in modulating other immune cells. Therefore, their therapeutic application to steer immune responses is considered in various disorders, including cancer. Due to differences in the cell source and manufacturing process, each DC medicinal product is unique. Consequently, release tests to ensure consistent quality need to be product-specific. Although general guidance concerning quality control testing of cell-based therapies is available, cell type-specific regulation is still limited. Especially guidance related to potency testing is needed, because developing an in vitro assay measuring cell properties relevant for in vivo functionality is challenging. In this review, we provide DC-specific guidance for development of in vitro potency assays for characterisation and release. We present a broad overview of in vitro potency assays suggested for DC products to determine their anti-tumor functionality. Several advantages and limitations of these assays are discussed. Also, we provide some points to consider for selection and design of a potency test. The ideal functionality assay for anti-tumor products evaluates the capacity of DCs to stimulate antigen-specific T cells. Because this approach may not be feasible for release, use of surrogate potency markers could be considered, provided that these markers are sufficiently linked to the in vivo DC biological activity and clinical response. Further elucidation of the involvement of specific DC subsets in anti-tumor responses will result in improved manufacturing processes for DC-based products and should be considered during potency assay development., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: Gait analysis of animal disease models can provide valuable insights into in vivo compound effects and thus help in preclinical drug development. The purpose of this paper is to establish a computational gait analysis approach for the Noldus Catwalk system, in which footprints are automatically captured and stored., Methods: We present a - to our knowledge - first machine learning based approach for the Catwalk system, which comprises a step decomposition, definition and extraction of meaningful features, multivariate step sequence alignment, feature selection, and training of different classifiers (gradient boosting machine, random forest, and elastic net)., Results: Using animal-wise leave-one-out cross validation we demonstrate that with our method we can reliable separate movement patterns of a putative Parkinson's disease animal model and several control groups. Furthermore, we show that we can predict the time point after and the type of different brain lesions and can even forecast the brain region, where the intervention was applied. We provide an in-depth analysis of the features involved into our classifiers via statistical techniques for model interpretation., Conclusion: A machine learning method for automated analysis of data from the Noldus Catwalk system was established., Significance: Our works shows the ability of machine learning to discriminate pharmacologically relevant animal groups based on their walking behavior in a multivariate manner. Further interesting aspects of the approach include the ability to learn from past experiments, improve with more data arriving and to make predictions for single animals in future studies.

The adaptive immune system is known to play an important role in anti-neoplastic responses via induction of several effector pathways, resulting in tumor cell death. Because of their ability to specifically recognize and kill tumor cells, the potential use of autologous tumor-derived and genetically engineered T cells as adoptive immunotherapy for cancer is currently being explored. Because of the variety of potential T cell-based medicinal products at the level of starting material and manufacturing process, product-specific functionality assays are needed to ensure quality for individual products. In this review, we provide an overview of in vitro potency assays suggested for characterization and release of different T cell-based anti-tumor products. We discuss functional assays, as presented in scientific advices and literature, highlighting specific advantages and limitations of the various assays. Because the anticipated in vivo mechanism of action for anti-tumor T cells involves tumor recognition and cell death, in vitro potency assays based on the cytotoxic potential of antigen-specific T cells are most evident. However, assays based on other T cell properties may be appropriate as surrogates for cytotoxicity. For all proposed assays, biological relevance of the tests and correlation of the read-outs with in vivo functionality need to be substantiated with sufficient product-specific (non-)clinical data. Moreover, further unraveling the complex interaction of immune cells with and within the tumor environment is expected to lead to further improvement of the T cell-based products. Consequently, increased knowledge will allow further optimized guidance for potency assay development., (Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

There is considerable interest in redeploying drugs for use in combination with other oncology therapeutics. The single-agent activity of statins in ovarian cancer has been widely reported, however the drug concentration required to cause cell death is considerably higher than that achieved in patients receiving statin treatment for hypercholesterolemia. Unfortunately, statins can cause myopathy when administered in high doses. One solution to this is to identify drugs that could be used in combination with statins to reduce the dose required and those that may potentially reduce the incidence of adverse side effects. When the BH3 mimetic ABT-737, or the phosphatidylinositol 3-kinase inhibitor pictilisib, were combined with pitavastatin in cell growth assays using Ovcar-3 and Igrov-1 cells, the drug combinations were more effective than pitavastatin alone. In support of this, ABT-737 or pictilisib markedly increased cell death induced by pitavastatin in several ovarian cancer cell lines. The drugs were also synergistic in apoptosis assays. These observations suggested that either BH3 mimetics or pictilisib in combination with pitavastatin could be used in a subset of ovarian tumours, particularly those sensitive to BH3 mimetics, and phosphatase and tensin homolog inhibition, in the treatment of ovarian cancer.

Mesenchymal stromal cells (MSCs) are multipotent cells derived from various tissues that can differentiate into several cell types. MSCs are able to modulate the response of immune cells of the innate and adaptive immune system. Because of these multimodal properties, the potential use of MSCs for immunotherapies is currently explored in various clinical indications. Due to the diversity of potential MSC medicinal products at the level of cell source, manufacturing process and indication, distinct functionality tests may be needed to ensure the quality for each of the different products. In this review, we focus on in vitro potency assays proposed for characterization and release of different MSC medicinal products. We discuss the most used functional assays, as presented in scientific advices and literature, highlighting specific advantages and limitations of the various assays. Currently, the most proposed and accepted potency assay for release is based on in vitro inhibition of T cell proliferation or other functionalities. However, for some products, assays based on other MSC or responder cell properties may be more appropriate. In all cases, the biological relevance of the proposed assay for the intended clinical activity should be substantiated with appropriate product-specific (non-)clinical data. In case practical considerations prevent the use of the ideal potency assay at release, use of a surrogate marker or test could be considered if correlation with functionality has been demonstrated. Nevertheless, as the field of MSC immunology is evolving, improvements can be expected in relevant assays and consequently in guidance related to potency testing., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Objective: We previously showed that mycobacterial Hsp70-derived peptide B29 induced B29-specific Treg cells that suppressed experimental arthritis in mice via cross-recognition of their mammalian Hsp70 homologs. The aim of the current study was to characterize B29 binding and specific CD4+ T cell responses in the context of human major histocompatibility complex (MHC) molecules., Methods: Competitive binding assays were performed to examine binding of peptide B29 and its mammalian homologs to HLA molecules. The effect of B29 immunization in HLA-DQ8-transgenic mice with proteoglycan-induced arthritis was assessed, followed by ex vivo restimulation with B29 to examine the T cell response. Human peripheral blood mononuclear cells were used to investigate the presence of B29-specific T cells with immunoregulatory potential., Results: The binding affinity of the B29 peptide was high to moderate for multiple HLA-DR and HLA-DQ molecules, including those highly associated with rheumatoid arthritis. This binding was considered to be functional, because B29 immunization resulted in the suppression of arthritis and T cell responses in HLA-DQ8-transgenic mice. In humans, we demonstrated the presence and expansion of B29-specific CD4+ T cells, which were cross-reactive with the mammalian homologs. Using HLA-DR4+ tetramers specific for B29 or the mammalian homolog mB29b, we showed expansion of cross-reactive T cells, especially the human FoxP3+ CD4+CD25+ T cell population, after in vitro stimulation with B29., Conclusion: These results demonstrated a conserved fine specificity and functionality of B29-induced Treg cell responses in the context of the human MHC. Based on these findings, a path for translation of the experimental findings for B29 into a clinical immunomodulatory therapeutic approach is within reach., (© 2016, American College of Rheumatology.)

In Parkinson's disease (PD), dopaminergic therapies are often associated with the development of motor complications. Attention has therefore been focused on the use of non-dopaminergic drugs. This study developed a new behavioural method capable of demonstrating the added value of combining adenosinergic and glutamatergic receptor antagonists in unilateral 6-OHDA lesioned rats. Rats were dosed orally with Tozadenant, a selective A2A receptor antagonist, and three different doses of Radiprodil, an NR2B-selective NMDA receptor antagonist. The drugs were given alone or in combination and rats were placed in an open-field for behavioural monitoring. Video recordings were automatically analysed. Five different behaviours were scored: distance traveled, ipsi- and contraversive turns, body position, and space occupancy. The results show that A2A or NR2B receptor antagonists given alone or in combination did not produce enhanced turning as observed with an active dose of L-Dopa/benserazide. Instead the treated rats maintained a straight body position, were able to shift from one direction to the other and occupied a significantly larger space in the arena. The highest "Tozadenant/Radiprodil" dose combination significantly increased all five behavioural parameters recorded compared to rats treated with vehicle or the same doses of the drugs alone. Our data suggest that the A2A/NR2B antagonist combination may be able to stimulate motor activity to a similar level as that achieved by L-Dopa but in the absence of the side-effects that are associated with dopaminergic hyperstimulation. If these results translate into the clinic, this combination could represent an alternative symptomatic treatment option for PD.

This paper describes the generation, characterisation and potential applications of a panel of novel anti-prion protein monoclonal antibodies (mAbs). The mAbs were generated by immunising PRNP null mice, using a variety of regimes, with a truncated form of recombinant ovine prion protein spanning residues 94-233. Epitopes of specific antibodies were mapped using solid-phase Pepscan analysis and clustered to four distinct regions within the PrP molecule. We have demonstrated the utility of these antibodies by use of Western blotting and immunohistochemistry in tissues from a range of different species affected by transmissible spongiform encephalopathy (TSE). In comparative tests against extensively-used and widely-published, commercially available antibodies, similar or improved results can be obtained using these new mAbs, specifically in terms of sensitivity of detection. Since many of these antibodies recognise native PrPC, they could also be applied to a broad range of immunoassays such as flow cytometry, DELFIA analysis or immunoprecipitation. We are using these reagents to increase our understanding of TSE pathogenesis and for use in potential diagnostic screening assays.

The susceptibility of sheep to prion infection is linked to variation in the PRNP gene, which encodes the prion protein. Common polymorphisms occur at codons 136, 154 and 171. Sheep which are homozygous for the A(136)R(154)Q(171) allele are the most susceptible to bovine spongiform encephalopathy (BSE). The effect of other polymorphisms on BSE susceptibility is unknown. We orally infected ARQ/ARQ Cheviot sheep with equal amounts of BSE brain homogenate and a range of incubation periods was observed. When we segregated sheep according to the amino acid (L or F) encoded at codon 141 of the PRNP gene, the shortest incubation period was observed in LL(141) sheep, whilst incubation periods in FF(141) and LF(141) sheep were significantly longer. No statistically significant differences existed in the expression of total prion protein or the disease-associated isoform in BSE-infected sheep within each genotype subgroup. This suggested that the amino acid encoded at codon 141 probably affects incubation times through direct effects on protein misfolding rates.

Objective: To estimate the cumulative risk of a false-positive screening result in European mammographic screening programmes, and examine the rates and procedures of further assessment., Methods: A literature review was conducted to identify studies of the cumulative risk of a false-positive result in European screening programmes (390,000 women). We then examined aggregate data, cross-sectional information about further assessment procedures among women with positive results in 20 mammographic screening programmes from 17 countries (1.7 million initial screens, 5.9 million subsequent screens), collected by the European Network for Information on Cancer project (EUNICE)., Results: The estimated cumulative risk of a false-positive screening result in women aged 50-69 undergoing 10 biennial screening tests varied from 8% to 21% in the three studies examined (pooled estimate 19.7%). The cumulative risk of an invasive procedure with benign outcome ranged from 1.8% to 6.3% (pooled estimate 2.9%). The risk of undergoing surgical intervention with benign outcome was 0.9% (one study only). From the EUNICE project, the proportions of all screening examinations in the programmes resulting in needle biopsy were 2.2% and 1.1% for initial and subsequent screens, respectively, though the rates differed between countries; the corresponding rates of surgical interventions among women without breast cancer were 0.19% and 0.07%., Conclusion: The specific investigative procedures following a recall should be considered when examining the cumulative risk of a false-positive screening result. Most women with a positive screening test undergo a non-invasive assessment procedure. Only a small proportion of recalled women undergo needle biopsy, and even fewer undergo surgical intervention.

Objectives: To summarize participation and coverage rates in population mammographic screening programmes for breast cancer in Europe., Methods: We used the European Network for Information on Cancer (EUNICE), a web-based data warehouse (EUNICE Breast Cancer Screening Monitoring, EBCSM) for breast cancer screening, to obtain information on programme characteristics, coverage and participation from its initial application in 10 national and 16 regional programmes in 18 European countries., Results: The total population targeted by the screening programme services covered in the report comprised 26.9 million women predominantly aged 50-69. Most of the collected data relates to 2005, 2006 and/or 2007. The average participation rate across all programmes was 53.4% (range 19.4-88.9% of personally invited); or 66.4% excluding Poland, a large programme that initiated personal invitations in 2007. Thirteen of the 26 programmes achieved the European Union benchmark of acceptable participation (>70%), nine achieved the desirable level (>75%). Despite considerable invitation coverage across all programmes (79.3%, range 50.9-115.2%) only 48.2% (range 28.4-92.1%) of the target population were actually screened. The overall invitation and examination coverage excluding Poland was 70.9% and 50.3%, respectively., Conclusions: The results demonstrate the feasibility of European-wide screening monitoring using the EBCSM data warehouse, although further efforts to refine the system and to harmonize standards and data collection practices will be required, to fully integrate all European countries. The more than three-fold difference in the examination coverage should be taken into account in the evaluation of service screening programmes.

Variant CJD (vCJD) is an incurable, infectious human disease, likely arising from the consumption of BSE-contaminated meat products. Whilst the epidemic appears to be waning, there is much concern that vCJD infection may be perpetuated in humans by the transfusion of contaminated blood products. Since 2004, several cases of transfusion-associated vCJD transmission have been reported and linked to blood collected from pre-clinically affected donors. Using an animal model in which the disease manifested resembles that of humans affected with vCJD, we examined which blood components used in human medicine are likely to pose the greatest risk of transmitting vCJD via transfusion. We collected two full units of blood from BSE-infected donor animals during the pre-clinical phase of infection. Using methods employed by transfusion services we prepared red cell concentrates, plasma and platelets units (including leucoreduced equivalents). Following transfusion, we showed that all components contain sufficient levels of infectivity to cause disease following only a single transfusion and also that leucoreduction did not prevent disease transmission. These data suggest that all blood components are vectors for prion disease transmission, and highlight the importance of multiple control measures to minimise the risk of human to human transmission of vCJD by blood transfusion.

Randomised controlled trials showed that breast cancer screening by mammography reduces breast cancer mortality in women over age 50 by 25-30%. However, it was not clear if this effect would persist outside the controlled trial environment or even could be enhanced. We review the current evidence of the impact of long-standing breast cancer screening programmes (Australia, Canada, Denmark, Finland, Iceland, Italy, the Netherlands, Spain, Sweden and the United Kingdom) on breast cancer mortality. The decrease observed in women invited to screening ranges from 16% to 36%. Breast cancer mortality reductions range from 24% to 48% in women having attended at least one screen after correcting for selection bias. Although evaluation design, time period studied, participation rates achieved differ, the trend in mortality reduction is consistent. Adjuvant therapy is estimated to contribute about one third to this decrease. We conclude that mammography screening programmes implemented for at least 10 years achieve a similar, but not greater mortality reduction as the randomised controlled trials. However, it may take some more years before the full impact of these mammography screening programmes can be assessed.

Background: Various centralised mammography screening programmes have shown to reduce breast cancer mortality at reasonable costs. However, mammography screening is not necessarily cost-effective in every situation. Opportunistic screening, the predominant screening modality in several European countries, may under certain circumstances be a cost-effective alternative. In this study, we compared the cost-effectiveness of both screening modalities in Switzerland., Methods: Using micro-simulation modelling, we predicted the effects and costs of biennial mammography screening for 50-69 years old women between 1999 and 2020, in the Swiss female population aged 30-70 in 1999. A sensitivity analysis on the test sensitivity of opportunistic screening was performed., Results: Organised mammography screening with an 80% participation rate yielded a breast cancer mortality reduction of 13%. Twenty years after the start of screening, the predicted annual breast cancer mortality was 25% lower than in a situation without screening. The 3% discounted cost-effectiveness ratio of organised mammography screening was euro11,512 per life year gained. Opportunistic screening with a similar participation rate was comparably effective, but at twice the costs: euro22,671-24,707 per life year gained. This was mainly related to the high costs of opportunistic mammography and frequent use of imaging diagnostics in combination with an opportunistic mammogram., Conclusion: Although data on the performance of opportunistic screening are limited, both opportunistic and organised mammography screening seem effective in reducing breast cancer mortality in Switzerland. However, for opportunistic screening to become equally cost-effective as organised screening, costs and use of additional diagnostics should be reduced.

We have studied the potential contribution of ABCG2 (breast cancer resistance protein) to resistance to nucleoside analogues. In cells transfected with DNA constructs resulting in overexpression of human or mouse ABCG2, we found resistance against cladribine, clofarabine, fludarabine, 6-mercaptopurine, and 6-mercaptopurine riboside in both MDCKII and HEK293 cells and against gemcitabine only in HEK293 cells. With Transwell studies in MDCK cells and transport experiments with vesicles from Sf9 and HEK293 cells, we show that ABCG2 is able to transport not only the nucleotide CdAMP, like several other ATP-binding cassette transporters of the ABCC (multidrug resistance protein) family, but also the nucleoside cladribine itself. Expression of ABCG2 in cells results in a substantial decrease of intracellular CdATP, explaining the resistance against cladribine. The high transport rate of cladribine and clofarabine by ABCG2 deduced from Transwell experiments raises the possibility that this transporter could affect the disposition of nucleoside analogues in patients or cause resistance in tumors.

Breast cancer is a major cause of suffering and death and is of significant concern to many women. Early detection of breast cancer by systematic mammography screening can find lesions for which treatment is more effective and generally more favourable for quality of life. The potential harm caused by mammography includes the creation of unnecessary anxiety and morbidity, inappropriate economic cost and the use of ionising radiation. It is for this reason that the strongest possible emphasis on quality control and quality assurance is required. Development of the European Guidelines for Quality Assurance in Breast Cancer Screening and Diagnosis has been an initiative within the Europe Against Cancer Programme. The fourth edition of the multidisciplinary guidelines was published in 2006 and comprises approximately 400 pages divided into 12 chapters prepared by >200 authors and contributors. The multidisciplinary editorial board has prepared a summary document to provide an overview of the fundamental points and principles that should support any quality screening or diagnostic service. This document includes a summary table of key performance indicators and is presented here in order to make these principles and standards known to a wider scientific community.

According to EUSOMA position paper 'The requirements of a specialist breast unit', each breast unit should have a core team made up of health professionals who have undergone specialist training in breast cancer. In this paper, on behalf of EUSOMA, authors have identified the standards of training in breast cancer, to harmonise and foster breast care training in Europe. The aim of this paper is to contribute to the increase in the level of care in a breast unit, as the input of qualified health professionals increases the quality of breast cancer patient care.

We summarize in this paper the recently published results on multidrug resistance-associated proteins 3, 4, and 5 (MRPs 3-5). MRP3 can transport organic compounds conjugated to glutathione, sulfate, or glucuronate, such as estradiol-17beta-glucuronide, bilirubin-glucuronides, and etoposide-glucuronide, and also bile salts and methotrexate. Studies in knockout mice have shown that Mrp3 contributes to the transport of morphine-3-glucuronide and acetaminophen-glucuronide from the liver into blood. There is no evidence for a major role of MRP3 in bile salt metabolism, at least in mice. The function of MRP3 in other tissues, notably the gut and the adrenal cortex, remains to be defined. MRP4 and MRP5 have attracted attention by their ability to transport cyclic nucleotides and many nucleotide analogs. The initial reports that MRP4 and 5 can transport cGMP with microM affinity have not been confirmed in recent work and the physiological importance of cyclic nucleotide transport by MRP4 and 5 remains to be determined. Transfected cells containing high concentrations of MRP4 and 5 are moderately resistant to base, nucleoside, and nucleotide analogs. The affinity of both transporters for nucleotide analogs is low (K (m) around 1 mM) and there is no evidence that the transport of these compounds results in resistance in vivo. The physiological function of MRP4 and 5 remains to be found.

Background: Increasing incidence of ductal carcinoma in situ (DCIS) confronts patients and clinicians with optimal treatment decisions. This multidisciplinary study investigates therapeutic modalities of DCIS in daily practice and provides recommendations on how to increase quality of care., Patients and Methods: All women (n = 116) with unilateral DCIS recorded in the Geneva Cancer Registry from 1995 to 1999 were considered. Information concerned patient and tumor characteristics, treatment and outcome. Factors linked to therapy were determined using a case-control approach. Cases were women with treatment of interest and controls other women on the study., Results: Most DCIS cases (62%) were discovered by mammography screening. Ninety (78%) women had breast-conserving surgery (BCS), 18 (16%) mastectomy and seven (6%) bilateral mastectomy. Eight (7%) patients had tumor-positive margins, 18 (16%) lymph node dissection and two (1.7%) chemotherapy. Twenty-five per cent of women with BCS had no radiotherapy, three had radiotherapy after mastectomy. Less than 50% underwent breast reconstruction after mastectomy. Method of discovery, multifocality, tumor localization, size and differentiation were linked to the use of BCS or lymph node dissection., Conclusions: Because of important disparities in DCIS management, recommendations are made to increase quality of care, in particular to prevent axillary dissection or bilateral mastectomy and to increase the use of radiotherapy after BCS.

In 1990, an international consensus was reached on the efficacy of adjuvant chemotherapy for lymph node positive (stage III) colon carcinoma (CC). This study evaluates the use and benefit of such therapy in routine health care practice. The study includes all patients with stage III CC treated by putative curative surgery (n = 182) recorded at the Geneva cancer registry between 1990 and 1996. Factors modifying chemotherapy use were determined by logistic regression, considering patients with chemotherapy as cases (n = 55) and others as controls (n = 127). The effect of chemotherapy on the 5-year survival was evaluated by the Cox model. Analyses were adjusted for possible confounders. The use of chemotherapy increased over the period (P(trend) < 0.001). Age strongly modulated chemotherapy use. In 1996, 54% of eligible patients received chemotherapy, this proportion fell to 13% after age 70. Decisions to use chemotherapy significantly depended on stage, grade and cancer site. The chance to be treated was non-significantly lower among individuals of low social class, widowed and foreigners. Chemotherapy significantly decreased mortality rates (Hazard ratio: 0.35, 95%CI: 0.18-0.68), independently of the prognostic factors and with similar benefit regardless of stage and age group. Strong beneficial effect of adjuvant chemotherapy on stage III CC can be achieved in routine practice. However, this study shows that it is probably not optimally utilised in Switzerland, particularly among the elderly., (Copyright 2001 Cancer Research Campaign)

The European Guidelines developed for mammography screening have contributed to the general discussion on quality assurance and the important tasks of the health professionals dealing with breast cancer screening. The cooperation of each medical discipline is of utmost importance in order to achieve optimal results and eventually a mortality reduction. The following guidelines are based on the British NHS quality assurance guidelines for surgeons in breast cancer screening and modified to meet the different needs in the European Countries. The term "surgeon" denotes a medical doctor trained and involved in the surgical treatment of breast diseases. The members of the working group who participated in order to adapt these guidelines are listed above. We hope that this document will contribute towards a more comprehensive approach of breast cancer screening-detected lesions throughout Europe.

We characterized on immuno- and enzymecytochemical level the lymphoid and non-lymphoid cells in the adenoid of children with upper respiratory tract infections (URI) and otitis media with effusion (OME) and compared these with the adenoid of children with URI without OME and with the adenoid of 'healthy' children and adults. Besides macrophages and dendritic cells we also showed the presence of MHC class II positive, ciliated, epithelial cells. These non-lymphoid cells were present in all adenoids. However, their number was less than 1% of all cells. We found no difference in lymphocyte subsets from children with URI + OME compared with those from children with URI alone. These two groups showed a significant decrease of CD8-positive (suppressor/cytotoxic) cells and a slight increase in CD22-positive B cells in comparison to 'healthy' children. No difference was found in percentages of CD4-positive (helper/inducer) cells. The localization of the lymphoid subsets in adenoids of children with URI and/or OME did not differ from those of 'healthy' children and adults.