2024-03-29T07:37:45Zhttp://repository.helmholtz-hzi.de/oai/requestoai:repository.helmholtz-hzi.de:10033/1189662019-08-30T11:32:41Zcom_10033_620652col_10033_620672
2011-01-07T15:26:15Z
urn:hdl:10033/118966
Toxin-antitoxin based transgene expression in mammalian cells.
Nehlsen, K
Herrmann, S
Zauers, J
Hauser, Hansjoerg
Wirth, D
Helmholtz Centre for Infection Research, Braunschweig, Germany.
Long-term, recombinant gene expression in mammalian cells depends on the nature of the transgene integration site and its inherent properties to modulate transcription (epigenetic effects). Here we describe a method by which high transgene expression is achieved and stabilized in extensively proliferating cultures. The method is based on strict co-expression of the transgene with an antitoxin in cells that express the respective toxin. Since the strength of antitoxin expression correlates with an advantage for cell growth, the cells with strong antitoxin expression are enriched over time in cultures of heterogeneous cells. This principle was applied to CHO cell lines that conditionally express the toxin kid and that are transduced to co-express the antitoxin kis together with different transgenes of interest. Cultivation of pools of transfectants that express the toxin steadily increase their transgene expression within several weeks to reach a maximum that is up to 120-fold over the initial status. In contrast, average transgene expression drops in the absence of toxin expression. Together, we show that cells conditionally expressing kid can be employed to create overexpressing cells by a simple coupling of kis to the transgene of interest, without further manipulation and in absence of selectable drugs.
2011-01-07T15:26:15Z
2011-01-07T15:26:15Z
2010-03
Article
Toxin-antitoxin based transgene expression in mammalian cells. 2010, 38 (5):e32 Nucleic Acids Res.
1362-4962
20007149
10.1093/nar/gkp1140
http://hdl.handle.net/10033/118966
Nucleic acids research
en
oai:repository.helmholtz-hzi.de:10033/1212722019-08-30T11:35:39Zcom_10033_620652col_10033_620672
2011-02-04T15:20:33Z
urn:hdl:10033/121272
Strict control of transgene expression in a mouse model for sensitive biological applications based on RMCE compatible ES cells.
Sandhu, U
Cebula, M
Behme, S
Riemer, P
Wodarczyk, C
Metzger, D
Reimann, J
Schirmbeck, R
Hauser, H
Wirth, D
Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany.
Recombinant mouse strains that harbor tightly controlled transgene expression proved to be indispensible tools to elucidate gene function. Different strategies have been employed to achieve controlled induction of the transgene. However, many models are accompanied by a considerable level of basal expression in the non-induced state. Thereby, applications that request tight control of transgene expression, such as the expression of toxic genes and the investigation of immune response to neo antigens are excluded. We developed a new Cre/loxP-based strategy to achieve strict control of transgene expression. This strategy was combined with RMCE (recombinase mediated cassette exchange) that facilitates the targeting of genes into a tagged site in ES cells. The tightness of regulation was confirmed using luciferase as a reporter. The transgene was induced upon breeding these mice to effector animals harboring either the ubiquitous (ROSA26) or liver-specific (Albumin) expression of CreER(T2), and subsequent feeding with Tamoxifen. Making use of RMCE, luciferase was replaced by Ovalbumin antigen. Mice generated from these ES cells were mated with mice expressing liver-specific CreER(T2). The transgenic mice were examined for the establishment of an immune response. They were fully competent to establish an immune response upon hepatocyte specific OVA antigen expression as indicated by a massive liver damage upon Tamoxifen treatment and did not show OVA tolerance. Together, this proves that this strategy supports strict control of transgenes that is even compatible with highly sensitive biological readouts.
2011-02-04T15:20:33Z
2011-02-04T15:20:33Z
2011-01-01
Article
Strict control of transgene expression in a mouse model for sensitive biological applications based on RMCE compatible ES cells. 2011, 39 (1):e1 Nucleic Acids Res.
1362-4962
20935052
10.1093/nar/gkq868
http://hdl.handle.net/10033/121272
Nucleic acids research
en
oai:repository.helmholtz-hzi.de:10033/2369952019-08-30T11:24:31Zcom_10033_620652col_10033_620672
2012-08-02T09:04:32Z
urn:hdl:10033/236995
Lentivirus production is influenced by SV40 large T-antigen and chromosomal integration of the vector in HEK293 cells.
Gama-Norton, Leonor
Botezatu, Lacramioara
Herrmann, Sabrina
Schweizer, Matthias
Alves, Paula Marques
Hauser, Hansjoerg
Wirth, Dagmar
Helmholtz Centre for Infection Research, Braunschweig, Germany .
Currently, lentiviral vectors for research and gene therapy are produced from 293-T cells that are transiently transfected with plasmids encoding the vector and helper functions. However, transiently transfected vectors as well as the presence of SV40 virus large T-antigen (T-Ag) cause serious technical and safety considerations. We aimed to exploit single copy integration sites in the HEK293 genome supporting lentiviral vector production. We found that lentiviral vectors result in minimal infectious particle production from single copy integrants in HEK293. Moreover, once this cell line harbors single copy integrations of lentiviral vectors, its ability to transiently produce lentiviral vectors becomes strongly impaired. T-Ag has a dramatic effect on virus production. Low levels of constitutive T-Ag expression can overcome the production restriction imposed by integrated lentiviral vectors copies. Interestingly, T-Ag does not exert its role at the level of transcriptional activity of the vector; rather, it seems to impose an indirect effect on the cell thereby enabling lentiviral vector production. Altogether, our study highlights the restrictions for integrated lentiviral vectors that are relevant for the establishment of stable and safe producer cell lines.
2012-08-02T09:04:32Z
2012-08-02T09:04:32Z
2011-10
Article
Lentivirus production is influenced by SV40 large T-antigen and chromosomal integration of the vector in HEK293 cells. 2011, 22 (10):1269-79 Hum. Gene Ther.
1557-7422
21554103
10.1089/hum.2010.143
http://hdl.handle.net/10033/236995
Human gene therapy
en
Archived with thanks to Human gene therapy
oai:repository.helmholtz-hzi.de:10033/2816722019-08-30T11:34:48Zcom_10033_620652col_10033_620672
2013-04-17T14:22:19Z
urn:hdl:10033/281672
The ROSA26-iPSC mouse: a conditional, inducible, and exchangeable resource for studying cellular (De)differentiation.
Haenebalcke, Lieven
Goossens, Steven
Dierickx, Pieterjan
Bartunkova, Sonia
D'Hont, Jinke
Haigh, Katharina
Hochepied, Tino
Wirth, Dagmar
Nagy, Andras
Haigh, Jody J
Vascular Cell Biology Unit, VIB Department for Molecular Biomedical Research, Ghent University, Technologiepark 927, 9052 Zwijnaarde Ghent, Belgium.
Control of cellular (de)differentiation in a temporal, cell-specific, and exchangeable manner is of paramount importance in the field of reprogramming. Here, we have generated and characterized a mouse strain that allows iPSC generation through the Cre/loxP conditional and doxycycline/rtTA-controlled inducible expression of the OSKM reprogramming factors entirely from within the ROSA26 locus. After reprogramming, these factors can be replaced by genes of interest-for example, to enhance lineage-directed differentiation-with the use of a trap-coupled RMCE reaction. We show that, similar to ESCs, Dox-controlled expression of the cardiac transcriptional regulator Mesp1 together with Wnt inhibition enhances the generation of functional cardiomyocytes upon in vitro differentiation of such RMCE-retargeted iPSCs. This ROSA26-iPSC mouse model is therefore an excellent tool for studying both cellular reprogramming and lineage-directed differentiation factors from the same locus and will greatly facilitate the identification and ease of functional characterization of the genetic/epigenetic determinants involved in these complex processes.
2013-04-17T14:22:19Z
2013-04-17T14:22:19Z
2013-02-21
Article
The ROSA26-iPSC mouse: a conditional, inducible, and exchangeable resource for studying cellular (De)differentiation. 2013, 3 (2):335-41 Cell Rep
2211-1247
23395636
10.1016/j.celrep.2013.01.016
http://hdl.handle.net/10033/281672
Cell reports
en
Archived with thanks to Cell reports
oai:repository.helmholtz-hzi.de:10033/2972272019-08-30T11:35:39Zcom_10033_620652col_10033_620672
2013-08-01T14:20:49Z
urn:hdl:10033/297227
An Inducible Transgenic Mouse Model for Immune Mediated Hepatitis Showing Clearance of Antigen Expressing Hepatocytes by CD8+ T Cells.
Cebula, Marcin
Ochel, Aaron
Hillebrand, Upneet
Pils, Marina C
Schirmbeck, Reinhold
Hauser, Hansjörg
Wirth, Dagmar
Model Systems for Infection and Immunity, Helmholtz Centre for Infection Research, Braunschweig, Germany.
The liver has the ability to prime immune responses against neo antigens provided upon infections. However, T cell immunity in liver is uniquely modulated by the complex tolerogenic property of this organ that has to also cope with foreign agents such as endotoxins or food antigens. In this respect, the nature of intrahepatic T cell responses remains to be fully characterized. To gain deeper insight into the mechanisms that regulate the CD8+ T cell responses in the liver, we established a novel OVA_X_CreER(T2) mouse model. Upon tamoxifen administration OVA antigen expression is observed in a fraction of hepatocytes, resulting in a mosaic expression pattern. To elucidate the cross-talk of CD8+ T cells with antigen-expressing hepatocytes, we adoptively transferred K(b)/OVA257-264-specific OT-I T cells to OVA_X_CreER(T2) mice or generated triple transgenic OVA_X CreER(T2)_X_OT-I mice. OT-I T cells become activated in OVA_X_CreER(T2) mice and induce an acute and transient hepatitis accompanied by liver damage. In OVA_X_CreER(T2)_X_OT-I mice, OVA induction triggers an OT-I T cell mediated, fulminant hepatitis resulting in 50% mortality. Surviving mice manifest a long lasting hepatitis, and recover after 9 weeks. In these experimental settings, recovery from hepatitis correlates with a complete loss of OVA expression indicating efficient clearance of the antigen-expressing hepatocytes. Moreover, a relapse of hepatitis can be induced upon re-induction of cured OVA_X_CreER(T2)_X_OT-I mice indicating absence of tolerogenic mechanisms. This pathogen-free, conditional mouse model has the advantage of tamoxifen inducible tissue specific antigen expression that reflects the heterogeneity of viral antigen expression and enables the study of intrahepatic immune responses to both de novo and persistent antigen. It allows following the course of intrahepatic immune responses: initiation, the acute phase and antigen clearance.
2013-08-01T14:20:49Z
2013-08-01T14:20:49Z
2013
Article
An Inducible Transgenic Mouse Model for Immune Mediated Hepatitis Showing Clearance of Antigen Expressing Hepatocytes by CD8+ T Cells. 2013, 8 (7):e68720 PLoS ONE
1932-6203
23869228
10.1371/journal.pone.0068720
http://hdl.handle.net/10033/297227
PloS one
en
Archived with thanks to PloS one
oai:repository.helmholtz-hzi.de:10033/3219722019-08-30T11:36:05Zcom_10033_620652col_10033_620672
2014-06-19T13:19:41Z
urn:hdl:10033/321972
Uncoupling of the dynamics of host-pathogen interaction uncovers new mechanisms of viral interferon antagonism at the single-cell level.
Rand, Ulfert
Hillebrand, Upneet
Sievers, Stephanie
Willenberg, Steffi
Köster, Mario
Hauser, Hansjörg
Wirth, Dagmar
Antiviral defence in mammals is mediated through type-I interferons (IFNs). Viruses antagonise this process through expression of IFN antagonist proteins (IAPs). Understanding and modelling of viral escape mechanisms and the dynamics of IAP action has the potential to facilitate the development of specific and safe drugs. Here, we describe the dynamics of interference by selected viral IAPs, NS1 from Influenza A virus and NS3/4A from Hepatitis C virus. We used Tet-inducible IAP gene expression to uncouple this process from virus-driven dynamics. Stochastic activation of the IFN-β gene required the use of single-cell live imaging to define the efficacy of the inhibitors during the virus-induced signalling processes. We found significant correlation between the onset of IAP expression and halted IFN-β expression in cells where IFN-β induction had already occurred. These data indicate that IAPs not only prevent antiviral signalling prior to IFN-β induction, but can also stop the antiviral response even after it has been activated. We found reduced NF-κB activation to be the underlying mechanism by which activated IFN expression can be blocked. This work demonstrates a new mechanism by which viruses can antagonise the IFN response.
2014-06-19T13:19:41Z
2014-06-19T13:19:41Z
2014-06-03
Article
Uncoupling of the dynamics of host-pathogen interaction uncovers new mechanisms of viral interferon antagonism at the single-cell level. 2014: Nucleic Acids Res.
1362-4962
24895433
10.1093/nar/gku492
http://hdl.handle.net/10033/321972
Nucleic acids research
Archived with thanks to Nucleic acids research
Oxford University Press
oai:repository.helmholtz-hzi.de:10033/3261962019-08-30T11:28:24Zcom_10033_620652col_10033_620672
2014-09-17T13:26:11Z
urn:hdl:10033/326196
Identification of molecular sub-networks associated with cell survival in a chronically SIVmac-infected human CD4+ T cell line.
He, Feng Q
Sauermann, Ulrike
Beer, Christiane
Winkelmann, Silke
Yu, Zheng
Sopper, Sieghart
Zeng, An-Ping
Wirth, Manfred
The deciphering of cellular networks to determine susceptibility to infection by HIV or the related simian immunodeficiency virus (SIV) is a major challenge in infection biology.
2014-09-17T13:26:11Z
2014-09-17T13:26:11Z
2014
Article
Identification of molecular sub-networks associated with cell survival in a chronically SIVmac-infected human CD4+ T cell line. 2014, 11 (1):152 Virol. J.
1743-422X
25163480
10.1186/1743-422X-11-152
http://hdl.handle.net/10033/326196
Virology journal
en
Archived with thanks to Virology journal
oai:repository.helmholtz-hzi.de:10033/3337022019-08-30T11:33:57Zcom_10033_620652col_10033_620672
2014-11-04T13:32:43Z
urn:hdl:10033/333702
Periostin secreted by mesenchymal stem cells supports tendon formation in an ectopic mouse model.
Noack, Sandra
Seiffart, Virginia
Willbold, Elmar
Laggies, Sandra
Winkel, Andreas
Shahab-Osterloh, Sandra
Flörkemeier, Thilo
Hertwig, Falk
Steinhoff, Christine
Nuber, Ulrike A
Gross, Gerhard
Hoffmann, Andrea
1 Department of Orthopaedic Trauma, Hannover Medical School (MHH), Hannover, Germany .
True tendon regeneration in human patients remains a vision of musculoskeletal therapies. In comparison to other mesenchymal lineages the biology of tenogenic differentiation is barely understood. Specifically, easy and efficient protocols are lacking that might enable tendon cell and tissue differentiation based on adult (stem) cell sources. In the murine mesenchymal progenitor cell line C3H10T½, overexpression of the growth factor bone morphogenetic protein 2 (BMP2) and a constitutively active transcription factor, Smad8 L+MH2, mediates tendon cell differentiation in vitro and the formation of tendon-like tissue in vivo. We hypothesized that during this differentiation secreted factors involved in extracellular matrix formation exert a major impact on tendon development. Gene expression analyses revealed four genes encoding secreted factors that are notably upregulated: periostin, C-type lectin domain family 3 (member b), RNase A4, and follistatin-like 1. These factors have not previously been implicated in tendon biology. Among these, periostin showed a specific expression in tenocytes of adult mouse Achilles tendon and in chondrocytes within the nonmineralized fibrocartilage zone of the enthesis with the calcaneus. Overexpression of periostin alone or in combination with constitutively active BMP receptor type in human mesenchymal stem cells and subsequent implantation into ectopic sites in mice demonstrated a reproducible moderate tenogenic capacity that has not been described before. Therefore, periostin may belong to the factors contributing to the development of tenogenic tissue.
2014-11-04T13:32:43Z
2014-11-04T13:32:43Z
2014-08-15
Article
Periostin secreted by mesenchymal stem cells supports tendon formation in an ectopic mouse model. 2014, 23 (16):1844-57 Stem Cells Dev.
1557-8534
24809660
10.1089/scd.2014.0124
http://hdl.handle.net/10033/333702
Stem cells and development
en
oai:repository.helmholtz-hzi.de:10033/6205672019-08-30T11:24:31Zcom_10033_620652col_10033_620672
2016-11-02T14:46:02Z
urn:hdl:10033/620567
Caveolin-1 influences human influenza A virus (H1N1) multiplication in cell culture
Sun, Lijing
Hemgård, Gun-Viol
Susanto, Sony A
Wirth, Manfred
Abstract Background The threat of recurring influenza pandemics caused by new viral strains and the occurrence of escape mutants necessitate the search for potent therapeutic targets. The dependence of viruses on cellular factors provides a weak-spot in the viral multiplication strategy and a means to interfere with viral multiplication. Results Using a motif-based search strategy for antiviral targets we identified caveolin-1 (Cav-1) as a putative cellular interaction partner of human influenza A viruses, including the pandemic influenza A virus (H1N1) strains of swine origin circulating from spring 2009 on. The influence of Cav-1 on human influenza A/PR/8/34 (H1N1) virus replication was determined in inhibition and competition experiments. RNAi-mediated Cav-1 knock-down as well as transfection of a dominant-negative Cav-1 mutant results in a decrease in virus titre in infected Madin-Darby canine kidney cells (MDCK), a cell line commonly used in basic influenza research as well as in virus vaccine production. To understand the molecular basis of the phenomenon we focussed on the putative caveolin-1 binding domain (CBD) located in the lumenal, juxtamembranal portion of the M2 matrix protein which has been identified in the motif-based search. Pull-down assays and co-immunoprecipitation experiments showed that caveolin-1 binds to M2. The data suggest, that Cav-1 modulates influenza virus A replication presumably based on M2/Cav-1 interaction. Conclusion As Cav-1 is involved in the human influenza A virus life cycle, the multifunctional protein and its interaction with M2 protein of human influenza A viruses represent a promising starting point for the search for antiviral agents.
2016-11-02T14:46:02Z
2016-11-02T14:46:02Z
2010-05-26
2015-09-04T08:22:03Z
Journal Article
Virology Journal. 2010 May 26;7(1):108
http://dx.doi.org/10.1186/1743-422X-7-108
http://hdl.handle.net/10033/620567
en
Sun et al.
oai:repository.helmholtz-hzi.de:10033/6210332019-08-30T11:32:16Zcom_10033_6823com_10033_6820com_10033_620652col_10033_6891col_10033_620672
2017-08-02T09:37:20Z
urn:hdl:10033/621033
Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci
Nehlsen, Kristina
da Gama-Norton, Leonor
Schucht, Roland
Hauser, Hansjörg
Wirth, Dagmar
2017-08-02T09:37:20Z
2017-08-02T09:37:20Z
2011-11-22
2015-09-04T08:22:40Z
BMC Proceedings. 2011 Nov 22;5(Suppl 8):O6
http://dx.doi.org/10.1186/1753-6561-5-S8-O6
http://hdl.handle.net/10033/621033
en
Nehlsen et al; licensee BioMed Central Ltd.
oai:repository.helmholtz-hzi.de:10033/6156752019-08-30T11:27:46Zcom_10033_620652col_10033_620672
2016-07-07T09:30:35Z
urn:hdl:10033/615675
The Role of Regulatory CD4 T Cells in Maintaining Tolerance in a Mouse Model of Autoimmune Hepatitis.
An Haack, Ira
Derkow, Katja
Riehn, Mathias
Rentinck, Marc-Nicolas
Kühl, Anja A
Lehnardt, Seija
Schott, Eckart
Dept. of Hepatology and Gastroenterology, Charité Universitätsmedizin Berlin, Berlin, Germany.
The role of regulatory CD4 T cells (Treg) in immune-mediated liver disease is still under debate. It remains disputed whether Treg suppress T cell-mediated hepatitis in vivo and whether hepatic regulatory T cells are functional in patients with autoimmune hepatitis.
2016-07-07T09:30:35Z
2016-07-07T09:30:35Z
2015
Article
The Role of Regulatory CD4 T Cells in Maintaining Tolerance in a Mouse Model of Autoimmune Hepatitis. 2015, 10 (11):e0143715 PLoS ONE
1932-6203
26599014
10.1371/journal.pone.0143715
http://hdl.handle.net/10033/615675
PloS one
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6201362019-08-30T11:25:11Zcom_10033_620652col_10033_620672
2016-09-14T11:42:11Z
urn:hdl:10033/620136
p120 Catenin-Mediated Stabilization of E-Cadherin Is Essential for Primitive Endoderm Specification.
Pieters, Tim
Goossens, Steven
Haenebalcke, Lieven
Andries, Vanessa
Stryjewska, Agata
De Rycke, Riet
Lemeire, Kelly
Hochepied, Tino
Huylebroeck, Danny
Berx, Geert
Stemmler, Marc P
Wirth, Dagmar
Haigh, Jody J
van Hengel, Jolanda
van Roy, Frans
Helmholtz Centre for infection researchz, Inhoffenstr. 7, 38124 Braunschweig.
E-cadherin-mediated cell-cell adhesion is critical for naive pluripotency of cultured mouse embryonic stem cells (mESCs). E-cadherin-depleted mESC fail to downregulate their pluripotency program and are unable to initiate lineage commitment. To further explore the roles of cell adhesion molecules during mESC differentiation, we focused on p120 catenin (p120ctn). Although one key function of p120ctn is to stabilize and regulate cadherin-mediated cell-cell adhesion, it has many additional functions, including regulation of transcription and Rho GTPase activity. Here, we investigated the role of mouse p120ctn in early embryogenesis, mESC pluripotency and early fate determination. In contrast to the E-cadherin-null phenotype, p120ctn-null mESCs remained pluripotent, but their in vitro differentiation was incomplete. In particular, they failed to form cystic embryoid bodies and showed defects in primitive endoderm formation. To pinpoint the underlying mechanism, we undertook a structure-function approach. Rescue of p120ctn-null mESCs with different p120ctn wild-type and mutant expression constructs revealed that the long N-terminal domain of p120ctn and its regulatory domain for RhoA were dispensable, whereas its armadillo domain and interaction with E-cadherin were crucial for primitive endoderm formation. We conclude that p120ctn is not only an adaptor and regulator of E-cadherin, but is also indispensable for proper lineage commitment.
2016-09-14T11:42:11Z
2016-09-14T11:42:11Z
2016-08
Article
p120 Catenin-Mediated Stabilization of E-Cadherin Is Essential for Primitive Endoderm Specification. 2016, 12 (8):e1006243 PLoS Genet.
1553-7404
27556156
10.1371/journal.pgen.1006243
http://hdl.handle.net/10033/620136
PLoS genetics
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6206632019-08-30T11:37:00Zcom_10033_620652col_10033_620672
2016-12-13T10:27:04Z
urn:hdl:10033/620663
Effective intrahepatic CD8+ T-cell immune responses are induced by low but not high numbers of antigen-expressing hepatocytes.
Ochel, Aaron
Cebula, Marcin
Riehn, Mathias
Hillebrand, Upneet
Lipps, Christoph
Schirmbeck, Reinhold
Hauser, Hansjoerg
Wirth, Dagmar
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Liver infections with hepatotropic viruses, such as hepatitis B virus and hepatitis C virus are accompanied by viral persistence and immune failure. CD8+ T cells are crucial mediators of the intrahepatic antiviral immune response. Chronic infections of the liver and other organs correlate with T-cell exhaustion. It was previously suggested that high antigen load could result in T-cell exhaustion. We aimed at elucidating the impact of different intrahepatic antigen loads on the quality of CD8+ T-cell-mediated immunity by employing an infection-free transgenic mouse model expressing ovalbumin (Ova) as the target antigen. Adoptive transfer of OT-I cells induced a transient intrahepatic immune response toward both high and low Ova levels. However, antigen clearance was achieved only in mice expressing low antigen levels. In contrast, T cells exposed to high antigen levels underwent exhaustion and became depleted, causing antigen persistence. Moreover, when functional T cells were exposed to high intrahepatic antigen levels, a complete transition toward exhaustion was observed. Thus, this study shows that the antigen expression level in the liver correlates inversely with T-cell immunity in vivo and governs the efficiency of immune responses upon antigen presentation.
2016-12-13T10:27:04Z
2016-12-13T10:27:04Z
2016-11
Article
Effective intrahepatic CD8+ T-cell immune responses are induced by low but not high numbers of antigen-expressing hepatocytes. 2016, 13 (6):805-815 Cell. Mol. Immunol.
2042-0226
26412123
10.1038/cmi.2015.80
http://hdl.handle.net/10033/620663
Cellular & molecular immunology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6210422019-08-30T11:30:58Zcom_10033_620652col_10033_620672
2017-08-04T08:32:52Z
urn:hdl:10033/621042
Isolation of F. novicida-Containing Phagosome from Infected Human Monocyte Derived Macrophages.
Marecic, Valentina
Shevchuk, Olga
Ozanic, Mateja
Mihelcic, Mirna
Steinert, Michael
Jurak Begonja, Antonija
Abu Kwaik, Yousef
Santic, Marina
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Francisella is a gram-negative bacterial pathogen, which causes tularemia in humans and animals. A crucial step of Francisella infection is its invasion of macrophage cells. Biogenesis of the Francisella-containing phagosome (FCP) is arrested for ~15 min at the endosomal stage, followed by gradual bacterial escape into the cytosol, where the microbe proliferates. The crucial step in pathogenesis of tularemia is short and transient presence of the bacterium within phagosome. Isolation of FCPs for further studies has been challenging due to the short period of time of bacterial residence in it and the characteristics of the FCP. Here, we will for the first time present the method for isolation of the FCPs from infected human monocytes-derived macrophages (hMDMs). For elimination of lysosomal compartment these organelles were pre-loaded with dextran coated colloidal iron particles prior infection and eliminated by magnetic separation of the post-nuclear supernatant (PNS). We encountered the challenge that mitochondria has similar density to the FCP. To separate the FCP in the PNS from mitochondria, we utilized iodophenylnitrophenyltetrazolium, which is converted by the mitochondrial succinate dehydrogenase into formazan, leading to increased density of the mitochondria and allowing separation by the discontinuous sucrose density gradient ultracentrifugation. The purity of the FCP preparation and its acquisition of early endosomal markers was confirmed by Western blots, confocal and transmission electron microscopy. Our strategy to isolate highly pure FCPs from macrophages should facilitate studies on the FCP and its biogenesis.
2017-08-04T08:32:52Z
2017-08-04T08:32:52Z
2017
Article
Isolation of F. novicida-Containing Phagosome from Infected Human Monocyte Derived Macrophages. 2017, 7:303 Front Cell Infect Microbiol
2235-2988
28725638
10.3389/fcimb.2017.00303
http://hdl.handle.net/10033/621042
Frontiers in cellular and infection microbiology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6210572019-08-30T11:32:17Zcom_10033_620652col_10033_620672
2017-08-16T11:44:18Z
urn:hdl:10033/621057
The CpG-sites of the CBX3 ubiquitous chromatin opening element are critical structural determinants for the anti-silencing function.
Kunkiel, Jessica
Gödecke, Natascha
Ackermann, Mania
Hoffmann, Dirk
Schambach, Axel
Lachmann, Nico
Wirth, Dagmar
Moritz, Thomas
Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Suppression of therapeutic transgene expression from retroviral gene therapy vectors by epigenetic defence mechanisms represents a problem that is particularly encountered in pluripotent stem cells (PSCs) and their differentiated progeny. Transgene expression in these cells, however, can be stabilised by CpG-rich ubiquitous chromatin opening elements (UCOEs). In this context we recently demonstrated profound anti-silencing properties for the small (679 bp) CBX3-UCO element and we now confirmed this observation in the context of the defined murine chromosomal loci ROSA26 and TIGRE. Moreover, since the structural basis for the anti-silencing activity of UCOEs has remained poorly defined, we interrogated various CBX3 subfragments in the context of lentiviral vectors and murine PSCs. We demonstrated marked though distinct anti-silencing activity in the pluripotent state and during PSC-differentiation for several of the CBX3 subfragments. This activity was significantly correlated with CpG content as well as endogenous transcriptional activity. Interestingly, also a scrambled CBX3 version with preserved CpG-sites retained the anti-silencing activity despite the lack of endogenous promoter activity. Our data therefore highlight the importance of CpG-sites and transcriptional activity for UCOE functionality and suggest contributions from different mechanisms to the overall anti-silencing function of the CBX3 element.
2017-08-16T11:44:18Z
2017-08-16T11:44:18Z
2017-08-11
Article
The CpG-sites of the CBX3 ubiquitous chromatin opening element are critical structural determinants for the anti-silencing function. 2017, 7 (1):7919 Sci Rep
2045-2322
28801671
10.1038/s41598-017-04212-8
http://hdl.handle.net/10033/621057
Scientific reports
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6210682019-08-30T11:27:16Zcom_10033_311624com_10033_6839com_10033_620652col_10033_620672col_10033_311625
2017-08-22T12:59:08Z
urn:hdl:10033/621068
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.
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.
2017-08-22T12:59:08Z
2017-08-22T12:59:08Z
2017
Article
Cell Polarization and Epigenetic Status Shape the Heterogeneous Response to Type III Interferons in Intestinal Epithelial Cells. 2017, 8:671 Front Immunol
1664-3224
28659914
10.3389/fimmu.2017.00671
http://hdl.handle.net/10033/621068
Frontiers in immunology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6211202019-08-30T11:34:22Zcom_10033_311624com_10033_6839com_10033_620652col_10033_620672col_10033_311625
2017-09-25T14:24:23Z
urn:hdl:10033/621120
TLR9-Mediated Conditioning of Liver Environment Is Essential for Successful Intrahepatic Immunotherapy and Effective Memory Recall.
Cebula, Marcin
Riehn, Mathias
Hillebrand, Upneet
Kratzer, Ramona F
Kreppel, Florian
Koutsoumpli, Georgia
Daemen, Toos
Hauser, Hansjoerg
Wirth, Dagmar
Helmholtz -Zentrum für Infektionsforschung GmbH. Inhoffenstr. 7, 38124 Braunschweig, Germany.
Immune defense against hepatotropic viruses such as hepatitis B (HBV) and hepatitis C (HCV) poses a major challenge for therapeutic approaches. Intrahepatic cytotoxic CD8 T cells that are crucial for an immune response against these viruses often become exhausted resulting in chronic infection. We elucidated the T cell response upon therapeutic vaccination in inducible transgenic mouse models in which variable percentages of antigen-expressing hepatocytes can be adjusted, providing mosaic antigen distribution and reflecting the varying viral antigen loads observed in patients. Vaccination-induced endogenous CD8 T cells could eliminate low antigen loads in liver but were functionally impaired if confronted with elevated antigen loads. Strikingly, only by conditioning the liver environment with TLR9 ligand prior and early after peripheral vaccination, successful immunization against high intrahepatic antigen density with its elimination was achieved. Moreover, TLR9 immunomodulation was also indispensable for functional memory recall after high frequency antigen challenge. Together, the results indicate that TLR9-mediated conditioning of liver environment during therapeutic vaccination or antigen reoccurrence is crucial for an efficacious intrahepatic T cell response.
2017-09-25T14:24:23Z
2017-09-25T14:24:23Z
2017-07-14
Article
TLR9-Mediated Conditioning of Liver Environment Is Essential for Successful Intrahepatic Immunotherapy and Effective Memory Recall. 2017 Mol. Ther.
1525-0024
28716576
10.1016/j.ymthe.2017.06.018
http://hdl.handle.net/10033/621120
Molecular therapy : the journal of the American Society of Gene Therapy
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6211302019-08-30T11:28:51Zcom_10033_311624com_10033_6839com_10033_620652col_10033_620672col_10033_311625
2017-10-09T13:56:28Z
urn:hdl:10033/621130
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.
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.
2017-10-09T13:56:28Z
2017-10-09T13:56:28Z
2017-09-19
Article
Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation. 2017, 45 (16):e147 Nucleic Acids Res.
1362-4962
28934472
10.1093/nar/gkx601
http://hdl.handle.net/10033/621130
Nucleic acids research
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6211652019-08-30T11:28:23Zcom_10033_620652col_10033_620672
2017-11-07T13:38:34Z
urn:hdl:10033/621165
CpG-ODN Facilitates Effective Intratracheal Immunization and Recall of Memory against Neoantigen-Expressing Alveolar Cells.
Riehn, Mathias
Cebula, Marcin
Hauser, Hansjörg
Wirth, Dagmar
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany.
Intrapulmonary immune reactions are impaired by the tolerogenic environment of the lung. This is manifested by the absence of effective endogenous T cell responses upon neoantigen expression. This tolerance is considered to contribute to lung cancer and inefficient immune therapeutic interventions. To investigate the mechanisms contributing to lung tolerance and to overcome these restrictions, we developed a transgenic mouse model with induction of a neoantigen (OVA) exclusively in alveolar type II epithelial cells. This model is characterized by the absence of functional endogenous T cell responses upon OVA neoantigen induction. Standard DNA and protein vaccination protocols resulted in the accumulation of high numbers of antigen-specific CD8 T cells in the lung. However, clearance of antigen-expressing cells was not achieved. To overcome this tolerance, we induced inflammatory conditions by coapplication of the TLR ligands LPS and CpG-ODN during intrapulmonary vaccinations. Both ligands induced high numbers of neoantigen-specific T cells in the lung. However, only coapplication of CpG-ODN was sufficient to establish functional cytotoxic responses resulting in the elimination of neoantigen presenting target cells. Remarkably, CpG-ODN was also crucial for functional memory responses upon re-induction of the neoantigen. The results highlight the need of TLR9 co-stimulation for overcoming tolerization, which might be a key factor for therapeutic interventions.
2017-11-07T13:38:34Z
2017-11-07T13:38:34Z
2017
Article
CpG-ODN Facilitates Effective Intratracheal Immunization and Recall of Memory against Neoantigen-Expressing Alveolar Cells. 2017, 8:1201 Front Immunol
1664-3224
29038654
10.3389/fimmu.2017.01201
http://hdl.handle.net/10033/621165
Frontiers in immunology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6212042019-08-30T11:26:42Zcom_10033_620659com_10033_620652com_10033_128109col_10033_621829col_10033_620672col_10033_620660
2017-12-13T09:08:17Z
urn:hdl:10033/621204
A mathematical model of the impact of insulin secretion dynamics on selective hepatic insulin resistance.
Zhao, Gang
Wirth, Dagmar
Schmitz, Ingo
Meyer-Hermann, Michael
Braunschweiger Zentrum für Systembiologie, Rebenring 56, 38106, Germany.
Physiological insulin secretion exhibits various temporal patterns, the dysregulation of which is involved in diabetes development. We analyzed the impact of first-phase and pulsatile insulin release on glucose and lipid control with various hepatic insulin signaling networks. The mathematical model suggests that atypical protein kinase C (aPKC) undergoes a bistable switch-on and switch-off, under the control of insulin receptor substrate 2 (IRS2). The activation of IRS1 and IRS2 is temporally separated due to the inhibition of IRS1 by aPKC. The model further shows that the timing of aPKC switch-off is delayed by reduced first-phase insulin and reduced amplitude of insulin pulses. Based on these findings, we propose a sequential model of postprandial hepatic control of glucose and lipid by insulin, according to which delayed aPKC switch-off contributes to selective hepatic insulin resistance, which is a long-standing paradox in the field.
2017-12-13T09:08:17Z
2017-12-13T09:08:17Z
2017-11-08
Article
A mathematical model of the impact of insulin secretion dynamics on selective hepatic insulin resistance. 2017, 8 (1):1362 Nat Commun
2041-1723
29118381
10.1038/s41467-017-01627-9
http://hdl.handle.net/10033/621204
Nature communications
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6213332019-08-30T11:28:51Zcom_10033_620652col_10033_620672
2018-03-23T15:32:45Z
urn:hdl:10033/621333
Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor.
Stichling, Nicole
Suomalainen, Maarit
Flatt, Justin W
Schmid, Markus
Pacesa, Martin
Hemmi, Silvio
Jungraithmayr, Wolfgang
Maler, Mareike D
Freudenberg, Marina A
Plückthun, Andreas
May, Tobias
Köster, Mario
Fejer, György
Greber, Urs F
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses.
2018-03-23T15:32:45Z
2018-03-23T15:32:45Z
2018-03
Article
Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor. 2018, 14 (3):e1006914 PLoS Pathog.
1553-7374
29522575
10.1371/journal.ppat.1006914
http://hdl.handle.net/10033/621333
PLoS pathogens
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6214122019-08-30T11:32:37Zcom_10033_620652col_10033_620672
2018-06-27T08:31:45Z
urn:hdl:10033/621412
Memory CD8 T cells support the maintenance of hematopoietic stem cells in the bone marrow
Geerman, Sulima
Brasser, Giso
Bhushal, Sudeep
Salerno, Fiamma
Kragten, Natasja A.
Hoogenboezem, Mark
de Haan, Gerald
Wolkers, Monika C.
Pascutti, María Fernanda
Nolte, Martijn A.
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Letter to the editor
2018-06-27T08:31:45Z
2018-06-27T08:31:45Z
Article
Other
0390-6078
1592-8721
10.3324/haematol.2017.169516
http://hdl.handle.net/10033/621412
http://www.haematologica.org/lookup/doi/10.3324/haematol.2017.169516
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
103
6
e230
e233
Haematologica
oai:repository.helmholtz-hzi.de:10033/6214322019-08-30T11:25:41Zcom_10033_620601com_10033_620652col_10033_620672col_10033_620602
2018-07-31T12:49:10Z
urn:hdl:10033/621432
Type I interferon receptor signaling delays Kupffer cell replenishment during acute fulminant viral hepatitis.
Borst, Katharina
Frenz, Theresa
Spanier, Julia
Tegtmeyer, Pia-Katharina
Chhatbar, Chintan
Skerra, Jennifer
Ghita, Luca
Namineni, Sukumar
Lienenklaus, Stefan
Köster, Mario
Heikenwaelder, Mathias
Sutter, Gerd
Kalinke, Ulrich
TWINCORE, Zentrum für experimentelle und klinischeInfektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany.
DNA virus infection
Innate immunity
Liver inflammation
Monocyte infiltration
Virus-induced fulminant hepatitis is a major cause of acute liver failure. During acute viral hepatitis the impact of type I interferon (IFN-I) on myeloid cells, including liver-resident Kupffer cells (KC), is only partially understood. Herein, we dissected the impact of locally induced IFN-I responses on myeloid cell function and hepatocytes during acute liver inflammation. Two different DNA-encoded viruses, vaccinia virus (VACV) and murine cytomegalovirus (MCMV), were studied. In vivo imaging was applied to visualize local IFN-β induction and IFN-I receptor (IFNAR) triggering in VACV-infected reporter mice. Furthermore, mice with a cell type-selective IFNAR ablation were analyzed to dissect the role of IFNAR signaling in myeloid cells and hepatocytes. Experiments with Cx3cr1 VACV infection induced local IFN-β responses, which lead to IFNAR signaling primarily within the liver. IFNAR triggering was needed to control the infection and prevent fulminant hepatitis. The severity of liver inflammation was independent of IFNAR triggering of hepatocytes, whereas IFNAR triggering of myeloid cells protected from excessive inflammation. Upon VACV or MCMV infection KC disappeared, whereas infiltrating monocytes differentiated to KC afterwards. During IFNAR triggering such replenished monocyte-derived KC comprised more IFNAR-deficient than -competent cells in mixed bone marrow chimeric mice, whereas after the decline of IFNAR triggering both subsets showed an even distribution. Upon VACV infection IFNAR triggering of myeloid cells, but not of hepatocytes, critically modulates acute viral hepatitis. During infection with DNA-encoded viruses IFNAR triggering of liver-infiltrating blood monocytes delays the development of monocyte-derived KC, pointing towards new therapeutic strategies for acute viral hepatitis.
2018-07-31T12:49:10Z
2018-07-31T12:49:10Z
2017-12-21
Article
1600-0641
29274730
10.1016/j.jhep.2017.11.029
http://hdl.handle.net/10033/621432
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
Journal of hepatology
oai:repository.helmholtz-hzi.de:10033/6214392019-08-30T11:34:45Zcom_10033_311624com_10033_6839com_10033_620601com_10033_620652col_10033_620672col_10033_311625col_10033_620602
2018-08-07T11:47:28Z
urn:hdl:10033/621439
Identification of a Predominantly Interferon-λ-Induced Transcriptional Profile in Murine Intestinal Epithelial Cells.
Selvakumar, Tharini A
Bhushal, Sudeep
Kalinke, Ulrich
Wirth, Dagmar
Hauser, Hansjörg
Köster, Mario
Hornef, Mathias W
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
gastrointestinal tract
interferon-lambda
interleukin 28 receptor
intestinal epithelium
transcription
Type I (α and β) and type III (λ) interferons (IFNs) induce the expression of a large set of antiviral effector molecules
2018-08-07T11:47:28Z
2018-08-07T11:47:28Z
2017-01-01
Article
1664-3224
29085367
10.3389/fimmu.2017.01302
http://hdl.handle.net/10033/621439
en
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
Frontiers in immunology
oai:repository.helmholtz-hzi.de:10033/6215392019-08-30T11:29:41Zcom_10033_620652col_10033_620672
2018-11-06T14:32:52Z
urn:hdl:10033/621539
Biocompatible Coatings from Smart Biopolymer Nanoparticles for Enzymatically Induced Drug Release.
Tolle, Christian
Riedel, Jan
Mikolai, Carina
Winkel, Andreas
Stiesch, Meike
Wirth, Dagmar
Menzel, Henning
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
X-ray photoelectron spectroscopy
alginate
cell adherence
cyto-compatibility
enzymatic cleavage
ionotropic gelation
nanogel
reflection-absorption infrared spectroscopy
smart drug delivery
Nanoparticles can be used as a smart drug delivery system, when they release the drug only upon degradation by specific enzymes. A method to create such responsive materials is the formation of hydrogel nanoparticles, which have enzymatically degradable crosslinkers. Such hydrogel nanoparticles were prepared by ionotropic gelation sodium alginate with lysine-rich peptide sequences-either α-poly-L-lysine (PLL) or the aggrecanase-labile sequence KKKK-GRD-ARGSV↓NITEGE-DRG-KKKK. The nanoparticle suspensions obtained were analyzed by means of dynamic light scattering and nanoparticle tracking analysis. Degradation experiments carried out with the nanoparticles in suspension revealed enzyme-induced lability. Drugs present in the polymer solution during the ionotropic gelation can be encapsulated in the nanoparticles. Drug loading was investigated for interferon-β (IFN-β) as a model, using a bioluminescence assay with MX2Luc2 cells. The encapsulation efficiency for IFN-β was found to be approximately 25%. The nanoparticles suspension can be used to spray-coat titanium alloys (Ti-6Al-4V) as a common implant material. The coatings were proven by ellipsometry, reflection-absorption infrared spectroscopy, and X-ray photoelectron spectroscopy. An enzyme-responsive decrease in layer thickness is observed due to the degradation of the coatings. The Alg/peptide coatings were cytocompatible for human gingival fibroblasts (HGFIB), which was investigated by CellTiterBlue and lactate dehydrogenase (LDH) assay. However, HGFIBs showed poor adhesion and proliferation on the Alg/peptide coatings, but these could be improved by modification of the alginate with a RGD-peptide sequence. The smart drug release system presented can be further tailored to have the right release kinetics and cell adhesion properties.
2018-11-06T14:32:52Z
2018-11-06T14:32:52Z
2018-09-28
Article
2218-273X
30274232
10.3390/biom8040103
http://hdl.handle.net/10033/621539
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
Biomolecules
oai:repository.helmholtz-hzi.de:10033/6215802019-08-30T11:29:43Zcom_10033_620601com_10033_620652col_10033_620672col_10033_620673col_10033_620602
2018-11-23T14:56:42Z
urn:hdl:10033/621580
Human monocyte-derived macrophages inhibit HCMV spread independent of classical antiviral cytokines.
Becker, Jennifer
Kinast, Volker
Döring, Marius
Lipps, Christoph
Duran, Veronica
Spanier, Julia
Tegtmeyer, Pia-Katharina
Wirth, Dagmar
Cicin-Sain, Luka
Alcamí, Antonio
Kalinke, Ulrich
TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany.; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig.
Human cytomegalovirus
epithelial cells
macrophages
plasmacytoid dendritic cells
type I interferons
Infection of healthy individuals with human cytomegalovirus (HCMV) is usually unnoticed and results in life-long latency, whereas HCMV reactivation as well as infection of newborns or immunocompromised patients can cause life-threatening disease. To better understand HCMV pathogenesis we studied mechanisms that restrict HCMV spread. We discovered that HCMV-infected cells can directly trigger plasmacytoid dendritic cells (pDC) to mount antiviral type I interferon (IFN-I) responses, even in the absence of cell-free virus. In contrast, monocyte-derived cells only expressed IFN-I when stimulated by cell-free HCMV, or upon encounter of HCMV-infected cells that already produced cell-free virus. Nevertheless, also in the absence of cell-free virus, i.e., upon co-culture of infected epithelial/endothelial cells and monocyte-derived macrophages (moMΦ) or dendritic cells (moDC), antiviral responses were induced that limited HCMV spread. The induction of this antiviral effect was dependent on cell-cell contact, whereas cell-free supernatants from co-culture experiments also inhibited virus spread, implying that soluble factors were critically needed. Interestingly, the antiviral effect was independent of IFN-γ, TNF-α, and IFN-I as indicated by cytokine inhibition experiments using neutralizing antibodies or the vaccinia virus-derived soluble IFN-I binding protein B18R, which traps human IFN-α and IFN-β. In conclusion, our results indicate that human macrophages and dendritic cells can limit HCMV spread by IFN-I dependent as well as independent mechanisms, whereas the latter ones might be particularly relevant for the restriction of HCMV transmission via cell-to-cell spread.
2018-11-23T14:56:42Z
2018-11-23T14:56:42Z
2018-01-01
Article
2150-5608
30403913
10.1080/21505594.2018.1535785
http://hdl.handle.net/10033/621580
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
Virulence
oai:repository.helmholtz-hzi.de:10033/6216292019-08-30T11:28:47Zcom_10033_311624com_10033_6839com_10033_620652com_10033_338554col_10033_621787col_10033_620672col_10033_311625
2019-01-04T12:26:50Z
urn:hdl:10033/621629
Macrophage entrapped silica coated superparamagnetic iron oxide particles for controlled drug release in a 3D cancer model.
Ullah, Sami
Seidel, Katja
Türkkan, Sibel
Warwas, Dawid Peter
Dubich, Tatyana
Rohde, Manfred
Hauser, Hansjörg
Behrens, Peter
Kirschning, Andreas
Köster, Mario
Wirth, Dagmar
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Cell based drug delivery
Controlled drug delivery, 3D tumour model
Hyperthermia
Macrophages
Magnetic silica nanoparticles
Targeted delivery of drugs is a major challenge in treatment of diverse diseases. Systemically administered drugs demand high doses and are accompanied by poor selectivity and side effects on non-target cells. Here, we introduce a new principle for targeted drug delivery. It is based on macrophages as transporters for nanoparticle-coupled drugs as well as controlled release of drugs by hyperthermia mediated disruption of the cargo cells and simultaneous deliberation of nanoparticle-linked drugs. Hyperthermia is induced by an alternating electromagnetic field (AMF) that induces heat from silica-coated superparamagnetic iron oxide nanoparticles (SPIONs). We show proof-of-principle of controlled release by the simultaneous disruption of the cargo cells and the controlled, AMF induced release of a toxin, which was covalently linked to silica-coated SPIONs via a thermo-sensitive linker. Cells that had not been loaded with SPIONs remain unaffected. Moreover, in a 3D co-culture model we demonstrate specific killing of associated tumour cells when employing a ratio as low as 1:40 (SPION-loaded macrophage: tumour cells). Overall, our results demonstrate that AMF induced drug release from macrophage-entrapped nanoparticles is tightly controlled and may be an attractive novel strategy for targeted drug release.
2019-01-04T12:26:50Z
2019-01-04T12:26:50Z
2018-12-23
Article
1873-4995
30586597
10.1016/j.jconrel.2018.12.040
http://hdl.handle.net/10033/621629
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Journal of controlled release : official journal of the Controlled Release Society
oai:repository.helmholtz-hzi.de:10033/6216362019-11-21T11:58:00Zcom_10033_620533com_10033_620652col_10033_620672col_10033_620534
2019-01-09T15:04:45Z
urn:hdl:10033/621636
Advances and Challenges of Biodegradable Implant Materials with a Focus on Magnesium-Alloys and Bacterial Infections
Rahim, Muhammad
Ullah, Sami
Mueller, Peter
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Medical implants made of biodegradable materials could be advantageous for temporary applications, such as mechanical support during bone-healing or as vascular stents to keep blood vessels open. After completion of the healing process, the implant would disappear, avoiding long-term side effects or the need for surgical removal. Various corrodible metal alloys based on magnesium, iron or zinc have been proposed as sturdier and potentially less inflammatory alternatives to degradable organic polymers, in particular for load-bearing applications. Despite the recent introduction of magnesium-based screws, the remaining hurdles to routine clinical applications are still challenging. These include limitations such as mechanical material characteristics or unsuitable corrosion characteristics. In this article, the salient features and clinical prospects of currently-investigated biodegradable implant materials are summarized, with a main focus on magnesium alloys. A mechanism of action for the stimulation of bone growth due to the exertion of mechanical force by magnesium corrosion products is discussed. To explain divergent in vitro and in vivo effects of magnesium, a novel model for bacterial biofilm infections is proposed which predicts crucial consequences for antibacterial implant strategies.
2019-01-09T15:04:45Z
2019-01-09T15:04:45Z
2018-07-10
Article
2075-4701
10.3390/met8070532
http://hdl.handle.net/10033/621636
Metals
http://www.mdpi.com/2075-4701/8/7/532
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
MPDI
8
7
532
Metals
oai:repository.helmholtz-hzi.de:10033/6216712019-08-30T11:30:30Zcom_10033_311624com_10033_6839com_10033_620857com_10033_620652com_10033_620618col_10033_620672col_10033_311625col_10033_620858col_10033_620619
2019-01-29T14:52:33Z
urn:hdl:10033/621671
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
Stadler, Marc
Schulz, Thomas F
Wirth, Dagmar
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
3D culture system
Drug validation
Humanized mouse model
KSHV
Novel anti-viral drugs
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.
2019-01-29T14:52:33Z
2019-01-29T14:52:33Z
2019-01-04
Article
1432-1440
30610257
10.1007/s00109-018-01733-1
http://hdl.handle.net/10033/621671
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Journal of molecular medicine (Berlin, Germany)
oai:repository.helmholtz-hzi.de:10033/6217752019-08-30T11:30:26Zcom_10033_620589com_10033_620652com_10033_311308col_10033_620672col_10033_620777col_10033_620608col_10033_559591
2019-05-15T07:43:02Z
urn:hdl:10033/621775
ER intrabody-mediated inhibition of interferon α secretion by mouse macrophages and dendritic cells.
Büssow, Konrad
Themann, Philipp
Luu, Sabine
Pentrowski, Paul
Harting, Claudia
Majewski, Mira
Vollmer, Veith
Köster, Mario
Grashoff, Martina
Zawatzky, Rainer
van den Heuvel, Joop
Kröger, Andrea
Böldicke, Thomas
HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Interferon α (IFNα) counteracts viral infections by activating various IFNα-stimulated genes (ISGs). These genes encode proteins that block viral transport into the host cell and inhibit viral replication, gene transcription and translation. Due to the existence of 14 different, highly homologous isoforms of mouse IFNα, an IFNα knockout mouse has not yet been established by genetic knockout strategies. An scFv intrabody for holding back IFNα isoforms in the endoplasmic reticulum (ER) and thus counteracting IFNα secretion is reported. The intrabody was constructed from the variable domains of the anti-mouse IFNα rat monoclonal antibody 4EA1 recognizing the 5 isoforms IFNα1, IFNα2, IFNα4, IFNα5, IFNα6. A soluble form of the intrabody had a KD of 39 nM to IFNα4. It could be demonstrated that the anti-IFNα intrabody inhibits clearly recombinant IFNα4 secretion by HEK293T cells. In addition, the secretion of IFNα4 was effectively inhibited in stably transfected intrabody expressing RAW 264.7 macrophages and dendritic D1 cells. Colocalization of the intrabody with IFNα4 and the ER marker calnexin in HEK293T cells indicated complex formation of intrabody and IFNα4 inside the ER. Intracellular binding of intrabody and antigen was confirmed by co-immunoprecipitation. Complexes of endogenous IFNα and intrabody could be visualized in the ER of Poly (I:C) stimulated RAW 264.7 macrophages and D1 dendritic cells. Infection of macrophages and dendritic cells with the vesicular stomatitis virus VSV-AV2 is attenuated by IFNα and IFNβ. The intrabody increased virus proliferation in RAW 264.7 macrophages and D1 dendritic cells under IFNβ-neutralizing conditions. To analyze if all IFNα isoforms are recognized by the intrabody was not in the focus of this study. Provided that binding of the intrabody to all isoforms was confirmed, the establishment of transgenic intrabody mice would be promising for studying the function of IFNα during viral infection and autoimmune diseases.
2019-05-15T07:43:02Z
2019-05-15T07:43:02Z
2019-01-01
Article
PLoS One. 2019 Apr 16;14(4):e0215062. doi: 10.1371/journal.pone.0215062. eCollection 2019.
1932-6203
30990863
10.1371/journal.pone.0215062
http://hdl.handle.net/10033/621775
PLOS ONE
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Plos
PloS one
oai:repository.helmholtz-hzi.de:10033/6218132019-08-30T11:29:12Zcom_10033_620652col_10033_620672
2019-06-12T08:45:32Z
urn:hdl:10033/621813
Model-based analysis of influenza A virus replication in genetically engineered cell lines elucidates the impact of host cell factors on key kinetic parameters of virus growth.
Laske, Tanja
Bachmann, Mandy
Dostert, Melanie
Karlas, Alexander
Wirth, Dagmar
Frensing, Timo
Meyer, Thomas F
Hauser, Hansjörg
Reichl, Udo
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
The best measure to limit spread of contagious diseases caused by influenza A viruses (IAVs) is annual vaccination. The growing global demand for low-cost vaccines requires the establishment of high-yield production processes. One possible option to address this challenge is the engineering of novel vaccine producer cell lines by manipulating gene expression of host cell factors relevant for virus replication. To support detailed characterization of engineered cell lines, we fitted an ordinary differential equation (ODE)-based model of intracellular IAV replication previously established by our group to experimental data obtained from infection studies in human A549 cells. Model predictions indicate that steps of viral RNA synthesis, their regulation and particle assembly and virus budding are promising targets for cell line engineering. The importance of these steps was confirmed in four of five single gene overexpression cell lines (SGOs) that showed small, but reproducible changes in early dynamics of RNA synthesis and virus release. Model-based analysis suggests, however, that overexpression of the selected host cell factors negatively influences specific RNA synthesis rates. Still, virus yield was rescued by an increase in the virus release rate. Based on parameter estimations obtained for SGOs, we predicted that there is a potential benefit associated with overexpressing multiple host cell genes in one cell line, which was validated experimentally. Overall, this model-based study on IAV replication in engineered cell lines provides a step forward in the dynamic and quantitative characterization of IAV-host cell interactions. Furthermore, it suggests targets for gene editing and indicates that overexpression of multiple host cell factors may be beneficial for the design of novel producer cell lines.
2019-06-12T08:45:32Z
2019-06-12T08:45:32Z
2019-01-01
Article
PLoS Comput Biol. 2019 Apr 11;15(4):e1006944. doi: 10.1371/journal.pcbi.1006944 eCollection 2019 Apr.
1553-7358
30973879
10.1371/journal.pcbi.1006944
http://hdl.handle.net/10033/621813
PLOS computational biology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
PLOS
PLoS computational biology
oai:repository.helmholtz-hzi.de:10033/6220942020-01-21T02:01:00Zcom_10033_620652col_10033_620672
2020-01-20T14:26:29Z
urn:hdl:10033/622094
Targeting the Kaposi Sarcoma Herpesvirus ORF 21 tyrosine kinase and viral lytic reactivation by tyrosine kinase inhibitors approved for clinical use.
Beauclair, Guillaume
Naimo, Eleonora
Dubich, Tatyana
Rückert, Jessica
Koch, Sandra
Dhingra, Akshay
Wirth, Dagmar
Schulz, Thomas F
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Kaposi's Sarcoma-associated herpesvirus (KSHV) is the cause of three human malignancies, Kaposi's Sarcoma, Primary Effusion Lymphoma and the plasma cell variant of Multicentric Castleman's Disease. Previous research has shown that several cellular tyrosine kinases play crucial roles during several steps in the virus replication cycle. Two KSHV proteins also have protein kinase function: open reading frame (ORF) 36 encodes a serin-threonine kinase, while ORF21 encodes a thymidine kinase (TK), which has recently been found to be an efficient tyrosine kinase. In this study, we explore the role of the ORF21 tyrosine kinase function in KSHV lytic replication. By generating a recombinant KSHV mutant with an enzymatically inactive ORF21 protein we show that the tyrosine kinase function of ORF21/TK is not required for the progression of the lytic replication in tissue culture, but that it is essential for the phosphorylation and activation to toxic moieties of the antiviral drugs zidovudine and brivudine. In addition, we identify several tyrosine kinase inhibitors, already in clinical use against human malignancies, which potently inhibit not only ORF21 TK kinase function, but also viral lytic reactivation and the development of KSHV-infected endothelial tumors in mice. As they target both cellular tyrosine kinases and a viral kinase, some of these compounds might find a use in the treatment of KSHV-associated malignancies.Importance: Our findings address the role of KSHV ORF21 as a tyrosine kinase during lytic replication and the activation of prodrugs in KSHV-infected cells. We also show the potential of selected clinically approved tyrosine kinase inhibitors to inhibit KSHV TK, KSHV lytic replication, infectious virions release and the development of an endothelial tumor. Since they target both cellular tyrosine kinases supporting productive viral replication and a viral kinase, these drugs, which are already approved for clinical use, may be suitable for repurposing for the treatment of KSHV-related tumors in AIDS patients or transplant recipients.
2020-01-20T14:26:29Z
2020-01-20T14:26:29Z
2019-12-11
Article
J Virol. 2019 Dec 11. pii: JVI.01791-19. doi: 10.1128/JVI.01791-19.
1098-5514
31826996
10.1128/JVI.01791-19
http://hdl.handle.net/10033/622094
Journal of Virology
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
American Society for Microbiology (ASM)
Journal of virology
oai:repository.helmholtz-hzi.de:10033/6220932020-02-27T08:46:24Zcom_10033_620652col_10033_620672
2020-01-20T14:22:18Z
urn:hdl:10033/622093
Targeting Kaposi's Sarcoma-Associated Herpesvirus ORF21 Tyrosine Kinase and Viral Lytic Reactivation by Tyrosine Kinase Inhibitors Approved for Clinical Use
Beauclair, Guillaume
Naimo, Eleonora
Dubich, Tatyana
Rückert, Jessica
Koch, Sandra
Dhingra, Akshay
Wirth, Dagmar
Schulz, Thomas F
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Kaposi's Sarcoma-associated herpesvirus (KSHV) is the cause of three human malignancies, Kaposi's Sarcoma, Primary Effusion Lymphoma and the plasma cell variant of Multicentric Castleman's Disease. Previous research has shown that several cellular tyrosine kinases play crucial roles during several steps in the virus replication cycle. Two KSHV proteins also have protein kinase function: open reading frame (ORF) 36 encodes a serin-threonine kinase, while ORF21 encodes a thymidine kinase (TK), which has recently been found to be an efficient tyrosine kinase. In this study, we explore the role of the ORF21 tyrosine kinase function in KSHV lytic replication. By generating a recombinant KSHV mutant with an enzymatically inactive ORF21 protein we show that the tyrosine kinase function of ORF21/TK is not required for the progression of the lytic replication in tissue culture, but that it is essential for the phosphorylation and activation to toxic moieties of the antiviral drugs zidovudine and brivudine. In addition, we identify several tyrosine kinase inhibitors, already in clinical use against human malignancies, which potently inhibit not only ORF21 TK kinase function, but also viral lytic reactivation and the development of KSHV-infected endothelial tumors in mice. As they target both cellular tyrosine kinases and a viral kinase, some of these compounds might find a use in the treatment of KSHV-associated malignancies.Importance: Our findings address the role of KSHV ORF21 as a tyrosine kinase during lytic replication and the activation of prodrugs in KSHV-infected cells. We also show the potential of selected clinically approved tyrosine kinase inhibitors to inhibit KSHV TK, KSHV lytic replication, infectious virions release and the development of an endothelial tumor. Since they target both cellular tyrosine kinases supporting productive viral replication and a viral kinase, these drugs, which are already approved for clinical use, may be suitable for repurposing for the treatment of KSHV-related tumors in AIDS patients or transplant recipients.
2020-01-20T14:22:18Z
2020-01-20T14:22:18Z
2019-12-11
Article
J Virol. 2019 Dec 11. pii: JVI.01791-19. doi: 10.1128/JVI.01791-19.
1098-5514
31826996
10.1128/JVI.01791-19
http://hdl.handle.net/10033/622093
Journal of Virology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
American Society for Microbiology (ASM)
Journal of virology
oai:repository.helmholtz-hzi.de:10033/6222672020-05-23T03:32:03Zcom_10033_620652col_10033_620672
2020-05-22T12:49:06Z
urn:hdl:10033/622267
Innate signalling molecules as genetic adjuvants do not alter the efficacy of a DNA-based influenza A vaccine.
Lapuente, Dennis
Stab, Viktoria
Storcksdieck Genannt Bonsmann, Michael
Maaske, Andre
Köster, Mario
Xiao, Han
Ehrhardt, Christina
Tenbusch, Matthias
HZI, Helmholtz Zentrum für Infektionsforschung, GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
In respect to the heterogeneity among influenza A virus strains and the shortcomings of current vaccination programs, there is a huge interest in the development of alternative vaccines that provide a broader and more long-lasting protection. Gene-based approaches are considered as promising candidates for such flu vaccines. In our study, innate signalling molecules from the RIG-I and the NALP3 pathways were evaluated as genetic adjuvants in intramuscular DNA immunizations. Plasmids encoding a constitutive active form of RIG-I (cRIG-I), IPS-1, IL-1β, or IL-18 were co-administered with plasmids encoding the hemagglutinin and nucleoprotein derived from H1N1/Puerto Rico/8/1934 via electroporation in BALB/c mice. Immunogenicity was analysed in detail and efficacy was demonstrated in homologous and heterologous influenza challenge experiments. Although the biological activities of the adjuvants have been confirmed by in vitro reporter assays, their single or combined inclusion in the vaccine did not result in superior vaccine efficacy. With the exception of significantly increased levels of antigen-specific IgG1 after the co-administration of IL-1β, there were only minor alterations concerning the immunogenicity. Since DNA electroporation alone induced substantial inflammation at the injection site, as demonstrated in this study using Mx2-Luc reporter mice, it might override the adjuvants´ contribution to the inflammatory microenvironment and thereby minimizes the influence on the immunogenicity. Taken together, the DNA immunization was protective against subsequent challenge infections but could not be further improved by the genetic adjuvants analysed in this study.
2020-05-22T12:49:06Z
2020-05-22T12:49:06Z
2020-04-03
Article
PLoS One. 2020;15(4):e0231138. Published 2020 Apr 3. doi:10.1371/journal.pone.0231138
32243477
10.1371/journal.pone.0231138
http://hdl.handle.net/10033/622267
1932-6203
PloS one
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
PLOS
15
4
e0231138
PloS one
United States
oai:repository.helmholtz-hzi.de:10033/6223002020-06-17T01:30:52Zcom_10033_620533com_10033_311624com_10033_6839com_10033_620652com_10033_620618col_10033_620672col_10033_620534col_10033_311625col_10033_620622
2020-06-16T12:57:22Z
urn:hdl:10033/622300
Expansion of functional personalized cells with specific transgene combinations.
Lipps, Christoph
Klein, Franziska
Wahlicht, Tom
Seiffert, Virginia
Butueva, Milada
Zauers, Jeannette
Truschel, Theresa
Luckner, Martin
Köster, Mario
MacLeod, Roderick
Pezoldt, Jörn
Hühn, Jochen
Yuan, Qinggong
Müller, Peter Paul
Kempf, Henning
Zweigerdt, Robert
Dittrich-Breiholz, Oliver
Pufe, Thomas
Beckmann, Rainer
Drescher, Wolf
Riancho, Jose
Sañudo, Carolina
Korff, Thomas
Opalka, Bertram
Rebmann, Vera
Göthert, Joachim R
Alves, Paula M
Ott, Michael
Schucht, Roland
Hauser, Hansjörg
Wirth, Dagmar
May, Tobias
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Fundamental research and drug development for personalized medicine necessitates cell cultures from defined genetic backgrounds. However, providing sufficient numbers of authentic cells from individuals poses a challenge. Here, we present a new strategy for rapid cell expansion that overcomes current limitations. Using a small gene library, we expanded primary cells from different tissues, donors, and species. Cell-type-specific regimens that allow the reproducible creation of cell lines were identified. In depth characterization of a series of endothelial and hepatocytic cell lines confirmed phenotypic stability and functionality. Applying this technology enables rapid, efficient, and reliable production of unlimited numbers of personalized cells. As such, these cell systems support mechanistic studies, epidemiological research, and tailored drug development.
2020-06-16T12:57:22Z
2020-06-16T12:57:22Z
2018-03-08
Article
Other
Nat Commun. 2018;9(1):994. Published 2018 Mar 8. doi:10.1038/s41467-018-03408-4.
29520052
10.1038/s41467-018-03408-4
http://hdl.handle.net/10033/622300
2041-1723
Nature communications
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Springer Nature
9
1
994
Nature communications
England
oai:repository.helmholtz-hzi.de:10033/6224122020-08-19T02:46:33Zcom_10033_620652col_10033_620672
2020-08-18T09:28:42Z
urn:hdl:10033/622412
Targeting cardiac fibrosis with engineered T cells.
Aghajanian, Haig
Kimura, Toru
Rurik, Joel G
Hancock, Aidan S
Leibowitz, Michael S
Li, Li
Scholler, John
Monslow, James
Lo, Albert
Han, Wei
Wang, Tao
Bedi, Kenneth
Morley, Michael P
Linares Saldana, Ricardo A
Bolar, Nikhita A
McDaid, Kendra
Assenmacher, Charles-Antoine
Smith, Cheryl L
Wirth, Dagmar
June, Carl H
Margulies, Kenneth B
Jain, Rajan
Puré, Ellen
Albelda, Steven M
Epstein, Jonathan A
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Fibrosis is observed in nearly every form of myocardial disease1. Upon injury, cardiac fibroblasts in the heart begin to remodel the myocardium by depositing excess extracellular matrix, resulting in increased stiffness and reduced compliance of the tissue. Excessive cardiac fibrosis is an important factor in the progression of various forms of cardiac disease and heart failure2. However, clinical interventions and therapies that target fibrosis remain limited3. Here we demonstrate the efficacy of redirected T cell immunotherapy to specifically target pathological cardiac fibrosis in mice. We find that cardiac fibroblasts that express a xenogeneic antigen can be effectively targeted and ablated by adoptive transfer of antigen-specific CD8+ T cells. Through expression analysis of the gene signatures of cardiac fibroblasts obtained from healthy and diseased human hearts, we identify an endogenous target of cardiac fibroblasts-fibroblast activation protein. Adoptive transfer of T cells that express a chimeric antigen receptor against fibroblast activation protein results in a significant reduction in cardiac fibrosis and restoration of function after injury in mice. These results provide proof-of-principle for the development of immunotherapeutic drugs for the treatment of cardiac disease.
2020-08-18T09:28:42Z
2020-08-18T09:28:42Z
2019-09-11
Article
Other
Nature. 2019;573(7774):430-433. doi:10.1038/s41586-019-1546-z.
31511695
10.1038/s41586-019-1546-z
http://hdl.handle.net/10033/622412
1476-4687
Nature
PMC6752964
en
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752964/
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Nature publishing group(NPG)
573
7774
430
433
Nature
United States
United States
United States
United States
United States
United States
England
oai:repository.helmholtz-hzi.de:10033/6226052020-11-24T01:42:33Zcom_10033_311624com_10033_6839com_10033_620652col_10033_620673col_10033_620672col_10033_311625
2020-11-23T15:22:40Z
urn:hdl:10033/622605
Synthetic rewiring and boosting type I interferon responses for visualization and counteracting viral infections.
Gödecke, Natascha
Riedel, Jan
Herrmann, Sabrina
Behme, Sara
Rand, Ulfert
Kubsch, Tobias
Cicin-Sain, Luka
Hauser, Hansjörg
Köster, Mario
Wirth, Dagmar
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Mammalian first line of defense against viruses is accomplished by the interferon (IFN) system. Viruses have evolved numerous mechanisms to reduce the IFN action allowing them to invade the host and/or to establish latency. We generated an IFN responsive intracellular hub by integrating the synthetic transactivator tTA into the chromosomal Mx2 locus for IFN-based activation of tTA dependent expression modules. The additional implementation of a synthetic amplifier module with positive feedback even allowed for monitoring and reacting to infections of viruses that can antagonize the IFN system. Low and transient IFN amounts are sufficient to trigger these amplifier cells. This gives rise to higher and sustained-but optionally de-activatable-expression even when the initial stimulus has faded out. Amplification of the IFN response induced by IFN suppressing viruses is sufficient to protect cells from infection. Together, this interfaced sensor/actuator system provides a toolbox for robust sensing and counteracting viral infections.
2020-11-23T15:22:40Z
2020-11-23T15:22:40Z
2020-11-18
Article
Nucleic Acids Res. 2020 Nov 18;48(20):11799-11811. doi: 10.1093/nar/gkaa961.
33137201
10.1093/nar/gkaa961
http://hdl.handle.net/10033/622605
1362-4962
Nucleic acids research
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Oxford Academic
48
20
11799
11811
Nucleic acids research
England
oai:repository.helmholtz-hzi.de:10033/6227352021-02-12T02:20:29Zcom_10033_620652col_10033_620672
2021-02-11T15:42:55Z
urn:hdl:10033/622735
3D culture conditions support Kaposi's sarcoma herpesvirus (KSHV) maintenance and viral spread in endothelial cells.
Dubich, Tatyana
Dittrich, Anne
Bousset, Kristine
Geffers, Robert
Büsche, Guntram
Köster, Mario
Hauser, Hansjörg
Schulz, Thomas F
Wirth, Dagmar
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
3D culture
Episomal viral genomes
KSHV infected endothelial cells
Viral maintenance
Xenograft model
Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumorigenic virus and the etiological agent of an endothelial tumor (Kaposi's sarcoma) and two B cell proliferative diseases (primary effusion lymphoma and multicentric Castleman's disease). While in patients with late stage of Kaposi's sarcoma the majority of spindle cells are KSHV-infected, viral copies are rapidly lost in vitro, both upon culture of tumor-derived cells or from newly infected endothelial cells. We addressed this discrepancy by investigating a KSHV-infected endothelial cell line in various culture conditions and in tumors of xenografted mice. We show that, in contrast to two-dimensional endothelial cell cultures, KSHV genomes are maintained under 3D cell culture conditions and in vivo. Additionally, an increased rate of newly infected cells was detected in 3D cell culture. Furthermore, we show that the PI3K/Akt/mTOR and ATM/γH2AX pathways are modulated and support an improved KSHV persistence in 3D cell culture. These mechanisms may contribute to the persistence of KSHV in tumor tissue in vivo and provide a novel target for KS specific therapeutic interventions. KEY MESSAGES: In vivo maintenance of episomal KSHV can be mimicked in 3D spheroid cultures 3D maintenance of KSHV is associated with an increased de novo infection frequency PI3K/Akt/mTOR and ATM/ γH2AX pathways contribute to viral maintenance.
2021-02-11T15:42:55Z
2021-02-11T15:42:55Z
2021-01-23
Article
J Mol Med (Berl). 2021 Jan 23. doi: 10.1007/s00109-020-02020-8. Epub ahead of print.
33484281
10.1007/s00109-020-02020-8
http://hdl.handle.net/10033/622735
1432-1440
Journal of molecular medicine (Berlin, Germany)
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
Springer International
Journal of molecular medicine (Berlin, Germany)
Germany
oai:repository.helmholtz-hzi.de:10033/6227892021-03-24T01:35:41Zcom_10033_620626com_10033_620652col_10033_620672col_10033_620627
2021-03-23T16:36:59Z
urn:hdl:10033/622789
Improved Functionality of Exhausted Intrahepatic CXCR5+ CD8+ T Cells Contributes to Chronic Antigen Clearance Upon Immunomodulation.
Kumashie, Kingsley Gideon
Cebula, Marcin
Hagedorn, Claudia
Kreppel, Florian
Pils, Marina C
Koch-Nolte, Friedrich
Rissiek, Björn
Wirth, Dagmar
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
CXCR5+ T cells
CpG oligonucleotide
T cell exhaustion
T cell reinvigoration
exhausted stem-like T cells
follicular helper-like T cells
liver
liver resident T cells
Chronic hepatotropic viral infections are characterized by exhausted CD8+ T cells in the presence of cognate antigen in the liver. The impairment of T cell response limits the control of chronic hepatotropic viruses. Immune-modulatory strategies are attractive options to re-invigorate exhausted T cells. However, in hepatotropic viral infections, the knowledge about immune-modulatory effects on the in-situ regulation of exhausted intrahepatic CD8+ T cells is limited. In this study, we elucidated the functional heterogeneity in the pool of exhausted CD8+ T cells in the liver of mice expressing the model antigen Ova in a fraction of hepatocytes. We found a subpopulation of intrahepatic CXCR5+ Ova-specific CD8+ T cells, which are profoundly cytotoxic, exhibiting efficient metabolic functions as well as improved memory recall and self-maintenance. The intrahepatic Ova-specific CXCR5+ CD8+ T cells are possibly tissue resident cells, which may rely largely on OXPHOS and glycolysis to fuel their cellular processes. Importantly, host conditioning with CpG oligonucleotide reinvigorates and promotes exhausted T cell expansion, facilitating complete antigen eradication. The CpG oligonucleotide-mediated reinvigoration may support resident memory T cell formation and the maintenance of CXCR5+ Ova-specific CD8+ T cells in the liver. These findings suggest that CpG oligodinucleotide may preferentially target CXCR5+ CD8+ T cells for expansion to facilitate the revival of exhausted T cells. Thus, therapeutic strategies aiming to expand CXCR5+ CD8+ T cells might provide a novel approach against chronic liver infection.
2021-03-23T16:36:59Z
2021-03-23T16:36:59Z
2021-02-03
Article
Front Immunol. 2021 Feb 3;11:592328. doi: 10.3389/fimmu.2020.592328.
33613516
10.3389/fimmu.2020.592328
http://hdl.handle.net/10033/622789
1664-3224
Frontiers in immunology
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
Frontiers
11
592328
Frontiers in immunology
Switzerland
oai:repository.helmholtz-hzi.de:10033/6228732021-05-18T01:59:49Zcom_10033_620652col_10033_620672
2021-05-17T12:08:28Z
urn:hdl:10033/622873
The Immunomodulatory CEA Cell Adhesion Molecule 6 (CEACAM6/CD66c) Is a Protein Receptor for the Influenza a Virus.
Rahman, Shah Kamranur
Ansari, Mairaj Ahmed
Gaur, Pratibha
Ahmad, Imtiyaz
Chakravarty, Chandrani
Verma, Dileep Kumar
Sharma, Anshika
Chhibber, Sanjay
Nehal, Naila
Wirth, Dagmar
Lal, Sunil K
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
CD66c
CEACAM6
IgG super family
carcinoembryonic antigen (CEA)
carcinoembryonic cell adhesion molecule (CEACAM)
flu
hemagglutinin
influenza A Virus
lipid raft
neuraminidase
receptor
sialic acid
virus
To establish a productive infection in host cells, viruses often use one or multiple host membrane glycoproteins as their receptors. For Influenza A virus (IAV) such a glycoprotein receptor has not been described, to date. Here we show that IAV is using the host membrane glycoprotein CD66c as a receptor for entry into human epithelial lung cells. Neuraminidase (NA), a viral spike protein, binds to CD66c on the cell surface during IAV entry into the host cells. Lung cells overexpressing CD66c showed an increase in virus binding and subsequent entry into the cell. Upon comparison, CD66c demonstrated higher binding capacity than other membrane glycoproteins (EGFR and DC-SIGN) reported earlier to facilitate IAV entry into host cells. siRNA mediated knockdown of CD66c from lung cells inhibited virus binding on cell surface and entry into cells. Blocking CD66c by antibody on the cell surface resulted in decreased virus entry. We found that CD66c is a specific glycoprotein receptor for influenza A virus that did not affect entry of non-IAV RNA virus (Hepatitis C virus). Finally, IAV pre-incubated with recombinant CD66c protein when administered intranasally in mice showed decreased cytopathic effects in mice lungs. This publication is the first to report CD66c (Carcinoembryonic cell adhesion molecule 6 or CEACAM6) as a glycoprotein receptor for Influenza A virus.
2021-05-17T12:08:28Z
2021-05-17T12:08:28Z
2021-04-21
Article
Viruses. 2021 Apr 21;13(5):726. doi: 10.3390/v13050726.
33919410
10.3390/v13050726
http://hdl.handle.net/10033/622873
1999-4915
Viruses
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
MDPI
13
5
Viruses
Switzerland
oai:repository.helmholtz-hzi.de:10033/6229742021-07-31T02:37:58Zcom_10033_620652com_10033_620601col_10033_620666col_10033_620672col_10033_620602
2021-07-30T13:33:26Z
urn:hdl:10033/622974
B cell depletion impairs vaccination-induced CD8 T cell responses in a type I interferon-dependent manner.
Graalmann, Theresa
Borst, Katharina
Manchanda, Himanshu
Vaas, Lea
Bruhn, Matthias
Graalmann, Lukas
Koster, Mario
Verboom, Murielle
Hallensleben, Michael
Guzmán, Carlos Alberto
Sutter, Gerd
Schmidt, Reinhold E
Witte, Torsten
Kalinke, Ulrich
TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany.; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Arthritis
B-Lymphocytes
Rheumatoid
Rituximab
T-Lymphocyte subsets
Vaccination
Objectives: The monoclonal anti-CD20 antibody rituximab is frequently applied in the treatment of lymphoma as well as autoimmune diseases and confers efficient depletion of recirculating B cells. Correspondingly, B cell-depleted patients barely mount de novo antibody responses during infections or vaccinations. Therefore, efficient immune responses of B cell-depleted patients largely depend on protective T cell responses.
Methods: CD8+ T cell expansion was studied in rituximab-treated rheumatoid arthritis (RA) patients and B cell-deficient mice on vaccination/infection with different vaccines/pathogens.
Results: Rituximab-treated RA patients vaccinated with Influvac showed reduced expansion of influenza-specific CD8+ T cells when compared with healthy controls. Moreover, B cell-deficient JHT mice infected with mouse-adapted Influenza or modified vaccinia virus Ankara showed less vigorous expansion of virus-specific CD8+ T cells than wild type mice. Of note, JHT mice do not have an intrinsic impairment of CD8+ T cell expansion, since infection with vaccinia virus induced similar T cell expansion in JHT and wild type mice. Direct type I interferon receptor signalling of B cells was necessary to induce several chemokines in B cells and to support T cell help by enhancing the expression of MHC-I.
Conclusions: Depending on the stimulus, B cells can modulate CD8+ T cell responses. Thus, B cell depletion causes a deficiency of de novo antibody responses and affects the efficacy of cellular response including cytotoxic T cells. The choice of the appropriate vaccine to vaccinate B cell-depleted patients has to be re-evaluated in order to efficiently induce protective CD8+ T cell responses.
2021-07-30T13:33:26Z
2021-07-30T13:33:26Z
2021-07-05
Article
Ann Rheum Dis. 2021 Jul 5:annrheumdis-2021-220435. doi: 10.1136/annrheumdis-2021-220435. Epub ahead of print.
34226189
10.1136/annrheumdis-2021-220435
http://hdl.handle.net/10033/622974
1468-2060
Annals of the rheumatic diseases
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
BMJ Publishing Group
Annals of the rheumatic diseases
England
oai:repository.helmholtz-hzi.de:10033/6230342021-09-17T13:48:14Zcom_10033_620652col_10033_620672
2021-09-15T14:47:21Z
urn:hdl:10033/623034
Defective interferon amplification and impaired host responses against influenza virus in obese mice.
Gaur, Pratibha
Riehn, Mathias
Zha, Lisha
Köster, Mario
Hauser, Hansjörg
Wirth, Dagmar
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Objective: Obesity is a major risk factor that increases morbidity and mortality upon infection. Although type I and type III interferon (IFN)-induced innate immune responses represent the first line of defense against viral infections, their functionality in the context of metabolic disorders remains largely obscure. This study aimed to investigate IFN responses upon respiratory viral infection in obese mice.
Methods: The activation of IFNs as well as IFN regulatory factors (IRFs) upon H3N2 influenza infection in mice upon high-fat-diet feeding was investigated.
Results: Influenza infection of obese mice was characterized by higher mortalities. In-depth analysis revealed impaired induction of both type I and type III IFNs as well as markedly reduced IFN responses. Notably, it was found that IRF7 gene expression in obese animals was reduced in homeostasis, and its induction by the virus was strongly attenuated.
Conclusions: The results suggest that the attenuated IRF7 expression and induction are responsible for the reduced expression levels of type I and III IFNs and, thus, for the higher susceptibility and severity of respiratory infections in obese mice.
2021-09-15T14:47:21Z
2021-09-15T14:47:21Z
2021-07-07
Review
Obesity (Silver Spring). 2021 Aug;29(8):1272-1278. doi: 10.1002/oby.23196.
34314110
10.1002/oby.23196
http://hdl.handle.net/10033/623034
1930-739X
Obesity (Silver Spring, Md.)
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
Wiley
29
8
1272
1278
Obesity (Silver Spring, Md.)
United States
oai:repository.helmholtz-hzi.de:10033/6230672021-10-09T01:58:40Zcom_10033_620652col_10033_620672
2021-10-08T09:22:11Z
urn:hdl:10033/623067
Defective interferon amplification and impaired host responses against influenza virus in obese mice.
Gaur, Pratibha
Riehn, Mathias
Zha, Lisha
Köster, Mario
Hauser, Hansjörg
Wirth, Dagmar
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Objective: Obesity is a major risk factor that increases morbidity and mortality upon infection. Although type I and type III interferon (IFN)-induced innate immune responses represent the first line of defense against viral infections, their functionality in the context of metabolic disorders remains largely obscure. This study aimed to investigate IFN responses upon respiratory viral infection in obese mice.
Methods: The activation of IFNs as well as IFN regulatory factors (IRFs) upon H3N2 influenza infection in mice upon high-fat-diet feeding was investigated.
Results: Influenza infection of obese mice was characterized by higher mortalities. In-depth analysis revealed impaired induction of both type I and type III IFNs as well as markedly reduced IFN responses. Notably, it was found that IRF7 gene expression in obese animals was reduced in homeostasis, and its induction by the virus was strongly attenuated.
Conclusions: The results suggest that the attenuated IRF7 expression and induction are responsible for the reduced expression levels of type I and III IFNs and, thus, for the higher susceptibility and severity of respiratory infections in obese mice.
© 2021 The Authors. Obesity published by Wiley Periodicals LLC on behalf of The Obesity Society (TOS).
2021-10-08T09:22:11Z
2021-10-08T09:22:11Z
2021-07-27
Article
Obesity (Silver Spring). 2021 Aug;29(8):1272-1278. doi: 10.1002/oby.23196.
34314110
10.1002/oby.23196
http://hdl.handle.net/10033/623067
1930-739X
Obesity (Silver Spring, Md.)
en
http://creativecommons.org/licenses/by-nc-nd/4.0/
Attribution-NonCommercial-NoDerivatives 4.0 International
Wiley
29
8
1272
1278
Obesity (Silver Spring, Md.)
United States