Braunschweig, Germany


Braunschweig, Germany
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Holzki J.K.,Helmholtz Center for Infection Research | Dag F.,Helmholtz Center for Infection Research | Dekhtiarenko I.,Helmholtz Center for Infection Research | Rand U.,Helmholtz Center for Infection Research | And 7 more authors.
Journal of Virology | Year: 2015

Cytomegalovirus (CMV) is a ubiquitous beta-herpesvirus whose reactivation from latency is a major cause of morbidity and mortality in immunocompromised hosts. Mouse CMV (MCMV) is a well-established model virus to study virus-host interactions. We showed in this study that the CD8-independent antiviral function of myeloid dendritic cells (mDC) is biologically relevant for the inhibition of MCMV replication in vivo and in vitro. In vivo ablation of CD11c+ DC resulted in higher viral titers and increased susceptibility to MCMV infection in the first 3 days postinfection. We developed in vitro coculture systems in which we cocultivated MCMV-infected endothelial cells or fibroblasts with T cell subsets and/or dendritic cells. While CD8 T cells failed to control MCMV replication, bone marrow-derived mDC reduced viral titers by a factor of up to 10,000. Contact of mDC with the infected endothelial cells was crucial for their antiviral activity. Soluble factors secreted by the mDC blocked MCMV replication at the level of immediate early (IE) gene expression, yet the viral lytic cycle reinitiated once the mDC were removed from the cells. On the other hand, the mDC did not impair MCMV replication in cells deficient for the interferon (IFN) alpha/beta receptor (IFNAR), arguing that type I interferons were critical for viral control by mDC. In light of our recent observation that type I IFN is sufficient for the induction of latency immediately upon infection, our results imply that IFN secreted by mDC may play an important role in the establishment of CMV latency. © 2015, American Society for Microbiology.

PubMed | University of Oslo, Saarland University, InSCREENeX GmbH, Helmholtz Institute for Pharmaceutical Research Saarland HIPS and 2 more.
Type: Journal Article | Journal: ALTEX | Year: 2016

This paper describes a new human alveolar epithelial cell line (hAELVi - human Alveolar Epithelial Lentivirus immortalized) with type I-like characteristics and functional tight junctions, suitable to model the air-blood barrier of the peripheral lung. Primary human alveolar epithelial cells were immortalized by a novel regimen, grown as monolayers on permeable filter supports and characterized morphologically, biochemically and biophysically. hAELVi cells maintain the capacity to form tight intercellular junctions, with high trans-epithelial electrical resistance (> 1000 *cm). The cells could be kept in culture over several days, up to passage 75, under liquid-liquid as well as air-liquid conditions. Ultrastructural analysis and real time PCR revealed type I-like cell properties, such as the presence of caveolae, expression of caveolin-1, and absence of surfactant protein C. Accounting for the barrier properties, inter-digitations sealed with tight junctions and desmosomes were also observed. Low permeability of the hydrophilic marker sodium fluorescein confirmed the suitability of hAELVi cells for in vitro transport studies across the alveolar epithelium. These results suggest that hAELVi cells reflect the essential features of the air-blood barrier, as needed for an alternative to animal testing to study absorption and toxicity of inhaled drugs, chemicals and nanomaterials.

Heiss M.,University of Heidelberg | Hellstrom M.,Uppsala University | Kalen M.,Uppsala University | May T.,InSCREENeX GmbH | And 5 more authors.
FASEB Journal | Year: 2015

Given the need for robust and costefficient in vitro models to study angiogenesis and reproducibly analyze potential pro- and antiangiogenic compounds in preclinical studies, we developed a 3- dimensional in vitro angiogenesis assay that is based on collagen gel-embedded, size-defined spheroids generated from cultured human umbilical vein endothelial cells (HUVECs). Despite its wide distribution, limitations, sensitivity, robustness, and improvements, the capacity of this assay for functional screening purposes has not been elucidated thus far. By using time-lapse videomicroscopy, we show that tip cells lead the formation of capillary-like and partially lumenizedsprouts originating from the spheroids. Angiogenic sprouting from spheroids generated from 5 different primary cultured human endothelial cell types was induced by physiologic concentrations of vascular endothelial cell growth factor 165.Based on this assay system, we determined the capacity of 880 approved drugs to interfere with or boost angiogenic sprouting, thereby assessing their putative angiogenesis-related side effects or novel applications. However, although this assay allowed for a rapid and reproducible determination of functional IC50 values of individual compounds, the sprouting results were partially affectedby theHUVECpassage number and donor variability. To overcome this limitation, immortalized HUVECs (iHUVECs) showing a more homogenous response in terms of proliferation and sprouting over multiple population doublings were used in the course of this study. Collectively, the spheroid-based angiogenesis assay provides a sensitive and versatile tool to study the impact of pro- and antiangiogenic determinants on multiple steps of the angiogenic cascade. It is compatible with different endothelial cell types and allows use of iHUVECs to improve its overall robustness.- Heiss, M., Hellstrom,M., Kaĺen,M.,May, T.,Weber,H., Hecker,M., Augustin, H. G., Korff, T. Endothelial cell spheroids as a versatile tool to study angiogenesis in vitro.

Agency: European Commission | Branch: FP7 | Program: BSG-SME | Phase: SME-2012-1 | Award Amount: 1.43M | Year: 2012

Silicone if combined with appropriate cell adherent regulatory compounds offers significant advantages as a micro cell carrier, effectively a 3D microtissue device. These benefits include a capacity for defined and controlled porosity, inner chamber size, density, flexible shape, friction resistant, and low toxicity. The lead SME partner holds the IP for a silicone cell carrier device, which met some of these criteria, but exhibited poor cell adherence and requires re-engineering and exploitation of current cell molecular knowledge. There is a strong commercial need for such fully functional 3D cell culture technologies (microtissue) that can be manufactured into robust and reproducible in vitro test systems for toxicity testing, drug testing, cosmetic testing as part of a projected 1.6b cell based test market. This device may also eventually function as an active component in bioartificial livers (BAL) and other organ devices. The market growth is driven by needs in drug screening for drug discovery and toxicity screening. There are also a number of current EU directives that is driving the demand for reliable in vitro 3D cell based test system to replace in vivo test systems. The bio artificial liver market is also expected to grow rapidly due to high rates of liver disease worldwide. This project will develop novel develop new 3D cell culture technology based on proprietary cell immortalisation and the owned cell immobilisation technology. The objectives will be to deliver: 1) Immortalised hepatocyte and endothelial cells that retain characteristics of primary liver cells in 3D co-culture. 2) A porous silicone carrier for 3D culture 3) A scaleable bioreactor systems for 3D culture The following products will be developed using this technology, 1) 3D liver cell multiwell plate assay system, 2) 3D liver microbioreactor system and 3) a bioreactor component for a BAL device. Minimal SME economic return is predicted at 12m within 3 years of project completion

May T.,InSCREENeX GmbH | Hauser H.,Helmholtz Center for Infection Research | Wirth D.,Helmholtz Center for Infection Research
BioSpektrum | Year: 2012

Cell lines are immortal cultures of cells that are used in research as well as for production of pharmaceuticals. Currently available cell lines are derived from normal and tumorous tissue of humans and diverse animal species, however, with considerable loss of specific properties. The development of new immortalization protocols has led to a significant improvement of the properties of the resulting cell lines. These retain specific properties and activities that include functional grafting. © Springer-Verlag 2012.

Schwerk J.,Helmholtz Center for Infection Research | Koster M.,Helmholtz Center for Infection Research | Hauser H.,Helmholtz Center for Infection Research | Rohde M.,Helmholtz Center for Infection Research | And 3 more authors.
PLoS ONE | Year: 2013

Cell lines derived from the small intestine that reflect authentic properties of the originating intestinal epithelium are of high value for studies on mucosal immunology and host microbial homeostasis. A novel immortalization procedure was applied to generate continuously proliferating cell lines from murine E19 embryonic small intestinal tissue. The obtained cell lines form a tight and polarized epithelial cell layer, display characteristic tight junction, microvilli and surface protein expression and generate increasing transepithelial electrical resistance during in vitro culture. Significant up-regulation of Cxcl2 and Cxcl5 chemokine expression upon exposure to defined microbial innate immune stimuli and endogenous cytokines is observed. Cell lines were also generated from a transgenic interferon reporter (Mx2-Luciferase) mouse, allowing reporter technology-based quantification of the cellular response to type I and III interferon. Thus, the newly created cell lines mimic properties of the natural epithelium and can be used for diverse studies including testing of the absorption of drug candidates. The reproducibility of the method to create such cell lines from wild type and transgenic mice provides a new tool to study molecular and cellular processes of the epithelial barrier. © 2013 Schwerk et al.

Riese P.,Helmholtz Center for Infection Research | Trittel S.,Helmholtz Center for Infection Research | May T.,Helmholtz Center for Infection Research | May T.,InSCREENeX GmbH | And 3 more authors.
European Journal of Immunology | Year: 2015

NK cells represent a vital component of the innate immune system. The recent discoveries demonstrating that the functionality of NK cells depends on their differentiation and education status underscore their potential as targets for immune intervention. However, to exploit their full potential, a detailed understanding of the cellular interactions involved in these processes is required. In this regard, the cross-talk between NKT cells and NK cells needs to be better understood. Our results provide strong evidence for NKT cell-induced effects on key biological features of NK cells. NKT-cell activation results in the generation of highly active CD27high NK cells with improved functionality. In this context, degranulation activity and IFNγ production were mainly detected in the educated subset. In a mCMV infection model, we also demonstrated that NKT-cell stimulation induced the generation of highly functional educated and uneducated NK cells, crucial players in viral control. Thus, our findings reveal new fundamental aspects of the NKT-NK cell axis that provide important hints for the manipulation of NK cells in clinical settings. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lipps C.,Helmholtz Center for Infection Research | May T.,InSCREENeX GmbH | Hauser H.,Helmholtz Center for Infection Research | Wirth D.,Helmholtz Center for Infection Research
Biological Chemistry | Year: 2013

In the first 50 years of cell culture, the development of new cell lines was mainly based on trial and error. Due to the understanding of the molecular networks of aging, senescence, proliferation, and adaption by mutation, the generation of new cell lines with physiologic properties has become more systematic. This endeavor has been supported by the availability of new technological achievements and increasing knowledge about the biology of cell differentiation and cell-cell communication. Here, we review some promising developments that are contributing toward this goal. These include molecular tools frequently used for the immortalization process. In addition to these broadly acting immortalization regimens, we focus on the developments of cell type-specific immortalization and on the methodologies of how to control the growth of newly established cell lines.

PubMed | Free University of Berlin, Inscreenex GmbH, Helmholtz Center for Infection Research, Rudolf Buchheim Institute for Pharmacology and Leibniz University of Hanover
Type: | Journal: Cellular and molecular life sciences : CMLS | Year: 2016

Homeostasis of solid tissue is characterized by a low proliferative activity of differentiated cells while special conditions like tissue damage induce regeneration and proliferation. For some cell types it has been shown that various tissue-specific functions are missing in the proliferating state, raising the possibility that their proliferation is not compatible with a fully differentiated state. While endothelial cells are important players in regenerating tissue as well as in the vascularization of tumors, the impact of proliferation on their features remains elusive. To examine cell features in dependence of proliferation, we established human endothelial cell lines in which proliferation is tightly controlled by a doxycycline-dependent, synthetic regulatory unit. We observed that uptake of macromolecules and establishment of cell-cell contacts was more pronounced in the growth-arrested state. Tube-like structures were formed in vitro in both proliferating and non-proliferating conditions. However, functional vessel formation upon transplantation into immune-compromised mice was restricted to the proliferative state. Kaposis sarcoma-associated herpes virus (KSHV) infection resulted in reduced expression of endothelial markers. Upon transplantation of infected cells, drastic differences were observed: proliferation arrested cells acquired a high migratory activity while the proliferating counterparts established a tumor-like phenotype, similar to Kaposi Sarcoma lesions. The study gives evidence that proliferation governs endothelial functions. This suggests that several endothelial functions are differentially expressed during angiogenesis. Moreover, since proliferation defines the functional properties of cells upon infection with KSHV, this process crucially affects the fate of virus-infected cells.

PubMed | University of Cantabria and InSCREENeX GmbH
Type: | Journal: Journal of bone and mineral metabolism | Year: 2016

Different model systems using osteoblastic cell lines have been developed to help understand the process of bone formation. Here, we report the establishment of two human osteoblastic cell lines obtained from primary cultures upon transduction of immortalizing genes. The resulting cell lines had no major differences to their parental lines in their gene expression profiles. Similar to primary osteoblastic cells, osteocalcin transcription increased following 1,25-dihydroxyvitamin D

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