• Catalytically Active Cas9 Mediates Transcriptional Interference to Facilitate Bacterial Virulence.

      Ratner, Hannah K; Escalera-Maurer, Andrés; Le Rhun, Anaïs; Jaggavarapu, Siddharth; Wozniak, Jessie E; Crispell, Emily K; Charpentier, Emmanuelle; Weiss, David S; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier; Cell Press, 2019-06-24)
      In addition to defense against foreign DNA, the CRISPR-Cas9 system of Francisella novicida represses expression of an endogenous immunostimulatory lipoprotein. We investigated the specificity and molecular mechanism of this regulation, demonstrating that Cas9 controls a highly specific regulon of four genes that must be repressed for bacterial virulence. Regulation occurs through a protospacer adjacent motif (PAM)-dependent interaction of Cas9 with its endogenous DNA targets, dependent on a non-canonical small RNA (scaRNA) and tracrRNA. The limited complementarity between scaRNA and the endogenous DNA targets precludes cleavage, highlighting the evolution of scaRNA to repress transcription without lethally targeting the chromosome. We show that scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-FnoCas9 machinery can also direct DNA cleavage. Natural Cas9 transcriptional interference likely represents a broad paradigm of regulatory functionality, which is potentially critical to the physiology of numerous Cas9-encoding pathogenic and commensal organisms.
    • Cell Polarization and Epigenetic Status Shape the Heterogeneous Response to Type III Interferons in Intestinal Epithelial Cells.

      Bhushal, Sudeep; Wolfsmüller, Markus; Selvakumar, Tharini A; Kemper, Lucas; Wirth, Dagmar; Hornef, Mathias W; Hauser, Hansjörg; Köster, Mario; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
      Type I and type III interferons (IFNs) are crucial components of the first-line antiviral host response. While specific receptors for both IFN types exist, intracellular signaling shares the same Jak-STAT pathway. Due to its receptor expression, IFN-λ responsiveness is restricted mainly to epithelial cells. Here, we display IFN-stimulated gene induction at the single cell level to comparatively analyze the activities of both IFN types in intestinal epithelial cells and mini-gut organoids. Initially, we noticed that the response to both types of IFNs at low concentrations is based on a single cell decision-making determining the total cell intrinsic antiviral activity. We identified histone deacetylase (HDAC) activity as a crucial restriction factor controlling the cell frequency of IFN-stimulated gene (ISG) induction upon IFN-λ but not IFN-β stimulation. Consistently, HDAC blockade confers antiviral activity to an elsewise non-responding subpopulation. Second, in contrast to the type I IFN system, polarization of intestinal epithelial cells strongly enhances their ability to respond to IFN-λ signaling and raises the kinetics of gene induction. Finally, we show that ISG induction in mini-gut organoids by low amounts of IFN is characterized by a scattered heterogeneous responsiveness of the epithelial cells and HDAC activity fine-tunes exclusively IFN-λ activity. This study provides a comprehensive description of the differential response to type I and type III IFNs and demonstrates that cell polarization in gut epithelial cells specifically increases IFN-λ activity.
    • Challenges in warranting access to prophylaxis and therapy for hepatitis B virus infection.

      Debarry, Jennifer; Cornberg, Markus; Manns, Michael P; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-01)
      Despite an available vaccine and efficient treatment for hepatitis B virus (HBV) infection, chronic HBV infection still remains a major global threat, and one of the top 20 causes of human mortality worldwide. One of the major challenges in controlling HBV infection is the high number of undiagnosed chronic carriers and the lack of access to prophylaxis and treatment in several parts of the world. We discuss relevant barriers that need to be overcome to achieve global control of HBV infection and make eradication possible. Most important, vaccination must be scaled-up to lower the risk of vertical transmission and decrease the number of new infections, and comprehensive screening programs must be linked to care to obtain a better rate of diagnosis and treatment. This can probably only be achieved if sustainable funding is available. We therefore emphasize the importance of making the management of viral hepatitis a global health priority.
    • Characterization of Populations by Multilocus Variable Number of Tandem Repeats (MLVA) Genotyping from Drinking Water and Biofilm in Hospitals from Different Regions of the West Bank.

      Zayed, Ashraf R; Pecellin, Marina; Salah, Alaa; Alalam, Hanna; Butmeh, Suha; Steinert, Michael; Lesnik, Rene; Brettar, Ingrid; Höfle, Manfred G; Bitar, Dina M; et al. (MDPI, 2020-10-22)
      The West Bank can be considered a high-risk area for Legionnaires' disease (LD) due to its hot climate, intermittent water supply and roof storage of drinking water. Legionella, mostly L. pneumophila, are responsible for LD, a severe, community-acquired and nosocomial pneumonia. To date, no extensive assessment of Legionella spp and L. pneumophila using cultivation in combination with molecular approaches in the West Bank has been published. Two years of environmental surveillance of Legionella in water and biofilms in the drinking water distribution systems (DWDS) of eight hospitals was carried out; 180 L. pneumophila strains were isolated, mostly from biofilms in DWDS. Most of the isolates were identified as serogroup (Sg) 1 (60%) and 6 (30%), while a minor fraction comprised Sg 8 and 10. Multilocus Variable number of tandem repeats Analysis using 13 loci (MLVA-8(12)) was applied as a high-resolution genotyping method and compared to the standard Sequence Based Typing (SBT). The isolates were genotyped in 27 MLVA-8(12) genotypes (Gt), comprising four MLVA clonal complexes (VACC 1; 2; 5; 11). The major fraction of isolates constituted Sequence Type (ST)1 and ST461. Most of the MLVA-genotypes were highly diverse and often unique. The MLVA-genotype composition showed substantial regional variability. In general, the applied MLVA-method made it possible to reproducibly genotype the isolates, and was consistent with SBT but showed a higher resolution. The advantage of the higher resolution was most evident for the subdivision of the large strain sets of ST1 and ST461; these STs were shown to be highly pneumonia-relevant in a former study. This shows that the resolution by MLVA is advantageous for back-tracking risk sites and for the avoidance of outbreaks of L. pneumophila. Overall, our results provide important insights into the detailed population structure of L. pneumophila, allowing for better risk assessment for DWDS.
    • Characterization of a transcriptional TPP riboswitch in the human pathogen Neisseriameningitidis.

      Righetti, Francesco; Materne, Solange Lise; Boss, John; Eichner, Hannes; Charpentier, Emmanuelle; Loh, Edmund; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Taylor & Francis, 2020-02-20)
      Increasing evidence has demonstrated that regulatory RNA elements such as riboswitches (RS) play a pivotal role in the fine-tuning of bacterial gene expression. In this study, we investigated and characterized a novel transcriptional thiamine pyrophosphate (TPP) RS in the obligate human pathogen N. meningitidis MC58 (serogroup B). This RS is located in the 5´ untranslated region upstream of thiC gene, encoding a protein involved in TPP biosynthesis, an essential cofactor for all living beings. Primer extension revealed the transcriptional start site of thiC. Northern blot analysis of thiC mRNA and reporter gene studies confirmed the presence of an active TPP-sensing RS. Expression patterns of the wild-type RS and site-specific mutants showed that it is an OFF switch that controls transcription elongation of thiC mRNA. Interestingly, the regulatory mechanism of the meningococcal thiC RS resembles the Gram-positive Bacillus subtilis thiC RS rather than the Gram-negative Escherichia coli thiC RS. Therefore, the meningococcal thiC RS represents a rare example of transcriptional RS in a Gram-negative bacterium. We further observed that the RS is actively involved in modulating gene expression in response to different growth media and to supplemented bacterial and eukaryotic cell lysates as possible sources of nutrients in the nasopharynx. Our results suggest that RS-mediated gene regulation could influence meningococcal fitness, through the fine-tuning of biosynthesis and scavenging of nutrients and cofactors, such as thiamine.
    • Chronic hepatitis C virus infection irreversibly impacts human natural killer cell repertoire diversity.

      Strunz, Benedikt; Hengst, Julia; Deterding, Katja; Manns, Michael P; Cornberg, Markus; Ljunggren, Hans-Gustaf; Wedemeyer, Heiner; Björkström, Niklas K; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-06-11)
      Diversity is a central requirement for the immune system's capacity to adequately clear a variety of different infections. As such, natural killer (NK) cells represent a highly diverse population of innate lymphocytes important in the early response against viruses. Yet, the extent to which a chronic pathogen affects NK cell diversity is largely unknown. Here we study NK cell functional diversification in chronic hepatitis C virus (HCV) infection. High-dimensional flow cytometer assays combined with stochastic neighbor embedding analysis reveal that chronic HCV infection induces functional imprinting on human NK cells that is largely irreversible and persists long after successful interventional clearance of the virus. Furthermore, HCV infection increases inter-individual, but decreases intra-individual, NK cell diversity. Taken together, our results provide insights into how the history of infections affects human NK cell diversity.
    • Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation.

      Gödecke, Natascha; Zha, Lisha; Spencer, Shawal; Behme, Sara; Riemer, Pamela; Rehli, Michael; Hauser, Hansjörg; Wirth, Dagmar; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, D38124 Braunschweig, Germany. (2017-09-19)
      Faithful expression of transgenes in cell cultures and mice is often challenged by locus dependent epigenetic silencing. We investigated silencing of Tet-controlled expression cassettes within the mouse ROSA26 locus. We observed pronounced DNA methylation of the Tet promoter concomitant with loss of expression in mES cells as well as in differentiated cells and transgenic animals. Strikingly, the ROSA26 promoter remains active and methylation free indicating that this silencing mechanism specifically affects the transgene, but does not spread to the host's chromosomal neighborhood. To reactivate Tet cassettes a synthetic fusion protein was constructed and expressed in silenced cells. This protein includes the enzymatic domains of ten eleven translocation methylcytosine dioxygenase 1 (TET-1) as well as the Tet repressor DNA binding domain. Expression of the synthetic fusion protein and Doxycycline treatment allowed targeted demethylation of the Tet promoter in the ROSA26 locus and in another genomic site, rescuing transgene expression in cells and transgenic mice. Thus, inducible, reversible and site-specific epigenetic modulation is a promising strategy for reactivation of silenced transgene expression, independent of the integration site.
    • Deconvolution of bulk blood eQTL effects into immune cell subpopulations.

      Aguirre-Gamboa, Raúl; de Klein, Niek; di Tommaso, Jennifer; Claringbould, Annique; van der Wijst, Monique Gp; de Vries, Dylan; Brugge, Harm; Oelen, Roy; Võsa, Urmo; Zorro, Maria M; et al. (BMC, 2020-06-12)
      A novel planctomycetal strain, designated Pla85_3_4T, was isolated from the surface of wood incubated at the discharge of a wastewater treatment plant in the Warnow river near Rostock, Germany. Cells of the novel strain have a cell envelope architecture resembling that of Gram-negative bacteria, are round to pear-shaped (length: 2.2 ± 0.4 µm, width: 1.2 ± 0.3 µm), form aggregates and divide by polar budding. Colonies have a cream colour. Strain Pla85_3_4T grows at ranges of 10-30 °C (optimum 26 °C) and at pH 6.5-10.0 (optimum 7.5), and has a doubling time of 26 h. Phylogenetically, strain Pla85_3_4T (DSM 103796T = LMG 29741T) is concluded to represent a novel species of a novel genus within the family Pirellulaceae, for which we propose the name Lignipirellula cremea gen. nov., sp. nov.
    • Degradable magnesium implant-associated infections by bacterial biofilms induce robust localized and systemic inflammatory reactions in a mouse model.

      Rahim, Muhammad Imran; Babbar, Anshu; Lienenklaus, Stefan; Pils, Marina; Rohde, M; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-06-01)
      Biomaterial-associated Pseudomonas aeruginosa biofilm infections constitute cascade of host immune reactions ultimately leading towards implant failure. Due to lack of relevant in vivo biofilm models, majority of the studies report host immune responses against free living or planktonic bacteria while bacteria in clinical situations live more frequently as biofilm communities than as single cells. Present study investigated host immune responses against biomaterial-associated P. aeruginosa biofilms in a clinically relevant mouse model. Previously, we reported metallic magnesium, a prospective biodegradable implant, to be permissive for bacterial biofilms in vivo even though it exhibits antibacterial properties in vitro. Therefore, magnesium was employed as biomaterial to investigate in vivo biofilm formation and associated host immune responses by using two P. aeruginosa strains and two mouse strains. P. aeruginosa formed biofilms on subcutaneously implanted magnesium discs. Non-invasive in vivo imaging indicated transient inflammatory responses at control sites whereas robust prolonged interferon-β (IFN-β) expression was observed from biofilms in a transgenic animal reporter. Further, immunohistology and electron microscopic results showed that bacterial biofilms were located in two dimensions immediately on the implant surface and at a short distance in the adjacent tissue. These biofilms were surrounded by inflammatory cells (mainly polymorphonuclear cells) as compared to controls. Interestingly, even though the number of live bacteria in various organs remained below detectable levels, splenomegaly indicated systemic inflammatory processes. Overall, these findings confirmed the resistance of biofilm infections in vivo to potentially antibacterial properties of magnesium degradation products. In vivo imaging and histology indicated the induction of both, local and systemic host inflammatory responses against P. aeruginosa biofilms. Even though the innate host immune defenses could not eliminate the local infection for up to two weeks, there was no apparent systemic bacteremia and all animals investigated survived the infection.
    • Dengue-specific subviral nanoparticles: design, creation and characterization.

      Khetarpal, Niyati; Poddar, Ankur; Nemani, Satish K; Dhar, Nisha; Patil, Aravind; Negi, Priyanka; Perween, Ashiya; Viswanathan, Ramaswamy; Lünsdorf, Heinrich; Tyagi, Poornima; et al. (2013)
      Dengue is today the most significant of arboviral diseases. Novel tools are necessary to effectively address the problem of dengue. Virus-like particles (VLP) offer a versatile nanoscale platform for developing tools with potential biomedical applications. From the perspective of a potentially useful dengue-specific tool, the dengue virus envelope protein domain III (EDIII), endowed with serotype-specificity, host receptor recognition and the capacity to elicit virus-neutralizing antibodies, is an attractive candidate.
    • Designation of type strains for seven species of the order Myxococcales and proposal for neotype strains of Cystobacter ferrugineus, Cystobacter minus and Polyangium fumosum.

      Lang, Elke; Reichenbach, Hans; Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, 38124 Braunschweig, Germany (2013-11)
      Ten species of the order Myxococcales with validly published names are devoid of living type strains. Four species of the genus Chondromyces are represented by dead herbarium samples as the type material. For a species of the genus Melittangium and two species of the genus Polyangium, no physical type material was assigned at the time of validation of the names or later on. In accordance with rule 18f of the International Code of Nomenclature of Bacteria the following type strains are designated for these species: strain Cm a14(T) ( = DSM 14605(T) = JCM 12615(T)) as the type strain of Chondromyces apiculatus, strain Cm c5(T) ( = DSM 14714(T) = JCM 12616(T)) as the type strain of Chondromyces crocatus, strain Sy t2(T) ( = DSM 14631(T) = JCM 12617(T)) as the type strain of Chondromyces lanuginosus, strain Cm p51(T) ( = DSM 14607(T) = JCM 12618(T)) as the type strain of Chondromyces pediculatus, strain Me b8(T) ( = DSM 14713(T) = JCM 12633(T)) as the type strain of Melittangium boletus, strain Pl s12(T) ( = DSM 14670(T) = JCM 12637(T)) as the type strain of Polyangium sorediatum and strain Pl sm5(T) ( = DSM 14734(T) = JCM 12638(T)) as the type strain of Polyangium spumosum. Furthermore, the type strains given for three species of the genera Cystobacter and Polyangium had been kept at one university institute and have been lost according to our investigations. In accordance with Rule 18c of the Bacteriological Code, we propose the following neotype strains: strain Cb fe18 ( = DSM 14716  = JCM 12624) as the neotype strain of Cystobacter ferrugineus, strain Cb m2 ( = DSM 14751 = JCM 12627) as the neotype strain of Cystobacter minus and strain Pl fu5 ( = DSM 14668 = JCM 12636) as the neotype strain of Polyangium fumosum. The proposals of the strains are based on the descriptions and strain proposals given in the respective chapters of Bergey's Manual of Systematic Bacteriology (2005).
    • The Deubiquitinating Enzyme Cylindromatosis Dampens CD8(+) T Cell Responses and Is a Critical Factor for Experimental Cerebral Malaria and Blood-Brain Barrier Damage.

      Schmid, Ursula; Stenzel, Werner; Koschel, Josephin; Raptaki, Maria; Wang, Xu; Naumann, Michael; Matuschewski, Kai; Schlüter, Dirk; Nishanth, Gopala; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
      Cerebral malaria is a severe complication of human malaria and may lead to death of Plasmodium falciparum-infected individuals. Cerebral malaria is associated with sequestration of parasitized red blood cells within the cerebral microvasculature resulting in damage of the blood-brain barrier and brain pathology. Although CD8(+) T cells have been implicated in the development of murine experimental cerebral malaria (ECM), several other studies have shown that CD8(+) T cells confer protection against blood-stage infections. Since the role of host deubiquitinating enzymes (DUBs) in malaria is yet unknown, we investigated how the DUB cylindromatosis (CYLD), an important inhibitor of several cellular signaling pathways, influences the outcome of ECM. Upon infection with Plasmodium berghei ANKA (PbA) sporozoites or PbA-infected red blood cells, at least 90% of Cyld(-/-) mice survived the infection, whereas all congenic C57BL/6 mice displayed signatures of ECM, impaired parasite control, and disruption of the blood-brain barrier integrity. Cyld deficiency prevented brain pathology, including hemorrhagic lesions, enhanced activation of astrocytes and microglia, infiltration of CD8(+) T cells, and apoptosis of endothelial cells. Furthermore, PbA-specific CD8(+) T cell responses were augmented in the blood of Cyld(-/-) mice with increased production of interferon-γ and granzyme B and elevated activation of protein kinase C-θ and nuclear factor "kappa light-chain enhancer" of activated B cells. Importantly, accumulation of CD8(+) T cells in the brain of Cyld(-/-) mice was significantly reduced compared to C57BL/6 mice. Bone marrow chimera experiments showed that the absence of ECM signatures in infected Cyld(-/-) mice could be attributed to hematopoietic and radioresistant parenchymal cells, most likely endothelial cells that did not undergo apoptosis. Together, we were able to show that host deubiqutinating enzymes play an important role in ECM and that CYLD promotes ECM supporting it as a potential therapeutic target for adjunct therapy to prevent cerebral complications of severe malaria.
    • Developmental induction of human T-cell responses against Candida albicans and Aspergillus fumigatus.

      Vogel, Katrin; Pierau, Mandy; Arra, Aditya; Lampe, Karen; Schlueter, Dirk; Arens, Christoph; Brunner-Weinzierl, Monika C; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Nature publishing group, 2018-11-15)
      The origin of human T-cell responses against fungal pathogens early in life is not clearly understood. Here, we show that antifungal T-cell responses are vigorously initiated within the first years of life against lysates and peptides of Candida albicans or Aspergillus fumigatus, presented by autologous monocytes. The neonatal responding T-cell pool consists of 20 different TCR-V
    • Die nicht-alkoholische Fettlebererkrankung : Die Rolle der Leber im metabolischen Syndrom

      Jupa, K.L.; Manns, M.P.; Jäckel, E; DZIF,Deutsches Zentrum für Infektionsforschung; HZI, Helmholtz-Zentrum für Infektiondforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig.
      Epidemiologie Die nicht-alkoholische Fettlebererkrankung (»non-alcoholic fatty liver disease«, NAFLD) ist mittlerweile eine der häufigsten Lebererkrankung weltweit mit einer mittleren Prävalenz von 20% weltweit und bis zu 30% in Europa [1]. Als Hauptrisikofaktoren gelten einerseits verschiedene Umweltfaktoren, insbesondere Bewegungsmangel und falsche Ernährung sowie andererseits die verschiedenen Aspekte des metabolischen Syndroms: Adipositas, Fettstoffwechselstörungen sowie Insulinresistenz und Diabetes mellitus Typ 2. Aus diesem Grund wird die Fettleber mittlerweile auch als hepatische Komponente des metabolischen Syndroms bezeichnet. Neben den Umweltfaktoren konnte auch gezeigt werden, dass eine genetische Prädisposition im Sinne von einem Adiponutrin Polymorphismus (»patatin-like phospholipase domain containing 3«, PNPLA3 [2, 3] zu einem gehäuften Auftreten einer Fettlebererkrankung führen kann.
    • Differential magnesium implant corrosion coat formation and contribution to bone bonding.

      Rahim, Muhammad Imran; Weizbauer, Andreas; Evertz, Florian; Hoffmann, Andrea; Rohde, M; Glasmacher, Birgit; Windhagen, Henning; Gross, Gerhard; Seitz, Jan-Marten; Mueller, Peter P; et al. (2017)
      Magnesium alloys are presently under investigation as promising biodegradable implant materials with osteoconductive properties. To study the molecular mechanisms involved, the potential contribution of soluble magnesium corrosion products to the stimulation of osteoblastic cell differentiation was examined. However, no evidence for the stimulation of osteoblast differentiation could be obtained when cultured mesenchymal precursor cells were differentiated in the presence of metallic magnesium or in cell culture medium containing elevated magnesium ion levels. Similarly, in soft tissue no bone induction by metallic magnesium or by the corrosion product magnesium hydroxide could be observed in a mouse model. Motivated by the comparatively rapid accumulation solid corrosion products physicochemical processes were examined as an alternative mechanism to explain the stimulation of bone growth by magnesium-based implants. During exposure to physiological solutions a structured corrosion coat formed on magnesium whereby the elements calcium and phosphate were enriched in the outermost layer which could play a role in the established biocompatible behavior of magnesium implants. When magnesium pins were inserted into avital bones, corrosion lead to increases in the pull out force, suggesting that the expanding corrosion layer was interlocking with the surrounding bone. Since mechanical stress is a well-established inducer of bone growth, volume increases caused by the rapid accumulation of corrosion products and the resulting force development could be a key mechanism and provide an explanation for the observed stimulatory effects of magnesium-based implants in hard tissue. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 697-709, 2017.
    • Divergent co-transcriptomes of different host cells infected with Toxoplasma gondii reveal cell type-specific host-parasite interactions.

      Swierzy, Izabela J; Händel, Ulrike; Kaever, Alexander; Jarek, Michael; Scharfe, Maren; Schlüter, Dirk; Lüder, Carsten G K; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-08-03)
      The apicomplexan parasite Toxoplasma gondii infects various cell types in avian and mammalian hosts including humans. Infection of immunocompetent hosts is mostly asymptomatic or benign, but leads to development of largely dormant bradyzoites that persist predominantly within neurons and muscle cells. Here we have analyzed the impact of the host cell type on the co-transcriptomes of host and parasite using high-throughput RNA sequencing. Murine cortical neurons and astrocytes, skeletal muscle cells (SkMCs) and fibroblasts differed by more than 16,200 differentially expressed genes (DEGs) before and after infection with T. gondii. However, only a few hundred of them were regulated by infection and these largely diverged in neurons, SkMCs, astrocytes and fibroblasts indicating host cell type-specific transcriptional responses after infection. The heterogeneous transcriptomes of host cells before and during infection coincided with ~5,400 DEGs in T. gondii residing in different cell types. Finally, we identified gene clusters in both T. gondii and its host, which correlated with the predominant parasite persistence in neurons or SkMCs as compared to astrocytes or fibroblasts. Thus, heterogeneous expression profiles of different host cell types and the parasites' ability to adapting to them may govern the parasite-host cell interaction during toxoplasmosis.
    • DSA are associated with more graft injury, more fibrosis and upregulation of rejection associated transcripts in subclinical rejection.

      Höfer, Anne; Jonigk, Danny; Hartleben, Björn; Verboom, Murielle; Hallensleben, Michael; Hübscher, Stefan G; Manns, Michael P; Jaeckel, Elmar; Taubert, Richard; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Lippincott, Williams & Wilkins , 2019-10-23)
      Background: Subclinical T cell-mediated rejection (subTCMR) is commonly found after liver transplantation and has a good short-term prognosis, even when it is left untreated. Donor-specific antibodies (DSA) are putatively associated with a worse prognosis for recipient and graft after liver transplantation. Methods: To assess the immune regulation in subTCMR grafts, gene expression of 93 transcripts for graft injury, tolerance and immune regulation was analyzed in 77 biopsies with “no histological rejection” (NHR; n=25), “clinical TCMR” (cTMCR; n=16) and subTCMR (n=36). In addition, all available subTCMR biopsies (n=71) were tested for DSA with bead assays. Results: SubTCMR showed heterogeneous and intermediate expression profiles of transcripts that were upregulated in cTCMR. Graft gene expression suggested a lower activation of effector lymphocytes and a higher activation of regulatory T cells in grafts with subTCMR compared to cTCMR.DSA positivity in subTCMR was associated with histological evidence of more severe graft inflammation and fibrosis. This more severe DSA+ associated graft injury in subTCMR was converged with an upregulation of cTCMR associated transcripts. In nonsupervised analysis DSA positive subTCMR mostly clustered together with cTCMR, while DSA negative subTCMR clustered together with NHR. Conclusion: T cell-mediated rejection seem to form a continuum of alloimmune activation. Although subTCMR exhibited less expression of TCMR associated transcript, DSA positivity in subTCMR was associated with an upregulation of rejection associated transcripts. The identification of DSA positive subclinical rejection might help to define patients with more inflammation in the graft and development of fibrosis.
    • Efficacy of rituximab in difficult-to-manage autoimmune hepatitis: Results from the International Autoimmune Hepatitis Group.

      Than, Nwe Ni; Hodson, James; Schmidt-Martin, Daniel; Taubert, Richard; Wawman, Rebecca E; Botter, Meemee; Gautam, Nishant; Bock, Kilian; Jones, Rebecca; Appanna, Gautham D; et al. (Elsevier, 2019-12-01)
      Twenty-two patients with type-1 AIH were included, with a median age of 40 years at diagnosis (range 19-79); 15/22 (68%) were female and 18/22 (82%) were Caucasian. The median period from diagnosis to the end of follow-up in these patients was 11 years (range 3-28). Values of alanine aminotransferase, aspartate aminotransferase and albumin improved significantly following rituximab therapy, and were sustained for up to 2 years (all p ≪0.001). Prednisolone doses were significantly reduced by 12 months post-treatment (p = 0.003), with 13/21 (62%) patients having a dose reduction. Over a median post-treatment follow-up period of 6 years (range 1-10), 5 patients developed AIH flares at a median of 22 months post-treatment, giving an estimated 71% freedom from AIH flare at 2 years. Four of these patients received a second course of treatment, of whom 2 had subsequent further flares. No serious adverse events attributable to rituximab were recorded.
    • An endothelial cell line infected by Kaposi's sarcoma-associated herpes virus (KSHV) allows the investigation of Kaposi's sarcoma and the validation of novel viral inhibitors in vitro and in vivo.

      Dubich, Tatyana; Lieske, Anna; Santag, Susann; Beauclair, Guillaume; Rückert, Jessica; Herrmann, Jennifer; Gorges, Jan; Büsche, Guntram; Kazmaier, Uli; Hauser, Hansjörg; et al. (2019-01-04)
      Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), a tumor of endothelial origin predominantly affecting immunosuppressed individuals. Up to date, vaccines and targeted therapies are not available. Screening and identification of anti-viral compounds are compromised by the lack of scalable cell culture systems reflecting properties of virus-transformed cells in patients. Further, the strict specificity of the virus for humans limits the development of in vivo models. In this study, we exploited a conditionally immortalized human endothelial cell line for establishment of in vitro 2D and 3D KSHV latency models and the generation of KS-like xenograft tumors in mice. Importantly, the invasive properties and tumor formation could be completely reverted by purging KSHV from the cells, confirming that tumor formation is dependent on the continued presence of KSHV, rather than being a consequence of irreversible transformation of the infected cells. Upon testing a library of 260 natural metabolites, we selected the compounds that induced viral loss or reduced the invasiveness of infected cells in 2D and 3D endothelial cell culture systems. The efficacy of selected compounds against KSHV-induced tumor formation was verified in the xenograft model. Together, this study shows that the combined use of anti-viral and anti-tumor assays based on the same cell line is predictive for tumor reduction in vivo and therefore allows faithful selection of novel drug candidates against Kaposi's sarcoma. KEY MESSAGES: Novel 2D, 3D, and xenograft mouse models mimic the consequences of KSHV infection. KSHV-induced tumorigenesis can be reverted upon purging the cells from the virus. A 3D invasiveness assay is predictive for tumor reduction in vivo. Chondramid B, epothilone B, and pretubulysin D diminish KS-like lesions in vivo.
    • Enteric Murine Ganglionitis Induced by Autoimmune CD8 T Cells Mimics Human Gastrointestinal Dysmotility.

      Sanchez-Ruiz, Monica; Brunn, Anna; Montesinos-Rongen, Manuel; Rudroff, Claudia; Hartmann, Melanie; Schlüter, Dirk; Pfitzer, Gabriele; Deckert, Martina; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2018-12-27)
      Inflammatory bowel diseases frequently cause gastrointestinal dysmotility, suggesting that they may also affect the enteric nervous system. So far, the precise mechanisms that lead to gastrointestinal dysmotility in inflammatory bowel diseases have not been elucidated. To determine the effect of CD8 T cells on gastrointestinal motility, transgenic mice expressing ovalbumin on enteric neurons were generated. In these mice, adoptive transfer of ovalbumin-specific OT-I CD8 T cells induced severe enteric ganglionitis. CD8 T cells homed to submucosal and myenteric plexus neurons, 60% of which were lost, clinically resulting in severely impaired gastrointestinal transition. Anti-interferon-γ treatment rescued neurons by preventing their up-regulation of major histocompatibility complex class I antigen, thus preserving gut motility. These preclinical murine data translated well into human gastrointestinal dysmotility. In a series of 30 colonic biopsy specimens from patients with gastrointestinal dysmotility, CD8 T cell-mediated ganglionitis was detected that was followed by severe loss of enteric neurons (74.8%). Together, the preclinical and clinical data support the concept that autoimmune CD8 T cells play an important pathogenetic role in gastrointestinal dysmotility and may destroy enteric neurons.