• Human-Relevant Sensitivity of iPSC-Derived Human Motor Neurons to BoNT/A1 and B1.

      Schenke, Maren; Prause, Hélène-Christine; Bergforth, Wiebke; Przykopanski, Adina; Rummel, Andreas; Klawonn, Frank; Seeger, Bettina; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2021-08-22)
      The application of botulinum neurotoxins (BoNTs) for medical treatments necessitates a potency quantification of these lethal bacterial toxins, resulting in the use of a large number of test animals. Available alternative methods are limited in their relevance, as they are based on rodent cells or neuroblastoma cell lines or applicable for single toxin serotypes only. Here, human motor neurons (MNs), which are the physiological target of BoNTs, were generated from induced pluripotent stem cells (iPSCs) and compared to the neuroblastoma cell line SiMa, which is often used in cell-based assays for BoNT potency determination. In comparison with the mouse bioassay, human MNs exhibit a superior sensitivity to the BoNT serotypes A1 and B1 at levels that are reflective of human sensitivity. SiMa cells were able to detect BoNT/A1, but with much lower sensitivity than human MNs and appear unsuitable to detect any BoNT/B1 activity. The MNs used for these experiments were generated according to three differentiation protocols, which resulted in distinct sensitivity levels. Molecular parameters such as receptor protein concentration and electrical activity of the MNs were analyzed, but are not predictive for BoNT sensitivity. These results show that human MNs from several sources should be considered in BoNT testing and that human MNs are a physiologically relevant model, which could be used to optimize current BoNT potency testing.
    • In Vivo Lentiviral Gene Delivery of HLA-DR and Vaccination of Humanized Mice for Improving the Human T and B Cell Immune Reconstitution.

      Kumar, Suresh; Koenig, Johannes; Schneider, Andreas; Wermeling, Fredrik; Boddul, Sanjaykumar; Theobald, Sebastian J; Vollmer, Miriam; Kloos, Doreen; Lachmann, Nico; Klawonn, Frank; et al. (MDPI, 2021-08-05)
      Humanized mouse models generated with human hematopoietic stem cells (HSCs) and reconstituting the human immune system (HIS-mice) are invigorating preclinical testing of vaccines and immunotherapies. We have recently shown that human engineered dendritic cells boosted bonafide human T and B cell maturation and antigen-specific responses in HIS-mice. Here, we evaluated a cell-free system based on in vivo co-delivery of lentiviral vectors (LVs) for expression of a human leukocyte antigen (HLA-DRA*01/ HLA-DRB1*0401 functional complex, "DR4"), and a LV vaccine expressing human cytokines (GM-CSF and IFN-α) and a human cytomegalovirus gB antigen (HCMV-gB). Humanized NOD/Rag1null/IL2Rγnull (NRG) mice injected by i.v. with LV-DR4/fLuc showed long-lasting (up to 20 weeks) vector distribution and expression in the spleen and liver. In vivo administration of the LV vaccine after LV-DR4/fLuc delivery boosted the cellularity of lymph nodes, promoted maturation of terminal effector CD4+ T cells, and promoted significantly higher development of IgG+ and IgA+ B cells. This modular lentigenic system opens several perspectives for basic human immunology research and preclinical utilization of LVs to deliver HLAs into HIS-mice.
    • Development of a data generator for multivariate numerical data with arbitrary correlations and distributions

      Vahldiek, Kai; Zhou, Libing; Zhu, Wenfeng; Klawonn, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (IOS Press, 2021-01-01)
      Artificial or simulated data are particularly relevant in tests and benchmarks for machine learning methods, in teaching for exercises and for setting up analysis workflows. They are relevant when real data may not be used for reasons of data protection, or when special distributions or effects should be present in the data to test certain machine learning methods. In this paper a generator for multivariate numerical data with arbitrary marginal distributions and – as far as possible – arbitrary correlations is presented. The data generator is implemented in the open source statistics software R. It can also be used for categorical variables, if data are generated separately for the corresponding characteristics of a categorical variable. Additionally, outliers can be integrated. The use of the data generator is demonstrated with a concrete example.
    • Pattern discovery in time series using autoencoder in comparison to nonlearning approaches

      Noering, Fabian Kai Dietrich; Schroeder, Yannik; Jonas, Konstantin; Klawonn, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (IOS Press, 2021-01-01)
      In technical systems the analysis of similar situations is a promising technique to gain information about the system's state, its health or wearing. Very often, situations cannot be defined but need to be discovered as recurrent patterns within time series data of the system under consideration. This paper addresses the assessment of different approaches to discover frequent variable-length patterns in time series. Because of the success of artificial neural networks (NN) in various research fields, a special issue of this work is the applicability of NNs to the problem of pattern discovery in time series. Therefore we applied and adapted a Convolutional Autoencoder and compared it to classical nonlearning approaches based on Dynamic Time Warping, based on time series discretization as well as based on the Matrix Profile. These nonlearning approaches have also been adapted, to fulfill our requirements like the discovery of potentially time scaled patterns from noisy time series. We showed the performance (quality, computing time, effort of parametrization) of those approaches in an extensive test with synthetic data sets. Additionally the transferability to other data sets is tested by using real life vehicle data. We demonstrated the ability of Convolutional Autoencoders to discover patterns in an unsupervised way. Furthermore the tests showed, that the Autoencoder is able to discover patterns with a similar quality like classical nonlearning approaches. © 2021 - IOS Press. All rights reserved.
    • Elevated Free Phosphatidylcholine Levels in Cerebrospinal Fluid Distinguish Bacterial from Viral CNS Infections.

      Al-Mekhlafi, Amani; Sühs, Kurt-Wolfram; Schuchardt, Sven; Kuhn, Maike; Müller-Vahl, Kirsten; Trebst, Corinna; Skripuletz, Thomas; Klawonn, Frank; Stangel, Martin; Pessler, Frank; et al. (MDPI, 2021-05-06)
      The identification of CSF biomarkers for bacterial meningitis can potentially improve diagnosis and understanding of pathogenesis, and the differentiation from viral CNS infections is of particular clinical importance. Considering that substantial changes in CSF metabolites in CNS infections have recently been demonstrated, we compared concentrations of 188 metabolites in CSF samples from patients with bacterial meningitis (n = 32), viral meningitis/encephalitis (n = 34), and noninflamed controls (n = 66). Metabolite reprogramming in bacterial meningitis was greatest among phosphatidylcholines, and concentrations of all 54 phosphatidylcholines were significantly (p = 1.2 × 10-25-1.5 × 10-4) higher than in controls. Indeed, all biomarkers for bacterial meningitis vs. viral meningitis/encephalitis with an AUC ≥ 0.86 (ROC curve analysis) were phosphatidylcholines. Four of the five most accurate (AUC ≥ 0.9) phosphatidylcholine biomarkers had higher sensitivity and negative predictive values than CSF lactate or cell count. Concentrations of the 10 most accurate phosphatidylcholine biomarkers were lower in meningitis due to opportunistic pathogens than in meningitis due to typical meningitis pathogens, and they correlated most strongly with parameters reflecting blood-CSF barrier dysfunction and CSF lactate (r = 0.73-0.82), less so with CSF cell count, and not with blood CRP. In contrast to the elevated phosphatidylcholine concentrations in CSF, serum concentrations remained relatively unchanged. Taken together, these results suggest that increased free CSF phosphatidylcholines are sensitive biomarkers for bacterial meningitis and do not merely reflect inflammation but are associated with local disease and a shift in CNS metabolism.
    • Towards the characterization of the hidden world of small proteins in Staphylococcus aureus, a proteogenomics approach.

      Fuchs, Stephan; Kucklick, Martin; Lehmann, Erik; Beckmann, Alexander; Wilkens, Maya; Kolte, Baban; Mustafayeva, Ayten; Ludwig, Tobias; Diwo, Maurice; Wissing, Josef; et al. (PLOS, 2021-06-01)
      Small proteins play essential roles in bacterial physiology and virulence, however, automated algorithms for genome annotation are often not yet able to accurately predict the corresponding genes. The accuracy and reliability of genome annotations, particularly for small open reading frames (sORFs), can be significantly improved by integrating protein evidence from experimental approaches. Here we present a highly optimized and flexible bioinformatics workflow for bacterial proteogenomics covering all steps from (i) generation of protein databases, (ii) database searches and (iii) peptide-to-genome mapping to (iv) visualization of results. We used the workflow to identify high quality peptide spectrum matches (PSMs) for small proteins (≤ 100 aa, SP100) in Staphylococcus aureus Newman. Protein extracts from S. aureus were subjected to different experimental workflows for protein digestion and prefractionation and measured with highly sensitive mass spectrometers. In total, 175 proteins with up to 100 aa (SP100) were identified. Out of these 24 (ranging from 9 to 99 aa) were novel and not contained in the used genome annotation.144 SP100 are highly conserved and were found in at least 50% of the publicly available S. aureus genomes, while 127 are additionally conserved in other staphylococci. Almost half of the identified SP100 were basic, suggesting a role in binding to more acidic molecules such as nucleic acids or phospholipids.
    • Proteogenomic Characterization of the Cement and Adhesive Gland of the Pelagic Gooseneck Barnacle .

      Domínguez-Pérez, Dany; Almeida, Daniela; Wissing, Josef; Machado, André M; Jänsch, Lothar; Antunes, Agostinho; Castro, Luís Filipe; Vasconcelos, Vitor; Campos, Alexandre; Cunha, Isabel; et al. (MDPI, 2021-03-25)
      We focus on the stalked goose barnacle L. anatifera adhesive system, an opportunistic less selective species for the substrate, found attached to a variety of floating objects at seas. Adhesion is an adaptative character in barnacles, ensuring adequate positioning in the habitat for feeding and reproduction. The protein composition of the cement multicomplex and adhesive gland was quantitatively studied using shotgun proteomic analysis. Overall, 11,795 peptide sequences were identified in the gland and 2206 in the cement, clustered in 1689 and 217 proteinGroups, respectively. Cement specific adhesive proteins (CPs), proteases, protease inhibitors, cuticular and structural proteins, chemical cues, and many unannotated proteins were found, among others. In the cement, CPs were the most abundant (80.5%), being the bulk proteins CP100k and -52k the most expressed of all, and CP43k-like the most expressed interfacial protein. Unannotated proteins comprised 4.7% of the cement proteome, ranking several of them among the most highly expressed. Eight of these proteins showed similar physicochemical properties and amino acid composition to known CPs and classified through Principal Components Analysis (PCA) as new CPs. The importance of PCA on the identification of unannotated non-conserved adhesive proteins, whose selective pressure is on their relative amino acid abundance, was demonstrated.
    • A Point Mutation in the Transcriptional Repressor PerR Results in a Constitutive Oxidative Stress Response in Clostridioides difficile 630Δ.erm

      Troitzsch, Daniel; Zhang, Hao; Dittmann, Silvia; Düsterhöft, Dorothee; Möller, Timon Alexander; Michel, Annika-Marisa; Jänsch, Lothar; Riedel, Katharina; Borrero-de Acuña, José Manuel; Jahn, Dieter; et al. (ASM, 2021-03-03)
      The human pathogen Clostridioides difficile has evolved into the leading cause of nosocomial diarrhea. The bacterium is capable of spore formation, which even allows survival of antibiotic treatment. Although C. difficile features an anaerobic lifestyle, we determined a remarkably high oxygen tolerance of the laboratory reference strain 630Δerm A mutation of a single nucleotide (single nucleotide polymorphism [SNP]) in the DNA sequence (A to G) of the gene encoding the regulatory protein PerR results in an amino acid substitution (Thr to Ala) in one of the helices of the helix-turn-helix DNA binding domain of this transcriptional repressor in C. difficile 630Δerm PerR is a sensor protein for hydrogen peroxide and controls the expression of genes involved in the oxidative stress response. We show that PerR of C. difficile 630Δerm has lost its ability to bind the promoter region of PerR-controlled genes. This results in a constitutive derepression of genes encoding oxidative stress proteins such as a rubrerythrin (rbr1) whose mRNA abundance under anaerobic conditions was increased by a factor of about 7 compared to its parental strain C. difficile 630. Rubrerythrin repression in strain 630Δerm could be restored by the introduction of PerR from strain 630. The permanent oxidative stress response of C. difficile 630Δerm observed here should be considered in physiological and pathophysiological investigations based on this widely used model strain.IMPORTANCE The intestinal pathogen Clostridioides difficile is one of the major challenges in medical facilities nowadays. In order to better combat the bacterium, detailed knowledge of its physiology is mandatory. C. difficile strain 630Δerm was generated in a laboratory from the patient-isolated strain C. difficile 630 and represents a reference strain for many researchers in the field, serving as the basis for the construction of insertional gene knockout mutants. In our work, we demonstrate that this strain is characterized by an uncontrolled oxidative stress response as a result of a single-base-pair substitution in the sequence of a transcriptional regulator. C. difficile researchers working with model strain 630Δerm should be aware of this permanent stress response.
    • Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo.

      Volckmar, Julia; Knop, Laura; Hirsch, Tatjana; Frentzel, Sarah; Erck, Christian; van Ham, Marco; Stegemann-Koniszewski, Sabine; Bruder, Dunja; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MyJove Corporation, 2021-02-05)
      Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation. Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (αDEC-205) for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting αDEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to αDEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA). We have successfully used chemically crosslinked αDEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.
    • Data Analysis Strategies for Microbiome Studies in Human Populations-a Systematic Review of Current Practice.

      Kleine Bardenhorst, Sven; Berger, Tom; Klawonn, Frank; Vital, Marius; Karch, André; Rübsamen, Nicole; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (ASM, 2021-02-23)
      Reproducibility is a major issue in microbiome studies, which is partly caused by missing consensus about data analysis strategies. The complex nature of microbiome data, which are high-dimensional, zero-inflated, and compositional, makes them challenging to analyze, as they often violate assumptions of classic statistical methods. With advances in human microbiome research, research questions and study designs increase in complexity so that more sophisticated data analysis concepts are applied. To improve current practice of the analysis of microbiome studies, it is important to understand what kind of research questions are asked and which tools are used to answer these questions. We conducted a systematic literature review considering all publications focusing on the analysis of human microbiome data from June 2018 to June 2019. Of 1,444 studies screened, 419 fulfilled the inclusion criteria. Information about research questions, study designs, and analysis strategies were extracted. The results confirmed the expected shift to more advanced research questions, as one-third of the studies analyzed clustered data. Although heterogeneity in the methods used was found at any stage of the analysis process, it was largest for differential abundance testing. Especially if the underlying data structure was clustered, we identified a lack of use of methods that appropriately addressed the underlying data structure while taking into account additional dependencies in the data. Our results confirm considerable heterogeneity in analysis strategies among microbiome studies; increasingly complex research questions require better guidance for analysis strategies.IMPORTANCE The human microbiome has emerged as an important factor in the development of health and disease. Growing interest in this topic has led to an increasing number of studies investigating the human microbiome using high-throughput sequencing methods. However, the development of suitable analytical methods for analyzing microbiome data has not kept pace with the rapid progression in the field. It is crucial to understand current practice to identify the scope for development. Our results highlight the need for an extensive evaluation of the strengths and shortcomings of existing methods in order to guide the choice of proper analysis strategies. We have identified where new methods could be designed to address more advanced research questions while taking into account the complex structure of the data.
    • Quantitative laboratory results: Normal or lognormal distribution?

      Klawonn, Frank; Hoffmann, Georg; Orth, Matthias; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Walter De Gruyter GmbH, 2020-06-01)
      The identification of a suitable distribution model is a prerequisite for the parametric estimation of reference intervals and other statistical laboratory tasks. Classification of normal vs. lognormal distributions from healthy populations is easy, but from mixed populations, containing unknown proportions of abnormal results, it is challenging. We demonstrate that Bowley’s skewness coefficient differentiates between normal and lognormal distributions. This classifier is robust and easy to calculate from the quartiles Q1–Q3 according to the formula (Q1 − 2 · Q2 + Q3)/(Q3 − Q1). We validate our algorithm with a more complex procedure, which optimizes the exponent λ of a power transformation. As a practical application, we show that Bowley’s skewness coefficient is suited selecting the adequate distribution model for the estimation of reference limits according to a recent International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) recommendation, especially if the data is right-skewed.
    • Crystal structure of bacterial cytotoxic necrotizing factor CNFy reveals molecular building blocks for intoxication.

      Chaoprasid, Paweena; Lukat, Peer; Mühlen, Sabrina; Heidler, Thomas; Gazdag, Emerich-Mihai; Dong, Shuangshuang; Bi, Wenjie; Rüter, Christian; Kirchenwitz, Marco; Steffen, Anika; et al. (Springer, 2021-01-07)
      Cytotoxic necrotizing factors (CNFs) are bacterial single-chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three-dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full-length Yersinia pseudotuberculosis CNFY . CNFY consists of five domains (D1-D5), and by integrating structural and functional data, we demonstrate that D1-3 act as export and translocation module for the catalytic unit (D4-5) and for a fused β-lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP-ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4-5 fragment. This liberates D5 from a semi-blocked conformation in full-length CNFY , leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad-specificity protein delivery tool.
    • Replicability, Repeatability, and Long-term Reproducibility of Cerebellar Morphometry.

      Sörös, Peter; Wölk, Louise; Bantel, Carsten; Bräuer, Anja; Klawonn, Frank; Witt, Karsten; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2021-01-09)
      To identify robust and reproducible methods of cerebellar morphometry that can be used in future large-scale structural MRI studies, we investigated the replicability, repeatability, and long-term reproducibility of three fully automated software tools: FreeSurfer, CEREbellum Segmentation (CERES), and automatic cerebellum anatomical parcellation using U-Net with locally constrained optimization (ACAPULCO). Replicability was defined as computational replicability, determined by comparing two analyses of the same high-resolution MRI data set performed with identical analysis software and computer hardware. Repeatability was determined by comparing the analyses of two MRI scans of the same participant taken during two independent MRI sessions on the same day for the Kirby-21 study. Long-term reproducibility was assessed by analyzing two MRI scans of the same participant in the longitudinal OASIS-2 study. We determined percent difference, the image intraclass correlation coefficient, the coefficient of variation, and the intraclass correlation coefficient between two analyses. Our results show that CERES and ACAPULCO use stochastic algorithms that result in surprisingly high differences between identical analyses for ACAPULCO and small differences for CERES. Changes between two consecutive scans from the Kirby-21 study were less than ± 5% in most cases for FreeSurfer and CERES (i.e., demonstrating high repeatability). As expected, long-term reproducibility was lower than repeatability for all software tools. In summary, CERES is an accurate, as demonstrated before, and reproducible tool for fully automated segmentation and parcellation of the cerebellum. We conclude with recommendations for the assessment of replicability, repeatability, and long-term reproducibility in future studies on cerebellar structure.
    • Dinoroseobacter shibae Outer Membrane Vesicles Are Enriched for the Chromosome Dimer Resolution Site dif.

      Wang, Hui; Beier, Nicole; Boedeker, Christian; Sztajer, Helena; Henke, Petra; Neumann-Schaal, Meina; Mansky, Johannes; Rohde, Manfred; Overmann, Jörg; Petersen, Jörn; et al. (American Society for Microbiology, 2021-01-12)
      Outer membrane vesicles (OMVs) are universally produced by prokaryotes and play important roles in symbiotic and pathogenic interactions. They often contain DNA, but a mechanism for its incorporation is lacking. Here, we show that Dinoroseobacter shibae, a dinoflagellate symbiont, constitutively secretes OMVs containing DNA. Time-lapse microscopy captured instances of multiple OMV production at the septum during cell division. DNA from the vesicle lumen was up to 22-fold enriched for the region around the terminus of replication (ter). The peak of coverage was located at dif, a conserved 28-bp palindromic sequence required for binding of the site-specific tyrosine recombinases XerC/XerD. These enzymes are activated at the last stage of cell division immediately prior to septum formation when they are bound by the divisome protein FtsK. We suggest that overreplicated regions around the terminus have been repaired by the FtsK-dif-XerC/XerD molecular machinery. The vesicle proteome was clearly dominated by outer membrane and periplasmic proteins. Some of the most abundant vesicle membrane proteins were predicted to be required for direct interaction with peptidoglycan during cell division (LysM, Tol-Pal, Spol, lytic murein transglycosylase). OMVs were 15-fold enriched for the saturated fatty acid 16:00. We hypothesize that constitutive OMV secretion in D. shibae is coupled to cell division. The footprint of the FtsK-dif-XerC/XerD molecular machinery suggests a novel potentially highly conserved route for incorporation of DNA into OMVs. Clearing the division site from small DNA fragments might be an important function of vesicles produced during exponential growth under optimal conditions.IMPORTANCE Gram-negative bacteria continually form vesicles from their outer membrane (outer membrane vesicles [OMVs]) during normal growth. OMVs frequently contain DNA, and it is unclear how DNA can be shuffled from the cytoplasm to the OMVs. We studied OMV cargo in Dinoroseobacter shibae, a symbiont of dinoflagellates, using microscopy and a multi-omics approach. We found that vesicles formed during undisturbed exponential growth contain DNA which is enriched for genes around the replication terminus, specifically, the binding site for an enzyme complex that is activated at the last stage of cell division. We suggest that the enriched genes are the result of overreplication which is repaired by their excision and excretion via membrane vesicles to clear the divisome from waste DNA.
    • PD-1 Blockade Aggravates Epstein-Barr Virus Post-Transplant Lymphoproliferative Disorder in Humanized Mice Resulting in Central Nervous System Involvement and CD4 T Cell Dysregulations.

      Volk, Valery; Theobald, Sebastian J; Danisch, Simon; Khailaie, Sahamoddin; Kalbarczyk, Maja; Schneider, Andreas; Bialek-Waldmann, Julia; Krönke, Nicole; Deng, Yun; Eiz-Vesper, Britta; et al. (Frontiers, 2021-01-12)
      Post-transplant lymphoproliferative disorder (PTLD) is one of the most common malignancies after solid organ or allogeneic stem cell transplantation. Most PTLD cases are B cell neoplasias carrying Epstein-Barr virus (EBV). A therapeutic approach is reduction of immunosuppression to allow T cells to develop and combat EBV. If this is not effective, approaches include immunotherapies such as monoclonal antibodies targeting CD20 and adoptive T cells. Immune checkpoint inhibition (ICI) to treat EBV+ PTLD was not established clinically due to the risks of organ rejection and graft-versus-host disease. Previously, blockade of the programmed death receptor (PD)-1 by a monoclonal antibody (mAb) during ex vivo infection of mononuclear cells with the EBV/M81+ strain showed lower xenografted lymphoma development in mice. Subsequently, fully humanized mice infected with the EBV/B95-8 strain and treated in vivo with a PD-1 blocking mAb showed aggravation of PTLD and lymphoma development. Here, we evaluated vis-a-vis in fully humanized mice after EBV/B95-8 or EBV/M81 infections the effects of a clinically used PD-1 blocker. Fifteen to 17 weeks after human CD34+ stem cell transplantation, Nod.Rag.Gamma mice were infected with two types of EBV laboratory strains expressing firefly luciferase. Dynamic optical imaging analyses showed systemic EBV infections and this triggered vigorous human CD8+ T cell expansion. Pembrolizumab administered from 2 to 5 weeks post-infections significantly aggravated EBV systemic spread and, for the M81 model, significantly increased the mortality of mice. ICI promoted Ki67+CD30+CD20+EBER+PD-L1+ PTLD with central nervous system (CNS) involvement, mirroring EBV+ CNS PTLD in humans. PD-1 blockade was associated with lower frequencies of circulating T cells in blood and with a profound collapse of CD4+ T cells in lymphatic tissues. Mice treated with pembrolizumab showed an escalation of exhausted T cells expressing TIM-3, and LAG-3 in tissues, higher levels of several human cytokines in plasma and high densities of FoxP3+ regulatory CD4+ and CD8+ T cells in the tumor microenvironment. We conclude that PD-1 blockade during acute EBV infections driving strong CD8+ T cell priming decompensates T cell development towards immunosuppression. Given the variety of preclinical models available, our models conferred a cautionary note indicating that PD-1 blockade aggravated the progression of EBV+ PTLD.
    • Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls.

      Wiesselmann, Milan; Hebecker, Stefanie; Borrero-de Acuña, José M; NIMTZ, MANFRED; Bollivar, David; Jänsch, Lothar; Moser, Jürgen; Jahn, Dieter; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Portland Press, 2020-11-19)
      During bacteriochlorophyll a biosynthesis, the oxygen-independent conversion of Mg-protoporphyrin IX monomethyl ester (Mg-PME) to protochlorophyllide (Pchlide) is catalyzed by the anaerobic Mg-PME cyclase termed BchE. Bioinformatics analyses in combination with pigment studies of cobalamin-requiring Rhodobacter capsulatus mutants indicated an unusual radical S-adenosylmethionine (SAM) and cobalamin-dependent BchE catalysis. However, in vitro biosynthesis of the isocyclic ring moiety of bacteriochlorophyll using purified recombinant BchE has never been demonstrated. We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. The reaction product was further converted to chlorophyllide in the presence of light-dependent Pchlide reductase. BchE activity was stimulated by increasing concentrations of NADPH or SAM, and inhibited by S-adenosylhomocysteine. Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. BchE catalysis was not sustained in chimeric experiments when a cytosolic extract from E. coli was used as a substitute. Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. A structure guided site-directed mutagenesis approach was performed on the basis of a three-dimensional homology model of BchE. A newly established in vivo complementation assay was used to investigate 24 BchE mutant proteins. Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified.
    • Pain drawings as a diagnostic tool for the differentiation between two pain-associated rare diseases (Ehlers-Danlos-Syndrome, Guillain-Barré-Syndrome).

      Wester, Larissa; Mücke, Martin; Bender, Tim Theodor Albert; Sellin, Julia; Klawonn, Frank; Conrad, Rupert; Szczypien, Natasza; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (BMC, 2020-11-17)
      Background: The diagnosis of rare diseases poses a particular challenge to clinicians. This study analyzes whether patients' pain drawings (PDs) help in the differentiation of two pain-associated rare diseases, Ehlers-Danlos Syndrome (EDS) and Guillain-Barré Syndrome (GBS). Method: The study was designed as a prospective, observational, single-center study. The sample comprised 60 patients with EDS (3 male, 52 female, 5 without gender information; 39.2 ± 11.4 years) and 32 patients with GBS (10 male, 20 female, 2 without gender information; 50.5 ± 13.7 years). Patients marked areas afflicted by pain on a sketch of a human body with anterior, posterior, and lateral views. PDs were electronically scanned and processed. Each PD was classified based on the Ružička similarity to the EDS and the GBS averaged image (pain profile) in a leave-one-out cross validation approach. A receiver operating characteristic (ROC) curve was plotted. Results: 60-80% of EDS patients marked the vertebral column with the neck and the tailbone and the knee joints as pain areas, 40-50% the shoulder-region, the elbows and the thumb saddle joint. 60-70% of GBS patients marked the dorsal and plantar side of the feet as pain areas, 40-50% the palmar side of the fingertips, the dorsal side of the left palm and the tailbone. 86% of the EDS patients and 96% of the GBS patients were correctly identified by computing the Ružička similarity. The ROC curve yielded an excellent area under the curve value of 0.95.
    • Effect of a strict hygiene bundle for the prevention of nosocomial transmission of SARS-CoV-2 in the hospital: a practical approach from the field.

      Ambrosch, Andreas; Rockmann, Felix; Klawonn, Frank; Lampl, Benedikt; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2020-10-20)
      Background: During the novel coronavirus disease (COVID-19) pandemic it is crucial for hospitals to implement infection prevention strategies to reduce nosocomial transmission to the lowest possible number. This is all the more important because molecular tests for identifying SARS-CoV-2 infected patients are uncertain, and the resources available for them are limited. In this view, a monocentric, retrospective study with an interventional character was conducted to investigate the extent to which the introduction of a strict hygiene bundle including a general mask requirement and daily screening for suspicious patients has an impact on the SARS-CoV-2 nosocomial rate in the pandemic environment. Methods: All inpatients from a maximum care hospital in Regensburg (Bavaria) between March 1st and June 10th 2020 were included. Patient with respiratory symptoms were tested for SARS-CoV-2 at admission, patients were managed according to a standard hygiene protocol. At the end of March a strict hygiene bundle was introduced including a general mask obligation and a daily clinical screening of inpatients for respiratory symptoms. Nosocomial infection rate for COVID-19 and the risk for infection transmission estimated by the nosocomial incidence density before and after introduction the hygiene bundle were compared. The infection pressure for the hospital during the entire observational period was characterized by the infection reports in the region in relation to the number of hospitalized COVID-19 patients and the number of infected employees. Results: In fact, after the introduction of a strict hygiene bundle including a general mouth and nose protection obligation and a daily clinical screening of suspicious patients, a significant reduction of the nosocomial rate from 0.28 to 0.06 (p = 0.026) was observed. Furthermore, the risk of spreading hospital-acquired infections also decreased dramatically from 0.0007 to 0.00018 (p = 0.031; rate ratio after/before 0.25 (95%CI 0.06, 1.07) despite a slow decrease of the hospital COVID 19-prevalence and an increase of infected employees. Conclusion: The available data underline that a strict hygiene bundle seem to be associated with a decrease of nosocomial SARS-CoV-2 transmission in the pandemic situation.
    • Sample size and performance estimation for biomarker combinations based on pilot studies with small sample sizes”

      Al-Mekhlafi, Amani; Becker, Tobias; Klawonn, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Taylor & Francis, 2020-01-01)
      High throughput technologies like microarrays, next generation sequencing and mass spectrometry enable the measurement of tens of thousands of biomarker candidates in pilot studies. Biological systems are often too complex to be based on simple single cause-effect associations and from the medical practice point of view, a single biomarker may not possess the desired sensitivity and/or specificity for disease classification and outcome prediction. Therefore, the efforts of researchers currently aims at combining biomarkers. The intention of biomarker pilot studies with small sample sizes is often to explore the possibility of finding good biomarker combinations and not to find and evaluate a final combination of biomarkers with high predictive value. The aim of the pilot study is to answer the question whether it is worthwhile to extend the study to a larger study and to obtain information about the required sample size. In this paper, we propose a method to judge the potential in a small biomarker pilot study without the need to explicitly identifying and confirming a specific subset of biomarkers. In addition, we provide a method for sample size estimation for an extended study when the results of the pilot study look promising.
    • A publicly accessible database for genome sequences supports tracing of transmission chains and epidemics.

      Frentrup, Martinique; Zhou, Zhemin; Steglich, Matthias; Meier-Kolthoff, Jan P; Göker, Markus; Riedel, Thomas; Bunk, Boyke; Spröer, Cathrin; Overmann, Jörg; Blaschitz, Marion; et al. (Microbiology Society, 2020-07-29)
      Clostridioides difficile is the primary infectious cause of antibiotic-associated diarrhea. Local transmissions and international outbreaks of this pathogen have been previously elucidated by bacterial whole-genome sequencing, but comparative genomic analyses at the global scale were hampered by the lack of specific bioinformatic tools. Here we introduce a publicly accessible database within EnteroBase (http://enterobase.warwick.ac.uk) that automatically retrieves and assembles C. difficile short-reads from the public domain, and calls alleles for core-genome multilocus sequence typing (cgMLST). We demonstrate that comparable levels of resolution and precision are attained by EnteroBase cgMLST and single-nucleotide polymorphism analysis. EnteroBase currently contains 18 254 quality-controlled C. difficile genomes, which have been assigned to hierarchical sets of single-linkage clusters by cgMLST distances. This hierarchical clustering is used to identify and name populations of C. difficile at all epidemiological levels, from recent transmission chains through to epidemic and endemic strains. Moreover, it puts newly collected isolates into phylogenetic and epidemiological context by identifying related strains among all previously published genome data. For example, HC2 clusters (i.e. chains of genomes with pairwise distances of up to two cgMLST alleles) were statistically associated with specific hospitals (P<10-4) or single wards (P=0.01) within hospitals, indicating they represented local transmission clusters. We also detected several HC2 clusters spanning more than one hospital that by retrospective epidemiological analysis were confirmed to be associated with inter-hospital patient transfers. In contrast, clustering at level HC150 correlated with k-mer-based classification and was largely compatible with PCR ribotyping, thus enabling comparisons to earlier surveillance data. EnteroBase enables contextual interpretation of a growing collection of assembled, quality-controlled C. difficile genome sequences and their associated metadata. Hierarchical clustering rapidly identifies database entries that are related at multiple levels of genetic distance, facilitating communication among researchers, clinicians and public-health officials who are combatting disease caused by C. difficile.