Publications by authors named "Thomas Winkler"

228 Publications

Targeting B cells in the pre-phase of systemic autoimmunity globally interferes with autoimmune pathology.

iScience 2021 Sep 1;24(9):103076. Epub 2021 Sep 1.

Chair of Genetics, Department of Biology, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erwin-Rommelstr. 3, 91058 Erlangen, Germany.

Systemic lupus erythematosus (SLE) is characterized by a loss of self-tolerance, systemic inflammation, and multi-organ damage. While a variety of therapeutic interventions are available, it has become clear that an early diagnosis and treatment may be key to achieve long lasting therapeutic responses and to limit irreversible organ damage. Loss of humoral tolerance including the appearance of self-reactive antibodies can be detected years before the actual onset of the clinical autoimmune disease, representing a potential early point of intervention. Not much is known, however, about how and to what extent this pre-phase of disease impacts the onset and development of subsequent autoimmunity. By targeting the B cell compartment in the pre-disease phase of a spontaneous mouse model of SLE we now show, that resetting the humoral immune system during the clinically unapparent phase of the disease globally alters immune homeostasis delaying the downstream development of systemic autoimmunity.
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http://dx.doi.org/10.1016/j.isci.2021.103076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8455742PMC
September 2021

Sorption of Neuropsychopharmaca in Microfluidic Materials for Studies.

ACS Appl Mater Interfaces 2021 Sep 16;13(38):45161-45174. Epub 2021 Sep 16.

Division of Micro- and Nanosystems, KTH Royal Institute of Technology, 10044 Stockholm, Sweden.

Sorption (, adsorption and absorption) of small-molecule compounds to polydimethylsiloxane (PDMS) is a widely acknowledged phenomenon. However, studies to date have largely been conducted under atypical conditions for microfluidic applications (lack of perfusion, lack of biological fluids, etc.), especially considering biological studies such as organs-on-chips where small-molecule sorption poses the largest concern. Here, we present an in-depth study of small-molecule sorption under relevant conditions for microphysiological systems, focusing on a standard geometry for biological barrier studies that find application in pharmacokinetics. We specifically assess the sorption of a broad compound panel including 15 neuropsychopharmaca at concentration levels. We consider devices constructed from PDMS as well as two material alternatives (off-stoichiometry thiol-ene-epoxy, or tape/polycarbonate laminates). Moreover, we study the much neglected impact of peristaltic pump tubing, an essential component of the recirculating systems required to achieve -like perfusion shear stresses. We find that the choice of the device material does not have a significant impact on the sorption behavior in our barrier-on-chip-type system. Our PDMS observations in particular suggest that excessive compound sorption observed in prior studies is not sufficiently described by compound hydrophobicity or other suggested predictors. Critically, we show that sorption by peristaltic tubing, including the commonly utilized PharMed BPT, dominates over device sorption even on an area-normalized basis, let alone at the typically much larger tubing surface areas. Our findings highlight the importance of validating compound dosages in organ-on-chip studies, as well as the need for considering tubing materials with equal or higher care than device materials.
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http://dx.doi.org/10.1021/acsami.1c07639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8485331PMC
September 2021

A pair of noncompeting neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model.

Eur J Immunol 2021 Aug 6. Epub 2021 Aug 6.

Division of Molecular Immunology, Internal Medicine III, Nikolaus-Fiebiger-Center of Molecular Medicine, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany.

TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS-CoV-2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralize SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2-induced weight loss. The two clusters of potent noncompeting SARS-CoV-2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID-19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives.
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http://dx.doi.org/10.1002/eji.202149374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8420377PMC
August 2021

Method for Multiplexed Dynamic Intravital Multiphoton Imaging.

Methods Mol Biol 2021 ;2350:145-156

Biophysical Analytics, Deutsches Rheumaforschungszentrum - a Leibniz Institute, Berlin, Germany.

Intravital two-photon microscopy enables monitoring of cellular dynamics and communication of complex systems, in genuine environment-the living organism. Particularly, its application in understanding the immune system brought unique insights into pathophysiologic processes in vivo. Here we present a method to achieve multiplexed dynamic intravital two-photon imaging by using a synergistic strategy combining a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an effective unmixing algorithm based on the calculation of spectral similarities with previously acquired fluorophore fingerprints. Our unmixing algorithm allows us to distinguish 7 fluorophore signals corresponding to various cellular and tissue compartments by using only four detector channels.
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http://dx.doi.org/10.1007/978-1-0716-1593-5_10DOI Listing
September 2021

Cost-Consequence Analysis of Using Cangrelor in High Angiographic Risk Percutaneous Coronary Intervention Patients: A US Hospital Perspective.

Am J Cardiovasc Drugs 2021 Jul 31. Epub 2021 Jul 31.

Brigham and Women's Hospital Heart and Vascular Center, and Harvard Medical School, Boston, MA, USA.

Objectives: The objective of this study was to evaluate a US hospital's cost implications and outcomes of cangrelor use in percutaneous coronary intervention (PCI) patients with two or more angiographic high-risk features (HRFs), including avoidance of oral P2Y inhibitor pretreatment in patients requiring cardiac surgery. Intravenous cangrelor provides direct, immediate onset and rapid-offset P2Y inhibition, which may reduce the necessity for oral P2Y pretreatment.

Methods: A decision analytic model was developed, estimating the annual impact over 3 years of cangrelor availability. Ischemic and bleeding events (48 h) from randomized clinical trial data were extrapolated to 30 days. Event costs were from the CHAMPION PHOENIX Economics substudy. Rates of coronary artery disease (CAD) presentation, PCI, oral P2Y pretreatment, and inpatient hospitalization costs were from published literature and clinical experts. Scenario analyses evaluated the impact of cangrelor availability on potential reduced P2Y pretreatment rates by 50-100%. Drug costs were 2019 wholesale acquisition costs and, where necessary, all costs were adjusted to 2019 dollars.

Results: In a hospital treating 1000 CAD PCI inpatients annually, increasing cangrelor use from 11 to 32% resulted in a reduction in 48-h ischemic events/year by 5.7%, while bleeding events increased by 2.9%. Total costs of $1,135,472 declined 12.8%, with a 50% reduction in P2Y pretreatment or 30% with no pretreatment. Savings were driven by a decrease in ischemic events, decrease in glycoprotein IIb/IIIa inhibitor use, and less need for and shorter oral P2Y inhibitor washout period for surgery patients.

Conclusion: Use of cangrelor in patients with two or more angiographic HRFs may improve outcomes and lower hospital budgets, mainly from avoiding surgery delays necessitated by oral P2Y inhibitor pretreatment.
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http://dx.doi.org/10.1007/s40256-021-00491-9DOI Listing
July 2021

CD22 Controls Germinal Center B Cell Receptor Signaling, Which Influences Plasma Cell and Memory B Cell Output.

J Immunol 2021 08 30;207(4):1018-1032. Epub 2021 Jul 30.

Division of Genetics, Department of Biology, University of Erlangen, Erlangen, Germany

Germinal center reactions are established during a thymus-dependent immune response. Germinal center (GC) B cells are rapidly proliferating and undergo somatic hypermutation in Ab genes. This results in the production of high-affinity Abs and establishment of long-lived memory cells. GC B cells show lower BCR-induced signaling when compared with naive B cells, but the functional relevance is not clear. CD22 is a member of the Siglec family and functions as an inhibitory coreceptor on B cells. Interestingly, GC B cells downregulate sialic acid forms that serve as high-affinity ligands for CD22, indicating a role for CD22 ligand binding during GC responses. We studied the role of CD22 in the GC with mixed bone marrow chimeric mice and found a disadvantage of CD22 GC B cells during the GC reaction. Mechanistic investigations ruled out defects in dark zone/light zone distribution and affinity maturation. Rather, an increased rate of apoptosis in CD22 GC B cells was responsible for the disadvantage, also leading to a lower GC output in plasma cells and memory B cells. CD22 GC B cells showed a clearly increased calcium response upon BCR stimulation, which was almost absent in wild-type GC B cells. We conclude that the differential expression of the low-affinity CD22 ligands in the GC normally results in a strong attenuation of BCR signaling in GC B cells, probably due to higher CD22-BCR interactions. Therefore, attenuation of BCR signaling by CD22 is involved in GC output and B cell fate.
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http://dx.doi.org/10.4049/jimmunol.2100132DOI Listing
August 2021

Impact of Orientational Glass Formation and Local Strain on Photo-Induced Halide Segregation in Hybrid Metal-Halide Perovskites.

J Phys Chem C Nanomater Interfaces 2021 Jul 30;125(27):15025-15034. Epub 2021 Jun 30.

Cavendish Laboratory, University of Cambridge, J. J. Thomson Avenue, Cambridge CB3 0HE, U.K.

Band gap tuning of hybrid metal-halide perovskites by halide substitution holds promise for tailored light absorption in tandem solar cells and emission in light-emitting diodes. However, the impact of halide substitution on the crystal structure and the fundamental mechanism of photo-induced halide segregation remain open questions. Here, using a combination of temperature-dependent X-ray diffraction and calorimetry measurements, we report the emergence of a disorder- and frustration-driven orientational glass for a wide range of compositions in CHNHPb(Cl Br ). Using temperature-dependent photoluminescence measurements, we find a correlation between halide segregation under illumination and local strains from the orientational glass. We observe no glassy behavior in CsPb(Cl Br ), highlighting the importance of the A-site cation for the structure and optoelectronic properties. Using first-principles calculations, we identify the local preferential alignment of the organic cations as the glass formation mechanism. Our findings rationalize the superior photostability of mixed-cation metal-halide perovskites and provide guidelines for further stabilization strategies.
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http://dx.doi.org/10.1021/acs.jpcc.1c03169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8287560PMC
July 2021

Discovery and prioritization of variants and genes for kidney function in >1.2 million individuals.

Nat Commun 2021 07 16;12(1):4350. Epub 2021 Jul 16.

Department of Genetic Epidemiology, University of Regensburg, Regensburg, Germany.

Genes underneath signals from genome-wide association studies (GWAS) for kidney function are promising targets for functional studies, but prioritizing variants and genes is challenging. By GWAS meta-analysis for creatinine-based estimated glomerular filtration rate (eGFR) from the Chronic Kidney Disease Genetics Consortium and UK Biobank (n = 1,201,909), we expand the number of eGFRcrea loci (424 loci, 201 novel; 9.8% eGFRcrea variance explained by 634 independent signal variants). Our increased sample size in fine-mapping (n = 1,004,040, European) more than doubles the number of signals with resolved fine-mapping (99% credible sets down to 1 variant for 44 signals, ≤5 variants for 138 signals). Cystatin-based eGFR and/or blood urea nitrogen association support 348 loci (n = 460,826 and 852,678, respectively). Our customizable tool for Gene PrioritiSation reveals 23 compelling genes including mechanistic insights and enables navigation through genes and variants likely relevant for kidney function in human to help select targets for experimental follow-up.
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http://dx.doi.org/10.1038/s41467-021-24491-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8285412PMC
July 2021

Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing Matrix.

Int J Mol Sci 2021 Jun 3;22(11). Epub 2021 Jun 3.

Biophysical Analytics, Deutsches Rheuma-Forschungszentrum, Berlin, a Leibniz Institute, 10117 Berlin, Germany.

Two-photon microscopy enables monitoring cellular dynamics and communication in complex systems, within a genuine environment, such as living tissues and, even, living organisms. Particularly, its application to understand cellular interactions in the immune system has brought unique insights into pathophysiologic processes in vivo. Simultaneous multiplexed imaging is required to understand the dynamic orchestration of the multiple cellular and non-cellular tissue compartments defining immune responses. Here, we present an improvement of our previously developed method, which allowed us to achieve multiplexed dynamic intravital two-photon imaging, by using a synergistic strategy. This strategy combines a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an unmixing algorithm based on the calculation of spectral similarities with previously measured fluorophore fingerprints. The improvement of the similarity spectral unmixing algorithm here described is based on dimensionality reduction of the mixing matrix. We demonstrate its superior performance in the correct pixel-based assignment of probes to tissue compartments labeled by single fluorophores with similar spectral fingerprints, as compared to the full-dimensional similarity spectral unmixing approach.
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http://dx.doi.org/10.3390/ijms22116046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8199868PMC
June 2021

Continuous Monitoring Reveals Protective Effects of N-Acetylcysteine Amide on an Isogenic Microphysiological Model of the Neurovascular Unit.

Small 2021 08 26;17(32):e2101785. Epub 2021 Jun 26.

Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Malvinas Väg 10 pl 5, Stockholm, 100 44, Sweden.

Microphysiological systems mimic the in vivo cellular ensemble and microenvironment with the goal of providing more human-like models for biopharmaceutical research. In this study, the first such model of the blood-brain barrier (BBB-on-chip) featuring both isogenic human induced pluripotent stem cell (hiPSC)-derived cells and continuous barrier integrity monitoring with <2 min temporal resolution is reported. Its capabilities are showcased in the first microphysiological study of nitrosative stress and antioxidant prophylaxis. Relying on off-stoichiometry thiol-ene-epoxy (OSTE+) for fabrication greatly facilitates assembly and sensor integration compared to the prevalent polydimethylsiloxane devices. The integrated cell-substrate endothelial resistance monitoring allows for capturing the formation and breakdown of the BBB model, which consists of cocultured hiPSC-derived endothelial-like and astrocyte-like cells. Clear cellular disruption is observed when exposing the BBB-on-chip to the nitrosative stressor linsidomine, and the barrier permeability and barrier-protective effects of the antioxidant N-acetylcysteine amide are reported. Using metabolomic network analysis reveals further drug-induced changes consistent with prior literature regarding, e.g., cysteine and glutathione involvement. A model like this opens new possibilities for drug screening studies and personalized medicine, relying solely on isogenic human-derived cells and providing high-resolution temporal readouts that can help in pharmacodynamic studies.
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http://dx.doi.org/10.1002/smll.202101785DOI Listing
August 2021

A Novel Strain-Specific Neutralizing Epitope on Glycoprotein H of Human Cytomegalovirus.

J Virol 2021 08 25;95(18):e0065721. Epub 2021 Aug 25.

Virologisches Institut, Klinische und Molekulare Virologie, Friedrich Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes severe clinical disease in immunosuppressed patients and congenitally infected newborn infants. Viral envelope glycoproteins represent attractive targets for vaccination or passive immunotherapy. To extend the knowledge of mechanisms of virus neutralization, monoclonal antibodies (MAbs) were generated following immunization of mice with HCMV virions. Hybridoma supernatants were screened for neutralization activity, yielding three potent MAbs, 6E3, 3C11, and 2B10. MAbs 6E3 and 3C11 blocked infection of all viral strains that were tested, while MAb 2B10 neutralized only 50% of the HCMV strains analyzed. Characterization of the MAbs using indirect immunofluorescence analyses demonstrated their reactivity with recombinantly derived gH. While MAbs 6E3 and 3C11 reacted with gH when expressed alone, 2B10 detected gH only when it was coexpressed with gB and gL. Recognition of gH by 3C11 was dependent on the expression of the entire ectodomain of gH, whereas 6E3 required residues 1 to 629 of gH. The strain-specific determinant for neutralization by Mab 2B10 was identified as a single Met→Ile amino acid polymorphism within gH, located within the central part of the protein. The polymorphism is evenly distributed among described HCMV strains. The 2B10 epitope thus represents a novel strain-specific antibody target site on gH of HCMV. The dependence of the reactivity of 2B10 on the simultaneous presence of gB/gH/gL will be of value in the structural definition of this tripartite complex. The 2B10 epitope may also represent a valuable tool for diagnostics to monitor infections/reinfections with different HCMV strains during pregnancy or after transplantation. HCMV infections are life threatening to people with compromised or immature immune systems. Understanding the antiviral antibody repertoire induced during HCMV infection is a necessary prerequisite to define protective antibody responses. Here, we report three novel anti-gH MAbs that potently neutralized HCMV infectivity. One of these MAbs (2B10) targets a novel strain-specific conformational epitope on gH that only becomes accessible upon coexpression of the minimal fusion machinery gB/gH/gL. Strain specificity is dependent on a single amino acid polymorphism within gH. Our data highlight the importance of strain-specific neutralizing antibody responses against HCMV. The 2B10 epitope may also represent a valuable tool for diagnostics to monitor infections/reinfections with different HCMV strains during pregnancy or after transplantation. In addition, the dependence of the reactivity of 2B10 on the simultaneous presence of gB/gH/gL will be of value in the structural definition of this tripartite complex.
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http://dx.doi.org/10.1128/JVI.00657-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387041PMC
August 2021

Manganese doping for enhanced magnetic brightening and circular polarization control of dark excitons in paramagnetic layered hybrid metal-halide perovskites.

Nat Commun 2021 Jun 9;12(1):3489. Epub 2021 Jun 9.

Walter Schottky Institut and Physik Department, Technische Universität München, Garching, Germany.

Materials combining semiconductor functionalities with spin control are desired for the advancement of quantum technologies. Here, we study the magneto-optical properties of novel paramagnetic Ruddlesden-Popper hybrid perovskites Mn:(PEA)PbI (PEA = phenethylammonium) and report magnetically brightened excitonic luminescence with strong circular polarization from the interaction with isolated Mn ions. Using a combination of superconducting quantum interference device (SQUID) magnetometry, magneto-absorption and transient optical spectroscopy, we find that a dark exciton population is brightened by state mixing with the bright excitons in the presence of a magnetic field. Unexpectedly, the circular polarization of the dark exciton luminescence follows the Brillouin-shaped magnetization with a saturation polarization of 13% at 4 K and 6 T. From high-field transient magneto-luminescence we attribute our observations to spin-dependent exciton dynamics at early times after excitation, with first indications for a Mn-mediated spin-flip process. Our findings demonstrate manganese doping as a powerful approach to control excitonic spin physics in Ruddlesden-Popper perovskites, which will stimulate research on this highly tuneable material platform with promise for tailored interactions between magnetic moments and excitonic states.
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http://dx.doi.org/10.1038/s41467-021-23602-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8190121PMC
June 2021

Charge Carrier Localization in Doped Perovskite Nanocrystals Enhances Radiative Recombination.

J Am Chem Soc 2021 Jun 16;143(23):8647-8653. Epub 2021 May 16.

Cavendish Laboratory, University of Cambridge, Cambridge CB30HE, U.K.

Nanocrystals based on halide perovskites offer a promising material platform for highly efficient lighting. Using transient optical spectroscopy, we study excitation recombination dynamics in manganese-doped CsPb(Cl,Br) perovskite nanocrystals. We find an increase in the intrinsic excitonic radiative recombination rate upon doping, which is typically a challenging material property to tailor. Supported by calculations, we can attribute the enhanced emission rates to increased charge carrier localization through lattice periodicity breaking from Mn dopants, which increases the overlap of electron and hole wave functions locally and thus the oscillator strength of excitons in their vicinity. Our report of a fundamental strategy for improving luminescence efficiencies in perovskite nanocrystals will be valuable for maximizing efficiencies in light-emitting applications.
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http://dx.doi.org/10.1021/jacs.1c01567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8297723PMC
June 2021

Multi-ancestry genome-wide gene-sleep interactions identify novel loci for blood pressure.

Mol Psychiatry 2021 Apr 15. Epub 2021 Apr 15.

Department of Epidemiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

Long and short sleep duration are associated with elevated blood pressure (BP), possibly through effects on molecular pathways that influence neuroendocrine and vascular systems. To gain new insights into the genetic basis of sleep-related BP variation, we performed genome-wide gene by short or long sleep duration interaction analyses on four BP traits (systolic BP, diastolic BP, mean arterial pressure, and pulse pressure) across five ancestry groups in two stages using 2 degree of freedom (df) joint test followed by 1df test of interaction effects. Primary multi-ancestry analysis in 62,969 individuals in stage 1 identified three novel gene by sleep interactions that were replicated in an additional 59,296 individuals in stage 2 (stage 1 + 2 P < 5 × 10), including rs7955964 (FIGNL2/ANKRD33) that increases BP among long sleepers, and rs73493041 (SNORA26/C9orf170) and rs10406644 (KCTD15/LSM14A) that increase BP among short sleepers (P < 5 × 10). Secondary ancestry-specific analysis identified another novel gene by long sleep interaction at rs111887471 (TRPC3/KIAA1109) in individuals of African ancestry (P = 2 × 10). Combined stage 1 and 2 analyses additionally identified significant gene by long sleep interactions at 10 loci including MKLN1 and RGL3/ELAVL3 previously associated with BP, and significant gene by short sleep interactions at 10 loci including C2orf43 previously associated with BP (P < 10). 2df test also identified novel loci for BP after modeling sleep that has known functions in sleep-wake regulation, nervous and cardiometabolic systems. This study indicates that sleep and primary mechanisms regulating BP may interact to elevate BP level, suggesting novel insights into sleep-related BP regulation.
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http://dx.doi.org/10.1038/s41380-021-01087-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8517040PMC
April 2021

SARS-CoV-2 in severe COVID-19 induces a TGF-β-dominated chronic immune response that does not target itself.

Nat Commun 2021 03 30;12(1):1961. Epub 2021 Mar 30.

Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany.

The pathogenesis of severe COVID-19 reflects an inefficient immune reaction to SARS-CoV-2. Here we analyze, at the single cell level, plasmablasts egressed into the blood to study the dynamics of adaptive immune response in COVID-19 patients requiring intensive care. Before seroconversion in response to SARS-CoV-2 spike protein, peripheral plasmablasts display a type 1 interferon-induced gene expression signature; however, following seroconversion, plasmablasts lose this signature, express instead gene signatures induced by IL-21 and TGF-β, and produce mostly IgG1 and IgA1. In the sustained immune reaction from COVID-19 patients, plasmablasts shift to the expression of IgA2, thereby reflecting an instruction by TGF-β. Despite their continued presence in the blood, plasmablasts are not found in the lungs of deceased COVID-19 patients, nor does patient IgA2 binds to the dominant antigens of SARS-CoV-2. Our results thus suggest that, in severe COVID-19, SARS-CoV-2 triggers a chronic immune reaction that is instructed by TGF-β, and is distracted from itself.
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http://dx.doi.org/10.1038/s41467-021-22210-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010106PMC
March 2021

Upregulation of CCR4 in activated CD8 T cells indicates enhanced lung homing in patients with severe acute SARS-CoV-2 infection.

Eur J Immunol 2021 06 19;51(6):1436-1448. Epub 2021 Apr 19.

Department of Internal Medicine 5, Hematology, and Oncology, University Hospital Erlangen and Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany.

COVID-19 is a life-threatening disease leading to bilateral pneumonia and respiratory failure. The underlying reasons why a smaller percentage of patients present with severe pulmonary symptoms whereas the majority is only mildly affected are to date not well understood. Comparing the immunological phenotype in healthy donors and patients with mild versus severe COVID-19 shows that in COVID-19 patients, NK-/B-cell activation and proliferation are enhanced independent of severity. As an important precondition for effective antibody responses, T-follicular helper cells and antibody secreting cells are increased both in patients with mild and severe SARS-CoV-2 infection. Beyond this, T cells in COVID-19 patients exhibit a stronger activation profile with differentiation toward effector cell phenotypes. Importantly, when looking at the rates of pulmonary complications in COVID-19 patients, the chemokine receptor CCR4 is higher expressed by both CD4 and CD8 T cells of patients with severe COVID-19. This raises the hypothesis that CCR4 upregulation on T cells in the pathogenesis of COVID-19 promotes stronger T-cell attraction to the lungs leading to increased immune activation with presumably higher pulmonary toxicity. Our study contributes significantly to the understanding of the immunological changes during COVID-19, as new therapeutic agents, preferentially targeting the immune system, are highly warranted.
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http://dx.doi.org/10.1002/eji.202049135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8250120PMC
June 2021

Visualization of dynamic polaronic strain fields in hybrid lead halide perovskites.

Nat Mater 2021 May 4;20(5):618-623. Epub 2021 Jan 4.

Stanford Institute for Materials and Energy Sciences, SLAC National Accelerator Laboratory, Menlo Park, CA, USA.

Excitation localization involving dynamic nanoscale distortions is a central aspect of photocatalysis, quantum materials and molecular optoelectronics. Experimental characterization of such distortions requires techniques sensitive to the formation of point-defect-like local structural rearrangements in real time. Here, we visualize excitation-induced strain fields in a prototypical member of the lead halide perovskites via femtosecond resolution diffuse X-ray scattering measurements. This enables momentum-resolved phonon spectroscopy of the locally distorted structure and reveals radially expanding nanometre-scale strain fields associated with the formation and relaxation of polarons in photoexcited perovskites. Quantitative estimates of the magnitude and shape of this polaronic distortion are obtained, providing direct insights into the dynamic structural distortions that occur in these materials. Optical pump-probe reflection spectroscopy corroborates these results and shows how these large polaronic distortions transiently modify the carrier effective mass, providing a unified picture of the coupled structural and electronic dynamics that underlie the optoelectronic functionality of the hybrid perovskites.
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http://dx.doi.org/10.1038/s41563-020-00865-5DOI Listing
May 2021

Eltrombopag monotherapy can improve hematopoiesis in patients with low to intermediate risk-1 myelodysplastic syndrome.

Haematologica 2020 12 1;105(12):2785-2794. Epub 2020 Dec 1.

National Heart, Lung, and Blood Institute.

Myelodysplastic syndromes (MDS) are a group of clonal myeloid disorders characterized by cytopenia and a propensity to develop acute myeloid leukemia (AML). The management of lower-risk (LR) MDS with persistent cytopenias remains suboptimal. Eltrombopag (EPAG), a thrombopoietin receptor agonist, can improve platelet counts in LR-MDS and tri-lineage hematopoiesis in aplastic anemia (AA). We conducted a phase 2 dose modification study to investigate the safety and efficacy of EPAG in LR-MDS. EPAG dose was escalated from 50 mg/day, to a maximum of 150 mg/day over a period of 16 weeks. The primary efficacy endpoint was hematologic response at 16-20 weeks. Eleven of 25 (44%) patients responded; five and six patients had uni- or bi-lineage hematologic responses, respectively. The predictors of response were presence of a PNH clone, marrow hypocellularity, thrombocytopenia with or without other cytopenia, and elevated plasma thrombopoietin levels at study entry. The safety profile was consistent with previous EPAG studies in AA; no patients discontinued drug due to adverse events. Three patients developed reversible grade-3 liver toxicity and one patient had increased reticulin fibrosis. Ten patients discontinued EPAG after achieving a robust response (median time 16 months); four of them reinitiated EPAG due to declining counts, and all attained a second robust response. Six patients had disease progression not associated with expansion of mutated clones and no patient progressed to AML on study. In conclusion, EPAG was well-tolerated and effective in restoring hematopoiesis in patients with low to intermediate-1 risk MDS. This study was registered at clinicaltrials.gov as #NCT00932156.
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http://dx.doi.org/10.3324/haematol.2020.249995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716353PMC
December 2020

Analysis of cell-free DNA in a consecutive series of 13,607 routine cases for the detection of fetal chromosomal aneuploidies in a single center in Germany.

Arch Gynecol Obstet 2021 06 5;303(6):1407-1414. Epub 2020 Nov 5.

Amedes Institut für Labormedizin und Klinische Genetik Rhein/Ruhr, Willy Brandt Platz 4, 45127, Essen, Germany.

Purpose: Noninvasive prenatal testing (NIPT) is a highly sensitive and specific method for detection of fetal chromosomal aneuploidies from maternal plasma. The objective of this study was to determine the performance of a new paired-end sequencing-based NIPT assay in 13,607 pregnancies from a single center in Germany.

Methods: Samples from 13,607 pregnant women who previously underwent NIPT were analyzed using VeriSeq NIPT Solution v2 assay for presence of common fetal trisomies and monosomy X. Follow-up to determine clinical truth was carried out.

Results: Of the 13,607 cases, 13,509 received a NIPT call resulting in a low study failure rate of 0.72%. There were 188 (1.4%) high-risk calls: 117 trisomy 21, 34 trisomy 18, 23 trisomy 13, one trisomy 21 + 13, and 13 monosomy X. High sensitivities and specificities of ≥ 98.89% were reported for all four aneuploidy conditions. Of the high-risk cases, clinical follow-up data were available for 77.1% (145/188). Clinical follow-up of high-risk calls revealed an overall positive predictive value of 84.8% (potential range 65.4-88.3%). NIPT results were provided for samples across a range of fetal fractions, down to 2% fetal fraction.

Conclusion: The VeriSeq NIPT Solution v2 assay detected fetal chromosomal aneuploidies across a range of fetal fractions with high sensitivities and specificities observed based on known clinical outcomes, a high overall PPV, and a low failure rate.
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http://dx.doi.org/10.1007/s00404-020-05856-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087552PMC
June 2021

Meta-analysis uncovers genome-wide significant variants for rapid kidney function decline.

Kidney Int 2021 04 31;99(4):926-939. Epub 2020 Oct 31.

Division of Nephrology, University of Washington, Seattle, Washington, USA; Kidney Research Institute, University of Washington, Seattle, Washington, USA.

Rapid decline of glomerular filtration rate estimated from creatinine (eGFRcrea) is associated with severe clinical endpoints. In contrast to cross-sectionally assessed eGFRcrea, the genetic basis for rapid eGFRcrea decline is largely unknown. To help define this, we meta-analyzed 42 genome-wide association studies from the Chronic Kidney Diseases Genetics Consortium and United Kingdom Biobank to identify genetic loci for rapid eGFRcrea decline. Two definitions of eGFRcrea decline were used: 3 mL/min/1.73m/year or more ("Rapid3"; encompassing 34,874 cases, 107,090 controls) and eGFRcrea decline 25% or more and eGFRcrea under 60 mL/min/1.73m at follow-up among those with eGFRcrea 60 mL/min/1.73m or more at baseline ("CKDi25"; encompassing 19,901 cases, 175,244 controls). Seven independent variants were identified across six loci for Rapid3 and/or CKDi25: consisting of five variants at four loci with genome-wide significance (near UMOD-PDILT (2), PRKAG2, WDR72, OR2S2) and two variants among 265 known eGFRcrea variants (near GATM, LARP4B). All these loci were novel for Rapid3 and/or CKDi25 and our bioinformatic follow-up prioritized variants and genes underneath these loci. The OR2S2 locus is novel for any eGFRcrea trait including interesting candidates. For the five genome-wide significant lead variants, we found supporting effects for annual change in blood urea nitrogen or cystatin-based eGFR, but not for GATM or LARP4B. Individuals at high compared to those at low genetic risk (8-14 vs. 0-5 adverse alleles) had a 1.20-fold increased risk of acute kidney injury (95% confidence interval 1.08-1.33). Thus, our identified loci for rapid kidney function decline may help prioritize therapeutic targets and identify mechanisms and individuals at risk for sustained deterioration of kidney function.
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http://dx.doi.org/10.1016/j.kint.2020.09.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010357PMC
April 2021

Mutant Isocitrate Dehydrogenase 1 Inhibitor Ivosidenib in Combination With Azacitidine for Newly Diagnosed Acute Myeloid Leukemia.

J Clin Oncol 2021 01 29;39(1):57-65. Epub 2020 Oct 29.

University of Oxford, Oxford, United Kingdom.

Purpose: Ivosidenib is an oral inhibitor of the mutant isocitrate dehydrogenase 1 (IDH1) enzyme, approved for treatment of -mutant (m) acute myeloid leukemia (AML). Preclinical work suggested that addition of azacitidine to ivosidenib enhances mIDH1 inhibition-related differentiation and apoptosis.

Patients And Methods: This was an open-label, multicenter, phase Ib trial comprising dose-finding and expansion stages to evaluate safety and efficacy of combining oral ivosidenib 500 mg once daily continuously with subcutaneous azacitidine 75 mg/m on days 1-7 in 28-day cycles in patients with newly diagnosed m AML ineligible for intensive induction chemotherapy (ClinicalTrials.gov identifier: NCT02677922).

Results: Twenty-three patients received ivosidenib plus azacitidine (median age, 76 years; range, 61-88 years). Treatment-related grade ≥ 3 adverse events occurring in > 10% of patients were neutropenia (22%), anemia (13%), thrombocytopenia (13%), and electrocardiogram QT prolongation (13%). Adverse events of special interest included all-grade IDH differentiation syndrome (17%), all-grade electrocardiogram QT prolongation (26%), and grade ≥ 3 leukocytosis (9%). Median treatment duration was 15.1 months (range, 0.3-32.2 months); 10 patients remained on treatment as of February 19, 2019. The overall response rate was 78.3% (18/23 patients; 95% CI, 56.3% to 92.5%), and the complete remission rate was 60.9% (14/23 patients; 95% CI, 38.5% to 80.3%). With median follow-up of 16 months, median duration of response in responders had not been reached. The 12-month survival estimate was 82.0% (95% CI, 58.8% to 92.8%). m clearance in bone marrow mononuclear cells by BEAMing (beads, emulsion, amplification, magnetics) digital polymerase chain reaction was seen in 10/14 patients (71.4%) achieving complete remission.

Conclusion: Ivosidenib plus azacitidine was well tolerated, with an expected safety profile consistent with monotherapy with each agent. Responses were deep and durable, with most complete responders achieving m mutation clearance.
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http://dx.doi.org/10.1200/JCO.20.01632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7771719PMC
January 2021

Rapid response flow cytometric assay for the detection of antibody responses to SARS-CoV-2.

Eur J Clin Microbiol Infect Dis 2021 Apr 20;40(4):751-759. Epub 2020 Oct 20.

Institute of Clinical and Molecular Virology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany.

SARS-CoV-2 has emerged as a previously unknown zoonotic coronavirus that spread worldwide causing a serious pandemic. While reliable nucleic acid-based diagnostic assays were rapidly available, only a limited number of validated serological assays were available in the early phase of the pandemic. Here, we evaluated a novel flow cytometric approach to assess spike-specific antibody responses.HEK 293T cells expressing SARS-CoV-2 spike protein in its natural confirmation on the surface were used to detect specific IgG and IgM antibody responses in patient sera by flow cytometry. A soluble angiotensin-converting-enzyme 2 (ACE-2) variant was developed as external standard to quantify spike-specific antibody responses on different assay platforms. Analyses of 201 pre-COVID-19 sera proved a high assay specificity in comparison to commercially available CLIA and ELISA systems, while also revealing the highest sensitivity in specimens from PCR-confirmed SARS-CoV-2-infected patients. The external standard allowed robust quantification of antibody responses among different assay platforms. In conclusion, our newly established flow cytometric assay allows sensitive and quantitative detection of SARS-CoV-2-specific antibodies, which can be easily adopted in different laboratories and does not rely on external supply of assay kits. The flow cytometric assay also provides a blueprint for rapid development of serological tests to other emerging viral infections.
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http://dx.doi.org/10.1007/s10096-020-04072-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572153PMC
April 2021

Siglec-15 on Osteoclasts Is Crucial for Bone Erosion in Serum-Transfer Arthritis.

J Immunol 2020 11 5;205(10):2595-2605. Epub 2020 Oct 5.

Division of Genetics, Department of Biology, University of Erlangen, 91058 Erlangen, Germany;

Siglec-15 is a conserved sialic acid-binding Ig-like lectin, which is expressed on osteoclasts. Deficiency of Siglec-15 leads to an impaired osteoclast development, resulting in a mild osteopetrotic phenotype. The role of Siglec-15 in arthritis is still largely unclear. To address this, we generated Siglec-15 knockout mice and analyzed them in a mouse arthritis model. We could show that Siglec-15 is directly involved in pathologic bone erosion in the K/BxN serum-transfer arthritis model. Histological analyses of joint destruction provided evidence for a significant reduction in bone erosion area and osteoclast numbers in Siglec-15 mice, whereas the inflammation area and cartilage destruction was comparable to wild-type mice. Thus, Siglec-15 on osteoclasts has a crucial function for bone erosion during arthritis. In addition, we generated a new monoclonal anti-Siglec-15 Ab to clarify its expression pattern on immune cells. Whereas this Ab demonstrated an almost exclusive Siglec-15 expression on murine osteoclasts and hardly any other expression on various other immune cell types, human Siglec-15 was more broadly expressed on human myeloid cells, including human osteoclasts. Taken together, our findings show a role of Siglec-15 as a regulator of pathologic bone resorption in arthritis and highlight its potential as a target for future therapies, as Siglec-15 blocking Abs are available.
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http://dx.doi.org/10.4049/jimmunol.2000472DOI Listing
November 2020

TFG is required for autophagy flux and to prevent endoplasmic reticulum stress in CH12 B lymphoma cells.

Autophagy 2021 Sep 22;17(9):2238-2256. Epub 2020 Sep 22.

Division of Molecular Immunology, Department of Internal Medicine 3, Nikolaus-Fiebiger-Zentrum, Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg, Erlangen, Germany.

Plasma cells depend on quality control of newly synthesized antibodies in the endoplasmic reticulum (ER) via macroautophagy/autophagy and proteasomal degradation. The cytosolic adaptor protein TFG (Trk-fused gene) regulates ER-Golgi transport, the secretory pathway and proteasome activity in non-immune cells. We show here that TFG is upregulated during lipopolysaccharide- and CpG-induced differentiation of B1 and B2 B cells into plasmablasts, with the highest expression of TFG in mature plasma cells. CRISPR-CAS9-mediated gene disruption of in the B lymphoma cell line CH12 revealed increased apoptosis, which was reverted by BCL2 but even more by ectopic TFG expression. Loss of TFG disrupted ER structure, leading to an expanded ER and increased expression of ER stress genes. When compared to wild-type CH12 cells, KO CH12 cells were more sensitive toward ER stress induced by tunicamycin, monensin and proteasome inhibition or by expression of an ER-bound immunoglobulin (Ig) μ heavy (µH) chain. CH12 KO B cells displayed more total LC3, lower LC3-II turnover and increased numbers and size of autophagosomes. Tandem-fluorescent-LC3 revealed less accumulation of GFP-LC3 in starved and chloroquine-treated CH12 KO B cells. The GFP:RFP ratio of tandem-fluorescent-LC3 was higher in tunicamycin-treated CH12 KO B cells, suggesting less autophagy flux during induced ER stress. Based on these data, we suggest that TFG controls autophagy flux in CH12 B cells and propose that TFG is a survival factor that alleviates ER stress through the support of autophagy flux in activated B cells and mature plasma cells.: Ab, antibody; Ag, antigen; ASC, antibody-secreting cells; ATG, autophagy-related; BCR, B cell receptor; COPII, coat protein complex II; CpG, non-methylated CpG oligonucleotide; ER, endoplasmic reticulum; ERAD, ER-associated degradation; FO, follicular; GFP, green fluorescent protein; HC, heavy chain; Ig, immunoglobulin; IRES, internal ribosomal entry site; LC, light chain; MZ, marginal zone; NFKB, nuclear factor of kappa light polypeptide gene enhancer in B cells; TLR, toll-like receptor; UPR, unfolded protein response.
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http://dx.doi.org/10.1080/15548627.2020.1821546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8496724PMC
September 2021

Genome-wide association meta-analysis for early age-related macular degeneration highlights novel loci and insights for advanced disease.

BMC Med Genomics 2020 08 26;13(1):120. Epub 2020 Aug 26.

Institute of Human Genetics, University of Regensburg, Regensburg, Germany.

Background: Advanced age-related macular degeneration (AMD) is a leading cause of blindness. While around half of the genetic contribution to advanced AMD has been uncovered, little is known about the genetic architecture of early AMD.

Methods: To identify genetic factors for early AMD, we conducted a genome-wide association study (GWAS) meta-analysis (14,034 cases, 91,214 controls, 11 sources of data including the International AMD Genomics Consortium, IAMDGC, and UK Biobank, UKBB). We ascertained early AMD via color fundus photographs by manual grading for 10 sources and via an automated machine learning approach for > 170,000 photographs from UKBB. We searched for early AMD loci via GWAS and via a candidate approach based on 14 previously suggested early AMD variants.

Results: Altogether, we identified 10 independent loci with statistical significance for early AMD: (i) 8 from our GWAS with genome-wide significance (P < 5 × 10), (ii) one previously suggested locus with experiment-wise significance (P < 0.05/14) in our non-overlapping data and with genome-wide significance when combining the reported and our non-overlapping data (together 17,539 cases, 105,395 controls), and (iii) one further previously suggested locus with experiment-wise significance in our non-overlapping data. Of these 10 identified loci, 8 were novel and 2 known for early AMD. Most of the 10 loci overlapped with known advanced AMD loci (near ARMS2/HTRA1, CFH, C2, C3, CETP, TNFRSF10A, VEGFA, APOE), except two that have not yet been identified with statistical significance for any AMD. Among the 17 genes within these two loci, in-silico functional annotation suggested CD46 and TYR as the most likely responsible genes. Presence or absence of an early AMD effect distinguished the known pathways of advanced AMD genetics (complement/lipid pathways versus extracellular matrix metabolism).

Conclusions: Our GWAS on early AMD identified novel loci, highlighted shared and distinct genetics between early and advanced AMD and provides insights into AMD etiology. Our data provide a resource comparable in size to the existing IAMDGC data on advanced AMD genetics enabling a joint view. The biological relevance of this joint view is underscored by the ability of early AMD effects to differentiate the major pathways for advanced AMD.
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http://dx.doi.org/10.1186/s12920-020-00760-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449002PMC
August 2020

Chances and challenges of machine learning-based disease classification in genetic association studies illustrated on age-related macular degeneration.

Genet Epidemiol 2020 10 2;44(7):759-777. Epub 2020 Aug 2.

Department of Genetic Epidemiology, University of Regensburg, Regensburg, Germany.

Imaging technology and machine learning algorithms for disease classification set the stage for high-throughput phenotyping and promising new avenues for genome-wide association studies (GWAS). Despite emerging algorithms, there has been no successful application in GWAS so far. We establish machine learning-based phenotyping in genetic association analysis as misclassification problem. To evaluate chances and challenges, we performed a GWAS based on automatically classified age-related macular degeneration (AMD) in UK Biobank (images from 135,500 eyes; 68,400 persons). We quantified misclassification of automatically derived AMD in internal validation data (4,001 eyes; 2,013 persons) and developed a maximum likelihood approach (MLA) to account for it when estimating genetic association. We demonstrate that our MLA guards against bias and artifacts in simulation studies. By combining a GWAS on automatically derived AMD and our MLA in UK Biobank data, we were able to dissect true association (ARMS2/HTRA1, CFH) from artifacts (near HERC2) and identified eye color as associated with the misclassification. On this example, we provide a proof-of-concept that a GWAS using machine learning-derived disease classification yields relevant results and that misclassification needs to be considered in analysis. These findings generalize to other phenotypes and emphasize the utility of genetic data for understanding misclassification structure of machine learning algorithms.
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http://dx.doi.org/10.1002/gepi.22336DOI Listing
October 2020

Paediatric Strategy Forum for medicinal product development for acute myeloid leukaemia in children and adolescents: ACCELERATE in collaboration with the European Medicines Agency with participation of the Food and Drug Administration.

Eur J Cancer 2020 09 17;136:116-129. Epub 2020 Jul 17.

FORMA Therapeutics, USA.

Purpose: The current standard-of-care for front-line therapy for acute myeloid leukaemia (AML) results in short-term and long-term toxicity, but still approximately 40% of children relapse. Therefore, there is a major need to accelerate the evaluation of innovative medicines, yet drug development continues to be adult-focused. Furthermore, the large number of competing agents in rare patient populations requires coordinated prioritisation, within the global regulatory framework and cooperative group initiatives.

Methods: The fourth multi-stakeholder Paediatric Strategy Forum focused on AML in children and adolescents.

Results: CD123 is a high priority target and the paediatric development should be accelerated as a proof-of-concept. Efforts must be coordinated, however, as there are a limited number of studies that can be delivered. Studies of FLT3 inhibitors in agreed paediatric investigation plans present challenges to be completed because they require enrolment of a larger number of patients than actually exist. A consensus was developed by industry and academia of optimised clinical trials. For AML with rare mutations that are more frequent in adolescents than in children, adult trials should enrol adolescents and when scientifically justified, efficacy data could be extrapolated. Methodologies and definitions of minimal residual disease need to be standardised internationally and validated as a new response criterion. Industry supported, academic sponsored platform trials could identify products to be further developed. The Leukaemia and Lymphoma Society PedAL/EUpAL initiative has the potential to be a major advance in the field.

Conclusion: These initiatives continue to accelerate drug development for children with AML and ultimately improve clinical outcomes.
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http://dx.doi.org/10.1016/j.ejca.2020.04.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789799PMC
September 2020

Low-Cost PVD Shadow Masks with Submillimeter Resolution from Laser-Cut Paper.

Micromachines (Basel) 2020 Jul 11;11(7). Epub 2020 Jul 11.

Division of Micro- and Nanosystems, KTH Royal Institute of Technology, 11428 Stockholm, Sweden.

We characterize an affordable method of producing stencils for submillimeter physical vapor deposition (PVD) by using paper and a benchtop laser cutter. Patterning electrodes or similar features on top of organic or biological substrates is generally not possible using standard photolithography. Shadow masks, traditionally made of silicon-based membranes, circumvent the need for aggressive solvents but suffer from high costs. Here, we evaluate shadow masks fabricated by CO laser processing from quantitative filter papers. Such papers are stiff and dimensionally stable, resilient in handling, and cut without melting or redeposition. Using two exemplary interdigitated electrode designs, we quantify the line resolution achievable with both high-quality and standard lenses, as well as the positional accuracy across multiple length scales. Additionally, we assess the gap between such laser-cut paper masks and a substrate, and quantify feature reproduction onto polycarbonate membranes. We find that ~100 µm line widths are achievable independent of lens type and that average positional accuracy is better than ±100 µm at 4"-wafer scale. Although this falls well short of the micron-size features achievable with typical shadow masks, resolution in the tenths to tens of millimeters is entirely sufficient for applications from contact pads to electrochemical cells, allowing new functionalities on fragile materials.
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http://dx.doi.org/10.3390/mi11070676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407583PMC
July 2020

Cell Fusion Induced by a Fusion-Active Form of Human Cytomegalovirus Glycoprotein B (gB) Is Inhibited by Antibodies Directed at Antigenic Domain 5 in the Ectodomain of gB.

J Virol 2020 08 31;94(18). Epub 2020 Aug 31.

Virologisches Institut, Klinische und Molekulare Virologie, Friedrich Alexander Universität Erlangen-Nürnberg, Erlangen, Germany

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause severe clinical disease in allograft recipients and infants infected Virus-neutralizing antibodies defined have been proposed to confer protection against HCMV infection, and the virion envelope glycoprotein B (gB) serves as a major target of neutralizing antibodies. The viral fusion protein gB is nonfusogenic on its own and requires glycoproteins H (gH) and L (gL) for membrane fusion, which is in contrast to requirements of related class III fusion proteins, including vesicular stomatitis virus glycoprotein G (VSV-G) or baculovirus gp64. To explore requirements for gB's fusion activity, we generated a set of chimeras composed of gB and VSV-G or gp64, respectively. These gB chimeras were intrinsically fusion active and led to the formation of multinucleated cell syncytia when expressed in the absence of other viral proteins. Utilizing a panel of virus-neutralizing gB-specific monoclonal antibodies (MAbs), we could demonstrate that syncytium formation of the fusogenic gB/VSV-G chimera can be significantly inhibited by only a subset of neutralizing MAbs which target antigenic domain 5 (AD-5) of gB. This observation argues for differential modes of action of neutralizing anti-gB MAbs and suggests that blocking the membrane fusion function of gB could be one mechanism of antibody-mediated virus neutralization. In addition, our data have important implications for the further understanding of the conformation of gB that promotes membrane fusion as well as the identification of structures in AD-5 that could be targeted by antibodies to block this early step in HCMV infection. HCMV is a major global health concern, and antiviral chemotherapy remains problematic due to toxicity of available compounds and the emergence of drug-resistant viruses. Thus, an HCMV vaccine represents a priority for both governmental and pharmaceutical research programs. A major obstacle for the development of a vaccine is a lack of knowledge of the nature and specificities of protective immune responses that should be induced by such a vaccine. Glycoprotein B of HCMV is an important target for neutralizing antibodies and, hence, is often included as a component of intervention strategies. By generation of fusion-active gB chimeras, we were able to identify target structures of neutralizing antibodies that potently block gB-induced membrane fusion. This experimental system provides an approach to screen for antibodies that interfere with gB's fusogenic activity. In summary, our data will likely contribute to both rational vaccine design and the development of antibody-based therapies against HCMV.
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http://dx.doi.org/10.1128/JVI.01276-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459561PMC
August 2020
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