Search

Neurology is focused on a model where patients receive their care through repeated visits to clinics and doctor’s offices. Diagnostic tests often require expensive and specialized equipment that are only available in clinics. However, this current model has significant drawbacks. First, diagnostic tests, such as daytime EEG and sleep studies, occur under artificial conditions in the clinic, which may mask or wrongly emphasize clinically important features. Second, early detection and high-quality management of chronic neurological disorders require repeat measurements to accurately capture the dynamics of the disease process, which is impractical to execute in the clinic for economical and logistical reasons. Third, clinic visits remain inaccessible to many patients due to geographical and economical circumstances. Fourth, global disruptions to daily life, such as the one caused by COVID-19, can seriously harm patients if access to in-person clinical visits for diagnostic and treatment purposes is throttled. Thus, translating diagnostic and treatment procedures to patients’ homes will convey multiple substantial benefits and has the potential to substantially improve clinical outcomes while reducing cost. NeuroTec was founded to accelerate the re-imagining of neurology and to promote the convergence of technological, scientific, medical and societal processes. The goal is to identify and validate new digital biomarkers that can close the last mile in neurology by enabling the translation of personalized diagnostics and therapeutic interventions from the clinic to the patient’s home.

Abstract The yellow fever 17D vaccine (YF17D) is highly effective but is frequently administered to individuals with pre-existing cross-reactive immunity, potentially impacting their immune responses. Here, we investigate the impact of pre-existing flavivirus immunity induced by the tick-borne encephalitis virus (TBEV) vaccine on the response to YF17D vaccination in 250 individuals up to 28 days post-vaccination (pv) and 22 individuals sampled one-year pv. Our findings indicate that previous TBEV vaccination does not affect the early IgM-driven neutralizing response to YF17D. However, pre-vaccination sera enhance YF17D virus infection in vitro via antibody-dependent enhancement (ADE). Following YF17D vaccination, TBEV-pre-vaccinated individuals develop high amounts of cross-reactive IgG antibodies with poor neutralizing capacity. In contrast, TBEV-unvaccinated individuals elicit a non-cross-reacting neutralizing response. Using YF17D envelope protein mutants displaying different epitopes, we identify quaternary dimeric epitopes as the primary target of neutralizing antibodies. Additionally, TBEV-pre-vaccination skews the IgG response towards the pan-flavivirus fusion loop epitope (FLE), capable of mediating ADE of dengue and Zika virus infections in vitro. Together, we propose that YF17D vaccination conceals the FLE in individuals without prior flavivirus exposure but favors a cross-reactive IgG response in TBEV-pre-vaccinated recipients directed to the FLE with potential to enhance dengue virus infection.

IntroductionPatients with primary adrenal insufficiency (PAI) suffer from increased risk of infection, adrenal crises and have a higher mortality rate. Such dismal outcomes have been inferred to immune cell dysregulation because of unphysiological cortisol replacement. As the immune landscape of patients with different types of PAI has not been systematically explored, we set out to immunophenotype PAI patients with different causes of glucocorticoid (GC) deficiency.MethodsThis cross-sectional single center study includes 28 patients with congenital adrenal hyperplasia (CAH), 27 after bilateral adrenalectomy due to Cushing’s syndrome (BADx), 21 with Addison’s disease (AD) and 52 healthy controls. All patients with PAI were on a stable GC replacement regimen with a median dose of 25 mg hydrocortisone per day. Peripheral blood mononuclear cells were isolated from heparinized blood samples. Immune cell subsets were analyzed using multicolor flow cytometry after four-hour stimulation with phorbol myristate acetate and ionomycin. Natural killer (NK-) cell cytotoxicity and clock gene expression were investigated.ResultsThe percentage of T helper cell subsets was downregulated in AD patients (Th1 p = 0.0024, Th2 p = 0.0157, Th17 p < 0.0001) compared to controls. Cytotoxic T cell subsets were reduced in AD (Tc1 p = 0.0075, Tc2 p = 0.0154) and CAH patients (Tc1 p = 0.0055, Tc2 p = 0.0012) compared to controls. NKCC was reduced in all subsets of PAI patients, with smallest changes in CAH. Degranulation marker CD107a expression was upregulated in BADx and AD, not in CAH patients compared to controls (BADx p < 0.0001; AD p = 0.0002). In contrast to NK cell activating receptors, NK cell inhibiting receptor CD94 was upregulated in BADx and AD, but not in CAH patients (p < 0.0001). Although modulation in clock gene expression could be confirmed in our patient subgroups, major interindividual-intergroup dissimilarities were not detected.DiscussionIn patients with different etiologies of PAI, distinct differences in T and NK cell-phenotypes became apparent despite the use of same GC preparation and dose. Our results highlight unsuspected differences in immune cell composition and function in PAI patients of different causes and suggest disease-specific alterations that might necessitate disease-specific treatment.

Background Melanoma is an immune sensitive disease, as demonstrated by the activity of immune check point blockade (ICB), but many patients will either not respond or relapse. More recently, tumor infiltrating lymphocyte (TIL) therapy has shown promising efficacy in melanoma treatment after ICB failure, indicating the potential of cellular therapies. However, TIL treatment comes with manufacturing limitations, product heterogeneity, as well as toxicity problems, due to the transfer of a large number of phenotypically diverse T cells. To overcome said limitations, we propose a controlled adoptive cell therapy approach, where T cells are armed with synthetic agonistic receptors (SAR) that are selectively activated by bispecific antibodies (BiAb) targeting SAR and melanoma-associated antigens.Methods Human as well as murine SAR constructs were generated and transduced into primary T cells. The approach was validated in murine, human and patient-derived cancer models expressing the melanoma-associated target antigens tyrosinase-related protein 1 (TYRP1) and melanoma-associated chondroitin sulfate proteoglycan (MCSP) (CSPG4). SAR T cells were functionally characterized by assessing their specific stimulation and proliferation, as well as their tumor-directed cytotoxicity, in vitro and in vivo.Results MCSP and TYRP1 expression was conserved in samples of patients with treated as well as untreated melanoma, supporting their use as melanoma-target antigens. The presence of target cells and anti-TYRP1 × anti-SAR or anti-MCSP × anti-SAR BiAb induced conditional antigen-dependent activation, proliferation of SAR T cells and targeted tumor cell lysis in all tested models. In vivo, antitumoral activity and long-term survival was mediated by the co-administration of SAR T cells and BiAb in a syngeneic tumor model and was further validated in several xenograft models, including a patient-derived xenograft model.Conclusion The SAR T cell-BiAb approach delivers specific and conditional T cell activation as well as targeted tumor cell lysis in melanoma models. Modularity is a key feature for targeting melanoma and is fundamental towards personalized immunotherapies encompassing cancer heterogeneity. Because antigen expression may vary in primary melanoma tissues, we propose that a dual approach targeting two tumor-associated antigens, either simultaneously or sequentially, could avoid issues of antigen heterogeneity and deliver therapeutic benefit to patients.

ABSTRACT Quick and accurate detection of neutralizing antibodies (nAbs) against yellow fever is essential in serodiagnosis during outbreaks for surveillance and to evaluate vaccine efficacy in population-wide studies. All of this requires serological assays that can process a large number of samples in a highly standardized format. Albeit being laborious, time-consuming, and limited in throughput, the classical plaque reduction neutralization test (PRNT) is still considered the gold standard for the detection and quantification of nAbs due to its sensitivity and specificity. Here, we report the development of an alternative fluorescence-based serological assay (SNTFLUO) with an equally high sensitivity and specificity that is fit for high-throughput testing with the potential for automation. Finally, our novel SNTFLUO was cross-validated in several reference laboratories and against international WHO standards, showing its potential to be implemented in clinical use. SNTFLUO assays with similar performance are available for the Japanese encephalitis, Zika, and dengue viruses amenable to differential diagnostics. IMPORTANCE Fast and accurate detection of neutralizing antibodies (nAbs) against yellow fever virus (YFV) is key in yellow fever serodiagnosis, outbreak surveillance, and monitoring of vaccine efficacy. Although classical PRNT remains the gold standard for measuring YFV nAbs, this methodology suffers from inherent limitations such as low throughput and overall high labor intensity. We present a novel fluorescence-based serum neutralization test (SNTFLUO) with equally high sensitivity and specificity that is fit for processing a large number of samples in a highly standardized manner and has the potential to be implemented for clinical use. In addition, we present SNTFLUO assays with similar performance for Japanese encephalitis, Zika, and dengue viruses, opening new avenues for differential diagnostics.

There is an urgent need for better diagnostic and analytical methods for vaccine research and infection control in virology. This has been highlighted by recently emerging viral epidemics and pandemics (Zika, SARS-CoV-2), and recurring viral outbreaks like the yellow fever outbreaks in Angola and the Democratic Republic of Congo (2016) and in Brazil (2016-2018). Current assays to determine neutralising activity against viral infections in sera are costly in time and equipment and suffer from high variability. Therefore, both basic infection research and diagnostic population screenings would benefit from improved methods to determine virus-neutralising activity in patient samples. Here we describe a robust, objective, and scalable Fluorescence Reduction Neutralisation Test (FluoRNT) for yellow fever virus, relying on flow cytometric detection of cells infected with a fluorescent Venus reporter containing variant of the yellow fever vaccine strain 17D (YF-17D-Venus). It accurately measures neutralising antibody titres in human serum samples within as little as 24 h. Samples from 32 vaccinees immunised with YF-17D were tested for neutralising activity by both a conventional focus reduction neutralisation test (FRNT) and FluoRNT. Both types of tests proved to be equally reliable for the detection of neutralising activity, however, FluoRNT is significantly more precise and reproducible with a greater dynamic range than conventional FRNT. The FluoRNT assay protocol is substantially faster, easier to control, and cheaper in per-assay costs. FluoRNT additionally reduces handling time minimising exposure of personnel to patient samples. FluoRNT thus brings a range of desirable features that can accelerate and standardise the measurement of neutralising anti-yellow fever virus antibodies. It could be used in applications ranging from vaccine testing to large cohort studies in systems virology and vaccinology. We also anticipate the potential to translate the methodology and analysis of FluoRNT to other flaviviruses such as West Nile, Dengue and Zika or to RNA viruses more generally.

Disease manifestations in COVID-19 range from mild to severe illness associated with a dysregulated innate immune response. Alterations in function and regeneration of dendritic cells (DCs) and monocytes may contribute to immunopathology and influence adaptive immune responses in COVID-19 patients. We analyzed circulating DC and monocyte subsets in 65 hospitalized COVID-19 patients with mild/moderate or severe disease from acute illness to recovery and in healthy controls. Persisting reduction of all DC subpopulations was accompanied by an expansion of proliferating Lineage-HLADR+ cells lacking DC markers. Increased frequency of CD163+ CD14+ cells within the recently discovered DC3 subpopulation in patients with more severe disease was associated with systemic inflammation, activated T follicular helper cells, and antibody-secreting cells. Persistent downregulation of CD86 and upregulation of programmed death-ligand 1 (PD-L1) in conventional DCs (cDC2 and DC3) and classical monocytes associated with a reduced capacity to stimulate naïve CD4+ T cells correlated with disease severity. Long-lasting depletion and functional impairment of DCs and monocytes may have consequences for susceptibility to secondary infections and therapy of COVID-19 patients.

Replication competent vesicular stomatitis virus (VSV) is the basis of a vaccine against Ebola and VSV strains are developed as oncolytic viruses. Both functions depend on the ability of VSV to induce adequate amounts of interferon-α/β. It is therefore important to understand how VSV triggers interferon responses. VSV activates innate immunity via retinoic acid-inducible gene I (RIG-I), a sensor for viral RNA. Our results show that VSV needs to replicate for a robust interferon response. Analysis of RIG-I-associated RNA identified a copy-back defective-interfering (DI) genome and full-length viral genomes as main trigger of RIG-I. VSV stocks depleted of DI genomes lost most of their interferon-stimulating activity. The remaining full-length genome and leader-N-read-through sequences, however, still triggered RIG-I. Awareness for DI genomes as trigger of innate immune responses will help to standardize DI genome content and to purposefully deplete or use DI genomes as natural adjuvants in VSV-based therapeutics.

Flavivirus outbreaks require fast and reliable diagnostics that can be easily adapted to newly emerging and re-emerging flaviviruses. Due to the serological cross-reactivity among flavivirus antibodies, neutralization tests (NT) are considered the gold standard for sero-diagnostics. Here, we first established wild-type single-round infectious virus replicon particles (VRPs) by packaging a yellow fever virus (YFV) replicon expressing Gaussia luciferase (Gluc) with YFV structural proteins in trans using a double subgenomic Sindbis virus (SINV) replicon. The latter expressed the YFV envelope proteins prME via the first SINV subgenomic promoter and the capsid protein via a second subgenomic SINV promoter. VRPs were produced upon co-electroporation of replicon and packaging RNA. Introduction of single restriction enzyme sites in the packaging construct flanking the prME sequence easily allowed to exchange the prME moiety resulting in chimeric VRPs that have the surface proteins of other flaviviruses including dengue virus 1–-4, Zika virus, West Nile virus, and tick-borne encephalitis virus. Besides comparing the YF-VRP based NT assay to a YF reporter virus NT assay, we analyzed the neutralization efficiencies of different human anti-flavivirus sera or a monoclonal antibody against all established VRPs. The assays were performed in a 96-well high-throughput format setting with Gluc as readout in comparison to classical plaque reduction NTs indicating that the VRP-based NT assays are suitable for high-throughput analyses of neutralizing flavivirus antibodies.

Abstract Objectives T follicular helper (Tfh) cells are the principal T helper cell subset that provides help to B cells for potent antibody responses against various pathogens. In this study, we took advantage of the live‐attenuated yellow fever virus (YFV) vaccine strain, YF‐17D, as a model system for studying human antiviral immune responses in vivo following exposure to an acute primary virus challenge under safe and highly controlled conditions, to comprehensively analyse the dynamics of circulating Tfh (cTfh) cells. Methods We tracked and analysed the response of cTfh and other T and B cell subsets in peripheral blood of healthy volunteers by flow cytometry over the course of 4 weeks after YF‐17D vaccination. Results Using surface staining of cell activation markers to track YFV‐specific T cells, we found increasing cTfh cell frequencies starting at day 3 and peaking around 2 weeks after YF‐17D vaccination. This kinetic was confirmed in a subgroup of donors using MHC multimer staining for four known MHC class II epitopes of YF‐17D. The subset composition of cTfh cells changed dynamically during the course of the immune response and was dominated by the cTfh1‐polarised subpopulation. Importantly, frequencies of cTfh1 cells correlated with the strength of the neutralising antibody response, whereas frequencies of cTfh17 cells were inversely correlated. Conclusion In summary, we describe detailed cTfh kinetics during YF‐17D vaccination. Our results suggest that cTfh expansion and polarisation can serve as a prognostic marker for vaccine success. These insights may be leveraged in the future to improve current vaccine design and strategies.

Background. Decreased organ function following liver resection is a major clinical issue. The practical method of ischemic postconditioning (IPostC) has been studied in heart diseases, but no data exist regarding fibrotic livers. Aims. We aimed to determine whether IPostC could protect healthy, fibrotic, and cirrhotic livers from ischemia reperfusion injury (IRI). Methods. Fibrosis was induced in male SD rats using bile duct ligation (BDL, 4 weeks), and cirrhosis was induced using thioacetamide (TAA, 18 weeks). Fibrosis and cirrhosis were histologically confirmed using HE and EvG staining. For healthy, fibrotic, and cirrhotic livers, isolated liver perfusion with 90 min of warm ischemia was performed in three groups (each with n=8): control, IPostC 8x20 sec, and IPostC 4x60 sec. additionally, healthy livers were investigated during a follow-up study. Lactate dehydrogenase (LDH) and thromboxane B2 (TXB2) in the perfusate, as well as bile flow (healthy/TAA) and portal perfusion pressure, were measured. Results. LDH and TXB2 were reduced, and bile flow was increased by IPostC, mainly in total and in the late phase of reperfusion. The follow-up study showed that the perfusate derived from a postconditioned group had much less damaging potential than perfusate derived from the nonpostconditioned group. Conclusion. IPostC following warm ischemia protects healthy, fibrotic, and cirrhotic livers against IRI. Reduced efflux of TXB2 is one possible mechanism for this effect of IPostC and increases sinusoidal microcirculation. These findings may help to improve organ function and recovery of patients after liver resection.

PURPOSE:To evaluate variability of central corneal thickness measurement (CCT) devices using a hitherto unprecedented number of CCT devices. METHODS:CCT was measured consecutively in 122 normal corneas of 61 subjects with seven different devices using three distinct measurement technologies: Scheimpflug, Ultrasound, and Optical Coherence Tomography (OCT). Per device deviation from the mean CCT value per eye was used to determine which of the devices performed best, compared to the mean value. RESULTS:Cirrus OCT yielded the lowest deviation. Deviations of the individual devices from the mean CCT of each eye were (OS/OD) 12.8±5.0/14.9±9.4 μm for Topcon noncontact specular microscopy (NCSM), 11.3±5.9/10.6±7.3 μm for Pentacam, 10.7±5.2/10.4±4.8 μm for Spectralis OCT, 6.0±3.9/6.2±4.9 μm for Topcon DRI OCT, 5.1±3.4/5.9±10.3 μm for AngioVue OCT, 4.8±4.1/5.7±4.6 μm for US pachymetry, and 4.2±3.2/5.7±4.6 μm for Cirrus OCT. The maximum differences between US pachymetry and the other devices were very high (up to 120 μm). CONCLUSION:Central corneal thickness may be under- or overestimated due to high interdevice variations. Measuring CCT with one device only may lead to inappropriate clinical and surgical recommendations. OCT showed superior results.

DNA-based nanostructures have received great attention as molecular vehicles for cellular delivery of biomolecules and cancer drugs. Here, we report on the cellular uptake of tubule-like DNA tile-assembled nanostructures 27 nm in length and 8 nm in diameter that carry siRNA molecules, folic acid and fluorescent dyes. In our observations, the DNA structures are delivered to the endosome and do not reach the cytosol of the GFP-expressing HeLa cells that were used in the experiments. Consistent with this observation, no elevated silencing of the GFP gene could be detected. Furthermore, the presence of up to six molecules of folic acid on the carrier surface did not alter the uptake behavior and gene silencing. We further observed several challenges that have to be considered when performing in vitro and in vivo experiments with DNA structures: (i) DNA tile tubes consisting of 42 nt-long oligonucleotides and carrying single- or double-stranded extensions degrade within one hour in cell medium at 37 °C, while the same tubes without extensions are stable for up to eight hours. The degradation is caused mainly by the low concentration of divalent ions in the media. The lifetime in cell medium can be increased drastically by employing DNA tiles that are 84 nt long. (ii) Dyes may get cleaved from the oligonucleotides and then accumulate inside the cell close to the mitochondria, which can lead to misinterpretation of data generated by flow cytometry and fluorescence microscopy. (iii) Single-stranded DNA carrying fluorescent dyes are internalized at similar levels as the DNA tile-assembled tubes used here.

Abstract Pigment cells and neuronal cells both are derived from the neural crest. Here, we describe the Pit‐Oct‐Unc (POU) domain transcription factor Brn3a, normally involved in neuronal development, to be frequently expressed in melanoma, but not in melanocytes and nevi. RNAi‐mediated silencing of Brn3a strongly reduced the viability of melanoma cell lines and decreased tumour growth in vivo. In melanoma cell lines, inhibition of Brn3a caused DNA double‐strand breaks as evidenced by Mre11/Rad50‐containing nuclear foci. Activated DNA damage signalling caused stabilization of the tumour suppressor p53, which resulted in cell cycle arrest and apoptosis. When Brn3a was ectopically expressed in primary melanocytes and fibroblasts, anchorage‐independent growth was increased. In tumourigenic melanocytes and fibroblasts, Brn3a accelerated tumour growth in vivo. Furthermore, Brn3a cooperated with proliferation pathways such as oncogenic BRAF, by reducing oncogene‐induced senescence in non‐malignant melanocytes. Together, these results identify Brn3a as a new factor in melanoma that is essential for melanoma cell survival and that promotes melanocytic transformation and tumourigenesis.

IL-1β is a potent player in cutaneous inflammation and central for the development of a Th17 micro-milieu in autoinflammatory diseases including psoriasis. Its production is controlled at the transcriptional level and by subsequent posttranslational processing via inflammatory caspases. In this study, we detected inflammatory caspase-5 active in epidermal keratinocytes and in psoriatic skin lesions. Further, interferon-γ and interleukin-17A synergistically induced caspase-5 expression in cultured keratinocytes, which was dependent on the antimicrobial peptide psoriasin (S100A7). However, diseases-relevant triggers for caspase-5 activity and IL-1β production remain unknown. Recently, extranuclear DNA has been identified as danger-signals abundant in the psoriatic epidermis. Here, we could demonstrate that cytosolic double-stranded (ds) DNA transfected into keratinocytes triggered the activation of caspase-5 and the release of IL-1β. Further, interleukin-17A promoted caspase-5 function via facilitation of the NLRP1-inflammasome. Anti-inflammatory vitamin D interfered with the IL-1β release and suppressed caspase-5 in keratinocytes and in psoriatic skin lesions. Our data link the disease-intrinsic danger signals psoriasin (S100A7) and dsDNA for NLPR1-dependent caspase-5 activity in psoriasis providing potential therapeutic targets in Th17-mediated skin autoinflammation.