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  • Heparin: role in protein purification and substitution with animal-component free material.

    Bolten, Svenja Nicolin; Rinas, Ursula; Scheper, Thomas; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2018-10-01)
    Heparin is a highly sulfated polysaccharide which belongs to the family of glycosaminoglycans. It is involved in various important biological activities. The major biological purpose is the inhibition of the coagulation cascade to maintain the blood flow in the vasculature. These properties are employed in several therapeutic drugs. Heparin's activities are associated with its interaction to various proteins. To date, the structural heparin-protein interactions are not completely understood. This review gives a general overview of specific patterns and functional groups which are involved in the heparin-protein binding. An understanding of the heparin-protein interactions at the molecular level is not only advantageous in the therapeutic application but also in biotechnological application of heparin for downstreaming. This review focuses on the heparin affinity chromatography. Diverse recombinant proteins can be successfully purified by this method. While effective, it is disadvantageous that heparin is an animal-derived material. Animal-based components carry the risk of contamination. Therefore, they are liable to strict quality controls and the validation of effective good manufacturing practice (GMP) implementation. Hence, adequate alternatives to animal-derived components are needed. This review examines strategies to avoid these disadvantages. Thereby, alternatives for the provision of heparin such as chemical synthesized heparin, chemoenzymatic heparin, and bioengineered heparin are discussed. Moreover, the usage of other chromatographic systems mimetic the heparin effect is reviewed.
  • Groundwater, soil and compost, as possible sources of virulent and antibiotic-resistant Pseudomonas aeruginosa.

    Kaszab, Edit; Radó, Júlia; Kriszt, Balázs; Pászti, Judit; Lesinszki, Virág; Szabó, Ádám; Tóth, Gergő; Khaledi, Ariane; Szoboszlay, Sándor; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Taylor & Francis, 2019-11-18)
    Pseudomonas aeruginosa is a major public health concern all around the world. In the frame of this work, a set of diverse environmental P. aeruginosa isolates with various antibiotic resistance profiles were examined in a Galleria mellonella virulence model. Motility, serotypes, virulence factors and biofilm-forming ability were also examined. Molecular types were determined by pulsed-field gel electrophoresis (PFGE). Based on our results, the majority of environmental isolates were virulent in the G. mellonella test and twitching showed a positive correlation with mortality. Resistance against several antibiotic agents such as Imipenem correlated with a lower virulence in the applied G. mellonella model. PFGE revealed that five examined environmental isolates were closely related to clinically detected pulsed-field types. Our study demonstrated that industrial wastewater effluents, composts, and hydrocarbon-contaminated sites should be considered as hot spots of high-risk clones of P. aeruginosa.
  • The role of epigenetics in the development of childhood asthma.

    Qi, Cancan; Xu, Cheng-Jian; Koppelman, Gerard H; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (2019-11-10)
    Introduction: The development of childhood asthma is caused by a combination of genetic factors and environmental exposures. Epigenetics describes mechanisms of (heritable) regulation of gene expression that occur without changes in DNA sequence. Epigenetics is strongly related to aging, is cell-type specific, and includes DNA methylation, noncoding RNAs, and histone modifications.Areas covered: This review summarizes recent epigenetic studies of childhood asthma in humans, which mostly involve studies of DNA methylation published in the recent five years. Environmental exposures, in particular cigarette smoking, have significant impact on epigenetic changes, but few of these epigenetic signals are also associated with asthma. Several asthma-associated genetic variants relate to DNA methylation. Epigenetic signals can be better understood by studying their correlation with gene expression, which revealed higher presence and activation of blood eosinophils in asthma. Strong associations of nasal methylation signatures and atopic asthma were identified, which were replicable across different populations.Expert commentary: Epigenetic markers have been strongly associated with asthma, and might serve as biomarker of asthma. The causal and longitudinal relationships between epigenetics and disease, and between environmental exposures and epigenetic changes need to be further investigated. Efforts should be made to understand cell-type-specific epigenetic mechanisms in asthma.
  • Quantitation of large, middle and small hepatitis B surface proteins in HBeAg-positive patients treated with peginterferon alfa-2a.

    Rinker, Franziska; Bremer, Corinna M; Schröder, Kathrin; Wiegand, Steffen B; Bremer, Birgit; Manns, Michael P; Kraft, Anke R; Wedemeyer, Heiner; Yang, Lei; Pavlovic, Vedran; et al. (Wiley, 2019-11-13)
    BACKGROUND & AIMS: Hepatitis B virus (HBV) contains three viral surface proteins, large, middle and small hepatitis B surface protein (LHBs, MHBs, SHBs). Proportions of LHBs and MHBs are lower in patients with inactive versus active chronic infection. Interferon alfa may convert HBeAg-positive chronic hepatitis B (CHB) to an inactive carrier state, but prediction of sustained response is unsatisfactory. The aim of this study was to test the hypothesis that quantification of MHBs and LHBs may allow for a better prognosis of therapeutic response than total hepatitis B surface antigen (HBsAg) concentration. METHODS: HBs proteins were measured before and during peginterferon alfa-2a therapy in serum from 127 Asian patients with HBeAg-positive CHB. Sustained response was defined as hepatitis B e antigen (HBeAg) seroconversion 24 weeks post-treatment. RESULTS: Mean total HBs levels were significantly lower in responders versus nonresponders at all time points (P<.05) and decreased steadily during the initial 24 weeks' treatment (by 1.16 versus 0.86 ng/mL in responders/nonresponders, respectively) with unchanged relative proportions. Genotype B had a twofold higher proportion of LHBs than genotype C (13% versus 6%). HBV DNA, HBeAg, HBsAg, and HBs protein levels predicted response equally well but not optimally (area under the ROC curve values >0.70). CONCLUSIONS: HBs proteins levels differ by HBV genotype. However, quantification of HBs proteins has no advantage over the already established HBsAg assays to predict response to peginterferon alfa-2a therapy in HBeAg-positive patients.
  • Unexpected roles for ADH1 and SORD in catalyzing the final step of erythritol biosynthesis.

    Schlicker, Lisa; Szebenyi, Doletha M E; Ortiz, Semira R; Heinz, Alexander; Hiller, Karsten; Field, Martha S; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (American Society for Biochemistry and Molecular Biology, 2019-11-01)
    The low-calorie sweetener erythritol is endogenously produced from glucose through the pentose phosphate pathway in humans. Erythritol is of medical interest because elevated plasma levels of this polyol are predictive for visceral adiposity gain and development of type 2 diabetes. However, the mechanisms behind these associations remain unknown because the erythritol biosynthesis pathway, particularly the enzyme catalyzing the final step of erythritol synthesis (reduction of erythrose to erythritol), is not characterized. In this study, we purified two enzymes from rabbit liver capable of catalyzing the conversion of erythrose to erythritol: alcohol dehydrogenase 1 (ADH1) and sorbitol dehydrogenase (SORD). Both recombinant human ADH1 and SORD reduce erythrose to erythritol, using NADPH as a co-factor, and cell culture studies indicate that this activity is primarily NADPH-dependent. We found that ADH1 variants vary markedly in both their affinity for erythrose and their catalytic capacity (turnover number). Interestingly, the recombinant protein produced from the ADH1B2 variant, common in Asian populations, is not active when NADPH is used as a co-factor in vitro We also confirmed SORD contributes to intracellular erythritol production in human A549 lung cancer cells, where ADH1 is minimally expressed. In summary, human ADH1 and SORD catalyze the conversion of erythrose to erythritol, pointing to novel roles for two dehydrogenase proteins in human glucose metabolism that may contribute to individual responses to diet. Proteomics data are available via ProteomeXchange with identifier PXD015178.
  • 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.
  • Functional design of pH-responsive folate-targeted polymer-coated gold nanoparticles for drug delivery and in vivo therapy in breast cancer

    Mahalunkar, Sneha; Yadav, Amit Singh; Gorain, Mahadeo; Pawar, Vinay; Braathen, Ranveig; Weiss, Siegfried; Bogen, Bjarne; Gosavi, Suresh W.; Kundu, Gopal C.; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2019-01-01)
    Background: Curcumin has been widely used owing to its various medicinal properties including antitumor effects. However, its clinical application is limited by its instability, poor solubility and low bioavailability. Folic acid (FA)-functionalized nanoformulations may enhance the sustained release of an anticancer drug (curcumin) by tumor-specific targeting to improve therapeutic benefit. This study aims to design a nanoconjugate (NC) comprised of folate–curcumin-loaded gold–polyvinylpyrrolidone nanoparticles (FA–CurAu-PVP NPs) for targeted delivery in breast cancer model systems. Methods: We developed curcumin-loaded FA-functionalized Au-PVP NCs by layer-by-layer assembly. The folic acid–curcumin Au-PVP NCs (FA–CurAu-PVP NCs) were characterized by ultraviolet–visible spectra, Fourier transform infrared spectroscopy, X-ray powder diffraction and thermogravimetric analysis. In vitro anticancer and antimigratory effects of NCs were examined by performing MTT and wound migration assays. The in vivo antitumor efficacy of NCs was investigated using a preclinical breast cancer orthotopic mouse model. Results: Curcumin (40 µg/mL) was loaded along with conjugation of folate onto Au-PVP NPs to form FA–CurAu-PVP NCs. The size and charge of the NCs were increased gradually through layer-by-layer assembly and showed 80% release of curcumin at acidic pH. The NC did not show aggregation when incubated with human serum and mimicked an intrinsic peroxidase-like property in the presence of 3,3ʹ,5,5ʹ-tetramethylbenzidine substrate. The MTT data using these NCs showed efficient anticancer activity at lower doses in estrogen/ progesterone receptor (ER/PR)-negative cells compared with ER/PR-positive cells. Furthermore, the NCs did not show cytotoxicity at the investigated concentration in human breast epithelial and mouse fibroblast cell lines. They showed inhibitory effects on cell migration and high antitumor efficacy in in vivo analysis. Conclusion: These results suggest that folate-based tumor targeting using CurAu-PVP NCs is a promising approach for tumor-specific therapy of breast cancer without harming normal cells.
  • Parasites in brains of wild rodents (Arvicolinae and Murinae) in the city of Leipzig, Germany

    Waindok, Patrick; Özbakış-Beceriklisoy, Gökben; Janecek-Erfurth, Elisabeth; Springer, Andrea; Pfeffer, Martin; Leschnik, Michael; Strube, Christina; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Elsevier, 2019-12-01)
    Small rodents serve as intermediate or paratenic hosts for a variety of parasites and may participate in thetransmission of these parasites into synanthropic cycles. Parasites with neuroinvasive stages, such asToxoplasmagondiiorToxocara canis, can cause detrimental damage in the brain of intermediate or paratenic hosts.Therefore, the occurrence of neuroinvasive parasite stages was evaluated in brains of wild rodents captured inthe city of Leipzig, Germany. In addition, a few specimens from the cities of Hanover, Germany, and Vienna,Austria were included, resulting in a total of 716 rodents collected between 2011 and 2016. Brains were in-vestigated for parasitic stages by microscopic examination of native tissue, artificially digested tissue as well asGiemsa-stained digestion solution to verify positive results. Infective stages of zoonotic ascarids or other hel-minths were not detected in any sample, while coccidian cysts were found in 10.1% (95% CI: 7.9–12.5%; 72/716) of examined brains. The most abundant rodent species in the study was the bank vole (Myodes glareolus;Arvicolinae), showing an infection rate with cerebral cysts of 13.9% (95% CI: 11.0–17.8%; 62/445), while 2.7%(95% CI: 1.0–5.8%; 6/222) of yellow-necked mice (Apodemusflavicollis; Murinae) were infected. Generalizedlinear modelling revealed a statistically significant difference in prevalence betweenM. glareolusandA.flavi-collis, significant local differences as well as an effect of increasing body mass on cyst prevalence. Coccidian cystswere differentiated by amplification of the18S rRNAgene and subsequent sequencing. The majority of iden-tifiable cysts (97.9%) were determined asFrenkelia glareoli, a coccidian species mainly circulating betweenM.glareolusas intermediate and buzzards (Buteospp.) as definitive hosts. The zoonotic pathogenToxoplasma gondiiwas confirmed in oneM. glareolusoriginating from the city of Leipzig. Overall, it can be concluded that neu-roinvasion of zoonotic parasites seems to be rare inM. glareolusandA.flavicollis.
  • Soluble immune markers in the different phases of chronic hepatitis B virus infection

    Wiegand, Steffen B.; Beggel, Bastian; Wranke, Anika; Aliabadi, Elmira; Jaroszewicz, Jerzy; Xu, Cheng Jian; Li, Yang; Manns, Michael P.; Lengauer, Thomas; Wedemeyer, Heiner; et al. (Nature publishing group, 2019-10-01)
    Chronic hepatitis B virus (HBV) infection may follow four different consecutive phases, which are defined by virology as well as biochemical markers and differ in terms of prognosis and need for antiviral treatment. Currently, host responses reflected by immune markers are not considered in this definition. We aimed to study soluble immune markers and their distribution in different phases of chronic HBV infection. In this cross-sectional retrospective study, we investigated a panel of 14 soluble immune markers (SIM) including CXCL10 in 333 patients with chronic HBV infection. In a small cohort of HBeAg positive patients we analyzed SIM before and after HBeAg seroconversion and compared seroconverters to patients with unknown outcome. Significant differences were documented in the levels of several SIM between the four phases of chronic HBV infection. The most pronounced difference among all investigated SIM was observed for CXCL10 concentrations with highest levels in patients with hepatitis. TGF-β and IL-17 revealed different levels between HBeAg negative patients. HBeAg positive patients with HBeAg seroconversion presented higher amounts of IL-12 before seroconversion compared to HBeAg positive patients with unknown follow up. SIM such as CXCL10 but also IL-12, TGF-β and IL-17 may be useful markers to further characterize the phase of chronic HBV infection.
  • Hepatitis E Virus (HEV)-Specific T Cell Receptor Cross-Recognition: Implications for Immunotherapy.

    Soon, Chai Fen; Zhang, Shihong; Suneetha, Pothakamuri Venkata; Antunes, Dinler Amaral; Manns, Michael Peter; Raha, Solaiman; Schultze-Florey, Christian; Prinz, Immo; Wedemeyer, Heiner; Sällberg Chen, Margaret; et al. (Frontiers, 2019-01-01)
    T cell immunotherapy is a concept developed for the treatment of cancer and infectious diseases, based on cytotoxic T lymphocytes to target tumor- or pathogen-specific antigens. Antigen-specificity of the T cell receptors (TCRs) is an important selection criterion in the developmental design of immunotherapy. However, off-target specificity is a possible autoimmunity concern if the engineered antigen-specific T cells are cross-reacting to self-peptides in-vivo. In our recent work, we identified several hepatitis E virus (HEV)-specific TCRs as potential candidates to be developed into T cell therapy to treat chronic hepatitis E. One of the identified TCRs, targeting a HLA-A2-restricted epitope at the RNA-dependent RNA polymerase (HEV-1527: LLWNTVWNM), possessed a unique multiple glycine motif in the TCR-β CDR3, which might be a factor inducing cross-reactivity. The aim of our study was to explore if this TCR could cross-recognize self-peptides to underlay autoimmunity. Indeed, we found that this HEV-1527-specific TCR could also cross-recognize an apoptosis-related epitope, Nonmuscle Myosin Heavy Chain 9 (MYH9-478: QLFNHTMFI). While this TCR had dual specificities to both viral epitope and a self-antigen by double Dextramer binding, it was selectively functional against HEV-1527 but not activated against MYH9-478. The consecutive glycine motif in β chain may be the reason promoting TCR binding promiscuity to recognize a secondary target, thereby facilitating cross-recognition. In conclusion, candidate TCRs for immunotherapy development should be screened for autoimmune potential, especially when the TCRs exhibit unique sequence pattern.
  • Commonly setting biological standards in rare diseases

    O’Connor, Daniel J.; Buckland, Jenny; Almond, Neil; Boyle, Jennifer; Coxon, Carmen; Gaki, Eleni; Martin, Javier; Mattiuzzo, Giada; Metcalfe, Clive; Page, Mark; et al. (Taylor& Francis, 2019-01-01)
    Introduction: Standardization is important across the life cycle of medicinal products, supporting the diagnosis, treatment, and prevention of a wide range of diseases. For rare diseases, standardization is even more important, as patient groups are small, presenting significant challenges in the design, conduct, analysis, and interpretation of clinical studies. It is here that standardization institutions, including the UK’s National Institute for Biological Standards and Control (NIBSC), can have a key role. Areas covered: A considerable proportion of NIBSC’s work supports the better understanding, diagnosis, treatment, and prevention of rare diseases. NIBSC is also part of the UK’s Medicines and Healthcare products Regulatory Agency (MHRA), creating an agency that is uniquely placed to combine scientific and regulatory expertize for the benefit of public health. This review provides an overview of NIBSC’s work in rare diseases and highlights the positive impact of the work of standardization institutions in this field. Expert opinion: Standardization in product development is key for patients with rare diseases. The work of standardization institutions is increasingly being recognized as crucial for supporting scientific and clinical advancements, and early and collaborative interactions can provide drug developers with the necessary expertize, when standards matter most.
  • Targeting Antitumoral Proteins to Breast Cancer by Local Administration of Functional Inclusion Bodies

    Pesarrodona, Mireia; Jauset, Toni; Díaz-Riascos, Zamira V.; Sánchez-Chardi, Alejandro; Beaulieu, Marie Eve; Seras-Franzoso, Joaquin; Sánchez-García, Laura; Baltà-Foix, Ricardo; Mancilla, Sandra; Fernández, Yolanda; et al. (Wiley-VCH, 2019-01-01)
    Two structurally and functionally unrelated proteins, namely Omomyc and p31, are engineered as CD44-targeted inclusion bodies produced in recombinant bacteria. In this unusual particulate form, both types of protein materials selectively penetrate and kill CD44+ tumor cells in culture, and upon local administration, promote destruction of tumoral tissue in orthotropic mouse models of human breast cancer. These findings support the concept of bacterial inclusion bodies as versatile protein materials suitable for application in chronic diseases that, like cancer, can benefit from a local slow release of therapeutic proteins
  • 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.
  • A combined in silico and in vitro study on mouse Serpina1a antitrypsin-deficiency mutants.

    Eggenschwiler, Reto; Patronov, Atanas; Hegermann, Jan; Fráguas-Eggenschwiler, Mariane; Wu, Guangming; Cortnumme, Leon; Ochs, Matthias; Antes, Iris; Cantz, Tobias; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Springer-Nature, 2019-05-16)
    Certain point-mutations in the human SERPINA1-gene can cause severe α1-antitrypsin-deficiency (A1AT-D). Affected individuals can suffer from loss-of-function lung-disease and from gain-of-function liver-disease phenotypes. However, age of onset and severity of clinical appearance is heterogeneous amongst carriers, suggesting involvement of additional genetic and environmental factors. The generation of authentic A1AT-D mouse-models has been hampered by the complexity of the mouse Serpina1-gene locus and a model with concurrent lung and liver-disease is still missing. Here, we investigate point-mutations in the mouse Serpina1a antitrypsin-orthologue, which are homolog-equivalent to ones known to cause severe A1AT-D in human. We combine in silico and in vitro methods and we find that analyzed mutations do introduce potential disease-causing properties into Serpina1a. Finally, we show that introduction of the King’s-mutation causes inactivation of neutrophil elastase inhibitory-function in both, mouse and human antitrypsin, while the mouse Z-mutant retains activity. This work paves the path to generation of better A1AT-D mouse-models.
  • Ten-year efficacy and safety of tenofovir disoproxil fumarate treatment for chronic hepatitis B virus infection.

    Marcellin, Patrick; Wong, Dave; Sievert, William; Buggisch, Peter; Petersen, Jörg; Flisiak, Robert; Manns, Michael; Kaita, Kelly; Krastev, Zahari; Lee, Samuel S; et al. (Wiley-Blackwell, 2019-05-28)
    Background & Aims Tenofovir disoproxil fumarate (TDF) is a first‐line treatment for chronic hepatitis B (CHB). We aimed to describe the efficacy and safety profiles of TDF treatment for up to 10 years in a well‐described cohort of CHB patients. Methods Hepatitis B e antigen (HBeAg)‐negative and HBeAg‐positive patients from two randomised, double‐blind trials (ClinicalTrials. gov: NCT00117676 and NCT00116805) completed 48 weeks of randomised treatment with TDF or adefovir dipivoxil. A subset of these patients was then eligible to receive open‐label TDF treatment for up to 10 years. At Year 10, patients were assessed for virological suppression, alanine aminotransferase (ALT) normalisation, serological response, safety, and tolerability. Results Of 641 randomised and treated patients, 585 (91%) entered the open‐label extension phase with 203 (32%) patients completing Year 10 of the study. At Year 10, 118/118 (100%) of HBeAg‐negative patients and 78/80 (98%) of HBeAg‐positive patients with available data achieved hepatitis B virus (HBV) DNA <69 IU/mL, while 88/106 (83%) and 60/77 (78%) patients achieved ALT normalisation, respectively. Of the 23 patients with HBeAg status available at Year 10, 12 (52%) and six (27%) experienced HBeAg loss and seroconversion, respectively. No resistance to TDF was documented up to Year 10. In the period between Year 8 and Year 10, the safety profile of TDF was similar to previous reports, with few patients experiencing renal‐ or bone‐related adverse events. Conclusions Over 10 years, TDF had a favourable safety profile, was well tolerated, and resulted in continued maintenance of virological suppression with no documented resistance.
  • TLR7 Controls VSV Replication in CD169 SCS Macrophages and Associated Viral Neuroinvasion.

    Solmaz, Gülhas; Puttur, Franz; Francozo, Marcela; Lindenberg, Marc; Guderian, Melanie; Swallow, Maxine; Duhan, Vikas; Khairnar, Vishal; Kalinke, Ulrich; Ludewig, Burkhard; et al. (Frontiers, 2019-01-01)
    Vesicular stomatitis virus (VSV) is an insect-transmitted rhabdovirus that is neurovirulent in mice. Upon peripheral VSV infection, CD169+ subcapsular sinus (SCS) macrophages capture VSV in the lymph, support viral replication, and prevent CNS neuroinvasion. To date, the precise mechanisms controlling VSV infection in SCS macrophages remain incompletely understood. Here, we show that Toll-like receptor-7 (TLR7), the main sensing receptor for VSV, is central in controlling lymph-borne VSV infection. Following VSV skin infection, TLR7-/- mice display significantly less VSV titers in the draining lymph nodes (dLN) and viral replication is attenuated in SCS macrophages. In contrast to effects of TLR7 in impeding VSV replication in the dLN, TLR7-/- mice present elevated viral load in the brain and spinal cord highlighting their susceptibility to VSV neuroinvasion. By generating novel TLR7 floxed mice, we interrogate the impact of cell-specific TLR7 function in anti-viral immunity after VSV skin infection. Our data suggests that TLR7 signaling in SCS macrophages supports VSV replication in these cells, increasing LN infection and may account for the delayed onset of VSV-induced neurovirulence observed in TLR7-/- mice. Overall, we identify TLR7 as a novel and essential host factor that critically controls anti-viral immunity to VSV. Furthermore, the novel mouse model generated in our study will be of valuable importance to shed light on cell-intrinsic TLR7 biology in future studies.
  • TGFβ-activation by dendritic cells drives Th17 induction and intestinal contractility and augments the expulsion of the parasite Trichinella spiralis in mice.

    Steel, Nicola; Faniyi, Aduragbemi A; Rahman, Sayema; Swietlik, Stefanie; Czajkowska, Beata I; Chan, Bethany T; Hardgrave, Alexander; Steel, Anthony; Sparwasser, Tim D; Assas, Mushref B; et al. (PLOS, 2019-01-01)
    Helminths are highly prevalent metazoan parasites that infect over a billion of the world’s population. Hosts have evolved numerous mechanisms to drive the expulsion of these parasites via Th2-driven immunity, but these responses must be tightly controlled to prevent equally devastating immunopathology. However, mechanisms that regulate this balance are still unclear. Here we show that the vigorous Th2 immune response driven by the small intestinal helminth Trichinella spiralis, is associated with increased TGFβ signalling responses in CD4+ T-cells. Mechanistically, enhanced TGFβ signalling in CD4+ T-cells is dependent on dendritic cell-mediated TGFβ activation which requires expression of the integrin αvβ8. Importantly, mice lacking integrin αvβ8 on DCs had a delayed ability to expel a T. spiralis infection, indicating an important functional role for integrin αvβ8-mediated TGFβ activation in promoting parasite expulsion. In addition to maintaining regulatory T-cell responses, the CD4+ T-cell signalling of this pleiotropic cytokine induces a Th17 response which is crucial in promoting the intestinal muscle hypercontractility that drives worm expulsion. Collectively, these results provide novel insights into intestinal helminth expulsion beyond that of classical Th2 driven immunity, and highlight the importance of IL-17 in intestinal contraction which may aid therapeutics to numerous diseases of the intestine.
  • miR-181a/b-1 controls thymic selection of Treg cells and tunes their suppressive capacity.

    Łyszkiewicz, Marcin; Winter, Samantha J; Witzlau, Katrin; Föhse, Lisa; Brownlie, Rebecca; Puchałka, Jacek; Verheyden, Nikita A; Kunze-Schumacher, Heike; Imelmann, Esther; Blume, Jonas; et al. (PLOS, 2019-03-01)
    The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1-deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte-associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function.
  • A Listeria monocytogenes ST2 clone lacking chitinase ChiB from an outbreak of non-invasive gastroenteritis.

    Halbedel, Sven; Prager, Rita; Banerji, Sangeeta; Kleta, Sylvia; Trost, Eva; Nishanth, Gopala; Alles, Georg; Hölzel, Christina; Schlesiger, Friederike; Pietzka, Ariane; et al. (Springer Nature, 2019-01-01)
    An outbreak with a remarkable Listeria monocytogenes clone causing 163 cases of non-invasive listeriosis occurred in Germany in 2015. Core genome multi locus sequence typing grouped non-invasive outbreak isolates and isolates obtained from related food samples into a single cluster, but clearly separated genetically close isolates obtained from invasive listeriosis cases. A comparative genomic approach identified a premature stop codon in the chiB gene, encoding one of the two L. monocytogenes chitinases, which clustered with disease outcome. Correction of this premature stop codon in one representative gastroenteritis outbreak isolate restored chitinase production, but effects in infection experiments were not found. While the exact role of chitinases in virulence of L. monocytogenes is still not fully understood, our results now clearly show that ChiB-derived activity is not required to establish L. monocytogenes gastroenteritis in humans. This limits a possible role of ChiB in human listeriosis to later steps of the infection.
  • Virulence of Agrobacterium tumefaciens requires lipid homeostasis mediated by the lysyl-phosphatidylglycerol hydrolase AcvB.

    Groenewold, Maike K; Hebecker, Stefanie; Fritz, Christiane; Czolkoss, Simon; Wiesselmann, Milan; Heinz, Dirk W; Jahn, Dieter; Narberhaus, Franz; Aktas, Meriyem; Moser, Jürgen; et al. (Wiley-Blackwell, 2019-01-01)
    Agrobacterium tumefaciens transfers oncogenic T-DNA via the type IV secretion system (T4SS) into plants causing tumor formation. The acvB gene encodes a virulence factor of unknown function required for plant transformation. Here we specify AcvB as a periplasmic lysyl-phosphatidylglycerol (L-PG) hydrolase, which modulates L-PG homeostasis. Through functional characterization of recombinant AcvB variants, we showed that the C-terminal domain of AcvB (residues 232-456) is sufficient for full enzymatic activity and defined key residues for catalysis. Absence of the hydrolase resulted in ~10-fold increase in L-PG in Agrobacterium membranes and abolished T-DNA transfer and tumor formation. Overproduction of the L-PG synthase gene (lpiA) in wild-type A. tumefaciens resulted in a similar increase in the L-PG content (~7-fold) and a virulence defect even in the presence of intact AcvB. These results suggest that elevated L-PG amounts (either by overproduction of the synthase or absence of the hydrolase) are responsible for the virulence phenotype. Gradually increasing the L-PG content by complementation with different acvB variants revealed that cellular L-PG levels above 3% of total phospholipids interfere with T-DNA transfer. Cumulatively, this study identified AcvB as a novel virulence factor required for membrane lipid homeostasis and T-DNA transfer.

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