2024-03-29T02:18:11Zhttp://repository.helmholtz-hzi.de/oai/requestoai:repository.helmholtz-hzi.de:10033/84032019-08-30T11:32:38Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Ben-Asouli, Yitzhak
author
Banai, Yona
author
Hauser, Hansjoerg
author
Kaempfer, Raymond
author
2000-02-15
Nucleic Acids Research 2000 28(4):1011-1018
0305-1048
1362-4962
10648795
http://hdl.handle.net/10033/8403
102579
Recognition of 5′-terminal TAR structure in human immunodeficiency virus-1 mRNA by eukaryotic translation initiation factor 2
oai:repository.helmholtz-hzi.de:10033/86072019-08-30T11:32:38Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Kollmus, H
author
Flohé, L
author
McCarthy, J E
author
1996-04-01
Nucleic Acids Research 1996 24(7):1195-1201
0305-1048
1362-4962
8614619
http://hdl.handle.net/10033/8607
145795
Analysis of eukaryotic mRNA structures directing cotranslational incorporation of selenocysteine.
oai:repository.helmholtz-hzi.de:10033/85212019-08-30T11:32:38Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Karreman, S
author
Hauser, Hansjoerg
author
Karreman, C
author
1996-05-01
Nucleic Acids Research 1996 24(9):1616-1624
0305-1048
1362-4962
8649977
http://hdl.handle.net/10033/8521
145857
On the use of double FLP recognition targets (FRTs) in the LTR of retroviruses for the construction of high producer cell lines.
oai:repository.helmholtz-hzi.de:10033/85912019-08-30T11:24:24Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Feng, Xuesheng
author
Guo, Zhong
author
Nourbakhsh, Mahtab
author
Hauser, Hansjorg
author
Ganster, Ray
author
Shao, Lifang
author
Geller, David A.
author
2002-10-29
Proceedings of the National Academy of Sciences of the United States of America 2002 99(22):14212-14217
0027-8424
1091-6490
12381793
10.1073/pnas.212306199
http://hdl.handle.net/10033/8591
137863
Identification of a negative response element in the human inducible nitric-oxide synthase (hiNOS) promoter: The role of NF-κB-repressing factor (NRF) in basal repression of the hiNOS gene
oai:repository.helmholtz-hzi.de:10033/85942019-08-30T11:24:31Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Wirth, M
author
Grannemann, R
author
Klehr, D
author
Hauser, Hansjoerg
author
1994-01
Images
Journal of Virology 1994 68(1):566-569
0022-538X
1098-5514
8254773
http://hdl.handle.net/10033/8594
236323
Screening retroviral packaging cells for highly efficient virus production by using a combined selection procedure.
oai:repository.helmholtz-hzi.de:10033/85952019-08-30T11:37:44Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Kollmus, H
author
Honigman, A
author
Panet, A
author
Hauser, H
author
1994-09
Images
Journal of Virology 1994 68(9):6087-6091
0022-538X
1098-5514
8057488
http://hdl.handle.net/10033/8595
237019
The sequences of and distance between two cis-acting signals determine the efficiency of ribosomal frameshifting in human immunodeficiency virus type 1 and human T-cell leukemia virus type II in vivo.
oai:repository.helmholtz-hzi.de:10033/85962019-08-30T11:25:43Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Reil, H
author
Kollmus, H
author
Weidle, U H
author
Hauser, Hansjoerg
author
1993-09
Images
Journal of Virology 1993 67(9):5579-5584
0022-538X
1098-5514
8350413
http://hdl.handle.net/10033/8596
237961
A heptanucleotide sequence mediates ribosomal frameshifting in mammalian cells.
oai:repository.helmholtz-hzi.de:10033/86512019-08-30T11:31:23Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Kirchhoff, S
author
Schaper, F
author
Hauser, Hansjoerg
author
1993-06-25
Images
Nucleic Acids Research 1993 21(12):2881-2889
0305-1048
1362-4962
8332497
http://hdl.handle.net/10033/8651
309674
Interferon regulatory factor 1 (IRF-1) mediates cell growth inhibition by transactivation of downstream target genes.
oai:repository.helmholtz-hzi.de:10033/86532019-08-30T11:26:13Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Oliveira, C C
author
Goossen, B
author
Zanchin, N I
author
McCarthy, J E
author
Hentze, M W
author
Stripecke, R
author
1993-11-25
Images
Nucleic Acids Research 1993 21(23):5316-5322
0305-1048
1362-4962
8265343
http://hdl.handle.net/10033/8653
310564
Translational repression by the human iron-regulatory factor (IRF) in Saccharomyces cerevisiae.
oai:repository.helmholtz-hzi.de:10033/86542019-08-30T11:26:13Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Hobom, G.
author
Schwarz, E.
author
Melzer, M.
author
Mayer, H.
author
1981-10-10
Images
Nucleic Acids Research 1981 9(19):4823-4832
0305-1048
1362-4962
6273787
http://hdl.handle.net/10033/8654
327482
Restriction endonuclease EcaI from Enterobacter cloacae
oai:repository.helmholtz-hzi.de:10033/86552019-08-30T11:26:13Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Rupp, E
author
Mayer, H
author
Wingender, E
author
1990-10-11
Images
Nucleic Acids Research 1990 18(19):5677-5683
0305-1048
1362-4962
1977134
http://hdl.handle.net/10033/8655
332300
The promoter of the human parathyroid hormone gene contains a functional cyclic AMP-response element.
oai:repository.helmholtz-hzi.de:10033/86932019-08-30T11:31:49Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Nourbakhsh, M
author
Hoffmann, K
author
Hauser, Hansjoerg
author
1993-02
Images
The EMBO Journal 1993 12(2):451-459
0261-4189
1460-2075
8440236
http://hdl.handle.net/10033/8693
413228
Interferon-beta promoters contain a DNA element that acts as a position-independent silencer on the NF-kappa B site.
oai:repository.helmholtz-hzi.de:10033/87012019-08-30T11:24:27Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Nourbakhsh, M
author
Hauser, Hansjoerg
author
1999-11-15
The EMBO Journal 1999 18(22):6415-6425
0261-4189
1460-2075
10562553
10.1093/emboj/18.22.6415
http://hdl.handle.net/10033/8701
1171704
Constitutive silencing of IFN-beta promoter is mediated by NRF (NF-kappaB-repressing factor), a nuclear inhibitor of NF-kappaB.
oai:repository.helmholtz-hzi.de:10033/87482019-08-30T11:25:09Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Kollmus, H
author
Hentze, M W
author
Hauser, Hansjoerg
author
1996-04
RNA 1996 2(4):316-323
1355-8382
1469-9001
8634912
http://hdl.handle.net/10033/8748
1369374
Regulated ribosomal frameshifting by an RNA-protein interaction.
oai:repository.helmholtz-hzi.de:10033/87542019-08-30T11:25:09Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Hoffmann, Andrea
author
Pelled, Gadi
author
Turgeman, Gadi
author
Eberle, Peter
author
Zilberman, Yoram
author
Shinar, Hadassah
author
Keinan-Adamsky, Keren
author
Winkel, Andreas
author
Shahab, Sandra
author
Navon, Gil
author
Gross, Gerhard
author
Gazit, Dan
author
2006-04-03
Journal of Clinical Investigation 2006 116(4):940-952
0021-9738
16585960
10.1172/JCI22689
http://hdl.handle.net/10033/8754
1421340
Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells
oai:repository.helmholtz-hzi.de:10033/123672019-08-30T11:25:43Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Mueller, Peter P
author
May, Tobias
author
Perz, Angela
author
Hauser, Hansjörg
author
Peuster, Matthias
author
2006-04-01
This study was conducted to determine the interaction of individual corrosion products from biodegradable iron stents with cells from the adjacent tissue. The response of human umbilical venous smooth muscle cells (SMCs) to an excess of ferrous ions was investigated in a cell culture model at the phenotypic and at the molecular level. When soluble ferrous ions were added to the cell culture medium the cell growth rate was reduced. Gene expression profiling indicated a reduction in the amounts of mRNA from genes that are required for cell proliferation. In addition, mRNA was regulated from multiple genes involved in iron homeostasis, DNA replication and lipid metabolism. In conclusion, ions released from iron stents could reduce the vascular SMC proliferation rate by influencing growth-related gene expression and may therefore play a beneficial role in antagonizing restenosis in vivo.
Biomaterials 2006, 27(10):2193-200
0142-9612
16310850
10.1016/j.biomaterials.2005.10.042
http://hdl.handle.net/10033/12367
Control of smooth muscle cell proliferation by ferrous iron.
oai:repository.helmholtz-hzi.de:10033/123992019-08-30T11:25:43Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Ma, Bin
author
Winkelbach, Simon
author
Lindenmaier, Werner
author
Dittmar, Kurt E J
author
2006
Multi-colour imaging of immunofluorescently labelled tissue using confocal microscopy was accomplished by using colour addition theory. This new technique includes several improvements for immunolabelling: (1) the co-localization of two or more markers on one cell for the identification of specific cell populations; (2) the co-localization of two fluorescent dyes from secondary reagents for the identification of the cells; (3) a multi-step staining protocol with two primary antibodies originating from the same host species or with two or three biotin-conjugated primary antibodies. After image acquisition, colour segmentation/unmixing are applied to the single multi-colour image to generate multi-pseudo-channels for individual or co-localized fluorescent dyes. With this new technique, we have been able to visualize six cell populations simultaneously in the mouse lymph node and intestine. The efficiency of this method has also been demonstrated in the three-dimensional reconstruction of thick sections from mouse ileum. Our method is simple, efficient, and may be indispensable in experimental cell and tissue studies requiring multiple immunolabelling.
Acta Histochem. 2006, 108(4):243-57
0065-1281
16730369
10.1016/j.acthis.2006.02.002
http://hdl.handle.net/10033/12399
Six-colour fluorescent imaging of lymphoid tissue based on colour addition theory.
oai:repository.helmholtz-hzi.de:10033/134582019-08-30T11:34:48Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Frahm, Thomas
author
Hauser, Hansjörg
author
Köster, Mario
author
2006-03-15
Signaling through the IFN type I receptor is mediated by assembly of the ISGF3 complex consisting of STAT1, STAT2 and IRF9. Whereas STAT1 is instrumentalized by many cytokines, STAT2 is specifically used by type I IFNs. Here, we report that the main regulatory mechanism of nuclear accumulation of STAT2 is nuclear export. We determined the kinetics of nucleocytoplasmic shuttling of STAT2 in living cells. In the absence of IFN, a virtually exclusive cytoplasmic localisation of STAT2 can be detected. Nevertheless, STAT2 is permanently and rapidly shuttling between the cytoplasm and the nucleus. The steady-state localization is explained by a very efficient nuclear export. Our studies indicate that at least two pathways (one of which is CRM1-dependent, the other not yet identified) are responsible for clearing the nucleus from STAT2. The constitutive nucleocytoplasmic shuttling of STAT2 does neither depend on the presence of IRF9 or STAT1, nor does it require tyrosine phosphorylation. Upon treatment with IFN type I, nuclear export of STAT2 is completely abolished in cells used within this study, whereas nuclear import is functioning. This explains the observed nuclear accumulation of STAT2. We have identified a region in the C-terminus of STAT2 that is essential for its almost exclusively cytoplasmic localization in the absence of IFN and responsible for CRM1-specific export. In comparative studies we show that nucleocytoplasmic shuttling of STAT2 is significantly different from that of STAT1. STAT1 is also shuttling in the absence of IFN, but the exchange rate in unstimulated cells is more than ten times lower. We further show that the latent STAT2 protein has stronger intrinsic nuclear-export activity than STAT1. Together, these observations lead to a model for IFN-type-I-induction in which the receptor-mediated heterodimerization overcomes the slow nuclear import of STAT1 and blocks the strong STAT2 export activity that leads to the accumulation of both signal transducers in the nucleus.
J. Cell. Sci. 2006, 119(Pt 6):1092-104
0021-9533
16507591
10.1242/jcs.02822
http://hdl.handle.net/10033/13458
IFN-type-I-mediated signaling is regulated by modulation of STAT2 nuclear export.
oai:repository.helmholtz-hzi.de:10033/142832019-08-30T11:34:48Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Yu, Zheng
author
Beer, Christiane
author
Koester, Mario
author
Wirth, Manfred
author
2006
BACKGROUND: Retroviral Gag determines virus assembly at the plasma membrane and the formation of virus-like particles in intracellular multivesicular bodies. Thereby, retroviruses exploit by interaction with cellular partners the cellular machineries for vesicular transport in various ways. RESULTS: The retroviral Gag precursor protein drives assembly of murine leukaemia viruses (MLV) at the plasma membrane (PM) and the formation of virus like particles in multivesicular bodies (MVBs). In our study we show that caveolin-1 (Cav-1), a multifunctional membrane-associated protein, co-localizes with Gag in a punctate pattern at the PM of infected NIH 3T3 cells. We provide evidence that Cav-1 interacts with the matrix protein (MA) of the Gag precursor. This interaction is mediated by a Cav-1 binding domain (CBD) within the N-terminus of MA. Interestingly, the CBD motif identified within MA is highly conserved among most other gamma-retroviruses. Furthermore, Cav-1 is incorporated into MLV released from NIH 3T3 cells. Overexpression of a GFP fusion protein containing the putative CBD of the retroviral MA resulted in a considerable decrease in production of infectious retrovirus. Moreover, expression of a dominant-negative Cav-1 mutant affected retroviral titres significantly. CONCLUSION: This study demonstrates that Cav-1 interacts with MLV Gag, co-localizes with Gag at the PM and affects the production of infectious virus. The results strongly suggest a role for Cav-1 in the process of virus assembly.
Virol. J. 2006, 3:73
1743-422X
16956408
10.1186/1743-422X-3-73
http://hdl.handle.net/10033/14283
Caveolin-1 interacts with the Gag precursor of murine leukaemia virus and modulates virus production.
oai:repository.helmholtz-hzi.de:10033/145942019-08-30T11:26:12Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Schucht, R
author
Coroadinha, A S
author
Zanta-Boussif, M A
author
Verhoeyen, E
author
Carrondo, M J T
author
Hauser, Hansjoerg
author
Wirth, Dagmar
author
2006-08-01
We developed a new strategy that provides well-defined high-titer producer cells for recombinant retroviruses in a minimum amount of time. The strategy involves the targeted integration of the retroviral vector into a chromosomal locus with favorable properties. For proof of concept we established a novel HEK293-based retroviral producer cell line, called Flp293A, with a single-copy retroviral vector integrated at a selected chromosomal locus. The vector was flanked by noninteracting Flp-recombinase recognition sites and was exchanged for different retroviral vectors via Flp-mediated cassette exchange. All analyzed cell clones showed correct integration and identical titers for each of the vectors, confirming that the expression characteristics from the parental cell were preserved. Titers up to 2.5 x 10(7) infectious particles/10(6) cells were obtained. Also, high-titer producer cells for a therapeutic vector that encodes the 8.9-kb collagen VII cDNA in a marker-free cassette were obtained within 3 weeks without screening. Thus, we provide evidence that the precise integration of viral vectors into a favorable chromosomal locus leads to high and predictable virus production. This method is compatible with other retroviral vectors, including self-inactivating vectors and marker-free vectors. Further, it provides a tool for evaluation of different retroviral vector designs.
Mol. Ther. 2006, 14(2):285-92
1525-0016
16697259
10.1016/j.ymthe.2005.12.003
http://hdl.handle.net/10033/14594
A new generation of retroviral producer cells: predictable and stable virus production by Flp-mediated site-specific integration of retroviral vectors.
oai:repository.helmholtz-hzi.de:10033/150152019-08-30T11:31:20Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Peuster, Matthias
author
Beerbaum, Phillip
author
Bach, Friedrich-Wilhelm
author
Hauser, Hansjoerg
author
2006-04
Are resorbable implants about to become a reality? 2006, 16 (2):107-16notCardiol Young
1047-9511
16553970
10.1017/S1047951106000011
http://hdl.handle.net/10033/15015
Cardiology in the young
Are resorbable implants about to become a reality?
oai:repository.helmholtz-hzi.de:10033/162742019-08-30T11:36:05Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Norgall, Susanne
author
Papoutsi, Maria
author
Rössler, Jochen
author
Schweigerer, Lothar
author
Wilting, Jörg
author
Weich, Herbert A
author
2007
BACKGROUND: Lymphangiomas are neoplasias of childhood. Their etiology is unknown and a causal therapy does not exist. The recent discovery of highly specific markers for lymphatic endothelial cells (LECs) has permitted their isolation and characterization, but expression levels and stability of molecular markers on LECs from healthy and lymphangioma tissues have not been studied yet. We addressed this problem by profiling LECs from normal dermis and two children suffering from lymphangioma, and also compared them with blood endothelial cells (BECs) from umbilical vein, aorta and myometrial microvessels. METHODS: Lymphangioma tissue samples were obtained from two young patients suffering from lymphangioma in the axillary and upper arm region. Initially isolated with anti-CD31 (PECAM-1) antibodies, the cells were separated by FACS sorting and magnetic beads using anti-podoplanin and/or LYVE-1 antibodies. Characterization was performed by FACS analysis, immunofluorescence staining, ELISA and micro-array gene analysis. RESULTS: LECs from foreskin and lymphangioma had an almost identical pattern of lymphendothelial markers such as podoplanin, Prox1, reelin, cMaf and integrin-alpha1 and -alpha9. However, LYVE-1 was down-regulated and VEGFR-2 and R-3 were up-regulated in lymphangiomas. Prox1 was constantly expressed in LECs but not in any of the BECs. CONCLUSION: LECs from different sources express slightly variable molecular markers, but can always be distinguished from BECs by their Prox1 expression. High levels of VEGFR-3 and -2 seem to contribute to the etiology of lymphangiomas.
Elevated expression of VEGFR-3 in lymphatic endothelial cells from lymphangiomas. 2007, 7:105 BMC Cancer
1471-2407
17584927
10.1186/1471-2407-7-105
http://hdl.handle.net/10033/16274
BMC cancer
Elevated expression of VEGFR-3 in lymphatic endothelial cells from lymphangiomas.
oai:repository.helmholtz-hzi.de:10033/172322019-08-30T11:36:05Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Adden, Nina
author
Hoffmann, Andrea
author
Gross, Gerhard
author
Windhagen, Henning
author
Thorey, Fritz
author
Menzel, Henning
author
2007
Surfaces of biomaterials often do not have the ideal properties for direct application in vivo. Although titanium and its alloys show a good biocompatibility, in some applications there is still need to improve the osteoblast adhesion to titanium implants. A polymeric surface coating is an ideal solution because the polymer can be adjusted to the needs of the application and can be bound to the surface by the photochemical grafting method. Therefore, 22 different polymers were tested for their compatibility using a murine mesenchymal progenitor cell line and three polymers were identified for which more elaborate investigations are reasonable. It was investigated whether or not the results of the cell culture test can be correlated with, e.g., the wetting properties. Indeed it was found that a contact angle above approx. 45 degrees was necessary for good cell adhesion and proliferation. However, otherwise no clear correlation between the contact angle hysteresis or the functionalities of the polymers and the cell growth was observed.
Screening of photochemically grafted polymer films for compatibility with osteogenic precursor cells. 2007, 18 (3):303-16notJ Biomater Sci Polym Ed
0920-5063
17471767
http://hdl.handle.net/10033/17232
Journal of biomaterials science. Polymer edition
Screening of photochemically grafted polymer films for compatibility with osteogenic precursor cells.
oai:repository.helmholtz-hzi.de:10033/192382019-08-30T11:37:00Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Reboll, Marc René
author
Oumard, André
author
Gazdag, Aniko Carla
author
Renger, Isabelle
author
Ritter, Birgit
author
Schwarzer, Michael
author
Hauser, Hansjoerg
author
Wood, Monika
author
Yamada, Michiyuki
author
Resch, Klaus
author
Nourbakhsh, Mahtab
author
2007-08
The mRNA of human NF-kappaB repressing factor (NRF) contains a long 5'-untranslated region (UTR) that directs ribosomes to the downstream start codon by a cap-independent mechanism. Comparison of the nucleotide (nt) sequences of human and mouse NRF mRNAs reveals a high degree of identity throughout a fragment of 150 nt proximal to the start codon. Here, we show that this region constitutes a minimal internal ribosome entry segment (IRES) module. Enzymatic RNA structure analysis reveals a secondary structure model of the NRF IRES module. Point mutation analysis of the module determines a short, 14-nt RNA element (nt 640-653) as a mediator of IRES function. Purification of IRES binding cellular proteins and subsequent ESI/MS/MS sequence analysis led to identification of the RNA-binding protein, JKTBP1. EMSA experiments show that JKTBP1 binds upstream to the 14-nt RNA element in the NRF IRES module (nt 579-639). Over-expression of JKTBP1 significantly enhances activity of the NRF IRES module in dicistronic constructs. Moreover, siRNA experiments demonstrate that down-regulation of endogenous JKTBP1 decreases NRF IRES activity and the level of endogenous NRF protein. The data of this study show that JKTBP1 and the 14-nt element act independently to mediate NRF IRES activity.
NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element. 2007, 13 (8):1328-40 RNA
1355-8382
17592041
10.1261/rna.545407
http://hdl.handle.net/10033/19238
RNA (New York, N.Y.)
NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element.
oai:repository.helmholtz-hzi.de:10033/194572019-08-30T11:37:00Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Jablonska, Jadwiga
author
Dittmar, Kurt E
author
Kleinke, Tanja
author
Buer, Jan
author
Weiss, Siegfried
author
2007-01
Early interactions between pathogens and host cells are often decisive for the subsequent course of infection. Here we investigated early events during infection by Listeria monocytogenes, a ubiquitously occurring facultative intracellular microorganism that exhibits severe pathogenicity, mainly in immunocompromised individuals. We show that the inflammatory chemokine CCL2 is highly up-regulated early after Listeria infection in spleens of BALB/c mice. ERTR-9+ macrophages of the marginal zone were identified as the only infected cells and exclusive producers of CCL2 at the early time point. Consequently, clusters of different cell types were formed around infected ERTR-9+ cells. Metallophilic MOMA-1+ marginal zone macrophages were, however, excluded from the clusters and migrated into the B-cell follicles. Depletion of CCL2 during infection resulted in a different composition of cell clusters in the spleen and increased the mortality rate of treated mice. Interestingly, ERTR-9+ macrophages no longer were part of clusters in such mice but remained at their original location in the marginal zone.
Essential role of CCL2 in clustering of splenic ERTR-9+ macrophages during infection of BALB/c mice by Listeria monocytogenes. 2007, 75 (1):462-70 Infect. Immun.
0019-9567
17074847
10.1128/IAI.00443-06
http://hdl.handle.net/10033/19457
Infection and immunity
Essential role of CCL2 in clustering of splenic ERTR-9+ macrophages during infection of BALB/c mice by Listeria monocytogenes.
oai:repository.helmholtz-hzi.de:10033/197732019-08-30T11:27:16Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Dror, Natalie
author
Alter-Koltunoff, Michal
author
Azriel, Aviva
author
Amariglio, Ninette
author
Jacob-Hirsch, Jasmine
author
Zeligson, Sharon
author
Morgenstern, Avigail
author
Tamura, Tomohiko
author
Hauser, Hansjörg
author
Rechavi, Gideon
author
Ozato, Keiko
author
Levi, Ben-Zion
author
2007-01
Interferon regulatory factor 1 (IRF-1) and IRF-8, also known as interferon consensus sequence binding protein (ICSBP), are important regulators of macrophage differentiation and function. These factors exert their activities through the formation of heterocomplexes. As such, they are coactivators of various interferon-inducible genes in macrophages. To gain better insights into the involvement of these two transcription factors in the onset of the innate immune response and to identify their regulatory network in activated macrophages, DNA microarray was employed. Changes in the expression profile were analyzed in peritoneal macrophages from wild type mice and compared to IRF-1 and IRF-8 null mice, before and following 4 h exposure to IFN-gamma and LPS. The expression pattern of 265 genes was significantly changed (up/down) in peritoneal macrophages extracted from wild type mice following treatment with IFN-gamma and LPS, while no changes in the expression levels of these genes were observed in samples of the same cell-type from both IRF-1 and IRF-8 null mice. Among these putative target genes, numerous genes are involved in macrophage activity during inflammation. The expression profile of 10 of them was further examined by quantitative RT-PCR. In addition, the promoter regions of three of the identified genes were analyzed by reporter gene assay for the ability to respond to IRF-1 and IRF-8. Together, our results suggest that both IRF-1 and IRF-8 are involved in the transcriptional regulation of these genes. We therefore suggest a broader role for IRF-1 and IRF-8 in macrophages differentiation and maturation, being important inflammatory mediators.
Identification of IRF-8 and IRF-1 target genes in activated macrophages. 2007, 44 (4):338-46 Mol. Immunol.
0161-5890
16597464
10.1016/j.molimm.2006.02.026
http://hdl.handle.net/10033/19773
Molecular immunology
Identification of IRF-8 and IRF-1 target genes in activated macrophages.
oai:repository.helmholtz-hzi.de:10033/230922019-08-30T11:31:23Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Schliephake, Henning
author
Weich, Herbert A
author
Dullin, Christian
author
Gruber, Rudolf
author
Frahse, Sarah
author
2008-01
The aim of the present study was to test the hypothesis that human recombinant bone morphogenic protein 2 (rhBMP-2) implanted in a slow release carrier of polylactic acid (PLA) can repair a non-healing defect in the rat mandible and maintain the thickness of an augmented volume. p-DL-lactic acid discs were produced and loaded with 48 and 96 microg rhBMP-2 and inserted into non-healing defects of the mandible of 45 Wistar rats. Fifteen rats received implants with 96 microg rhBMP-2 (Group 2), 48 microg rhBMP-2 (Group 1) and blank implants without BMP (Group 0) each on one side of the mandible. Unfilled defects of the same size on the contralateral sides of the mandibles served as empty controls. After 6, 13 and 26 weeks, implants of each group were retrieved from five animals each and submitted to flat panel detector computed tomography. Bone formation and thickness of augmentation was assessed by computer-assisted histomorphometry. In Group 2 significantly more bone was produced than in Group 1. Implants of Group 1 induced significantly more bone than the blank controls only after 6 weeks, whereas the difference was not significant after 13 and 26 weeks. Differences between Group 2 and Group 1 were clearly significant after 26 weeks. The thickness of bone tissue was maintained in Group 2 whereas it decreased in Group 1 and was negligible in Group 0. It is concluded that the PLA implants with 96 microg rhBMP-2 were able to bridge a non-healing defect in the rat mandible and maintained the thickness of an augmented volume. However, continuous supply of osteogenic signals appears to be required to compensate for adverse effects during polymer degradation.
Mandibular bone repair by implantation of rhBMP-2 in a slow release carrier of polylactic acid--an experimental study in rats. 2008, 29 (1):103-10 Biomaterials
0142-9612
17936352
10.1016/j.biomaterials.2007.09.019
http://hdl.handle.net/10033/23092
Biomaterials
Mandibular bone repair by implantation of rhBMP-2 in a slow release carrier of polylactic acid--an experimental study in rats.
oai:repository.helmholtz-hzi.de:10033/257142019-08-30T11:28:51Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Klöpper, Jonas
author
Lindenmaier, Werner
author
Fiedler, Ulrike
author
Mehlhorn, Alexander
author
Stark, G Björn
author
Finkenzeller, Günter
author
2008-01
Survival of ex vivo constructed tissues after transplantation is limited by insufficient oxygen and nutrient supply. Therefore, strategies aiming at improvement of neovascularization of engineered tissues are a key issue in tissue engineering applications. This in vitro study aimed at exploring the usability of osteogenically differentiated human mesenchymal stem cells (MSCs) as carriers of the angiogenic growth factor genes vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) for therapeutic angiogenesis in bone tissue engineering. The ex vivo adenoviral vector mediated transduction into osteogenically differentiated MSCs revealed a highly efficient and long lasting expression of the transgenes. Biological activity of VEGF and Ang-1 secreted from transduced cells was confirmed by analyzing the sprouting, proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) in response to conditioned medium obtained from transduced cells. The transduced osteogenically differentiated MSCs described in this report may be suitable for inducing neovascularization in bone tissue engineering applications.
High efficient adenoviral-mediated VEGF and Ang-1 gene delivery into osteogenically differentiated human mesenchymal stem cells. 2008, 75 (1):83-90 Microvasc. Res.
0026-2862
17603084
10.1016/j.mvr.2007.04.010
http://hdl.handle.net/10033/25714
Microvascular research
High efficient adenoviral-mediated VEGF and Ang-1 gene delivery into osteogenically differentiated human mesenchymal stem cells.
oai:repository.helmholtz-hzi.de:10033/847562019-08-30T11:31:46Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Garritsen, Henk S.P.
author
Fan, Alex Xiu-Cheng
author
Lenz, Daniela
author
Hanning, Horst
author
Zhong, Xiao Yan
author
Geffers, Robert
author
Lindenmaier, Werner
author
Dittmar, Kurt E.J.
author
2009-05-18
Molecular diagnostics in transfusion medicine: In capillary, on a chip, in silico, or in flight? 2009 36 (3): 181-187 Transfusion Medicine and Hemotherapy.
16603796
16603818
10.1159/000217719
http://hdl.handle.net/10033/84756
Transfusion Medicine and Hemotherapy
Molecular Diagnostics in Transfusion Medicine: In Capilary, on a Chip, in Silico, or in Flight?
oai:repository.helmholtz-hzi.de:10033/901542019-08-30T11:35:13Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Hoffmann, Andrea
author
Gross, Gerhard
author
2009
Our aim is to review the recent progress in the management of musculoskeletal disorders. We will cover novel therapeutic approaches based on growth factors, gene therapy and cells, including stem cells, which may be combined with each other as appropriate. We focus mainly on the treatment of soft tissue injuries - muscle, cartilage, and tendon/ligament for both human and animal athletes. The need for innovative strategies results from the fact that despite all efforts, the current strategies for cartilage and tendon/ligament still result in the formation of functionally and biomechanically inferior tissues after injury (a phenomenon called 'repair' as opposed to proper 'regeneration'), whereas the outcome for muscle is more favorable. Innovative approaches are urgently needed not only to enhance the outcome of conservative or surgical procedures but also to speed up the healing process from the very long disabling periods, which is of special relevance for athletes.
Innovative strategies for treatment of soft tissue injuries in human and animal athletes. 2009, 54:150-65 Med Sport Sci
0254-5020
19696513
10.1159/000235702
http://hdl.handle.net/10033/90154
Medicine and sport science
Innovative strategies for treatment of soft tissue injuries in human and animal athletes.
oai:repository.helmholtz-hzi.de:10033/1189092019-08-30T11:33:27Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Dittmar, Kurt E.J.
author
Simann, Meike
author
Zghoul, Nadia
author
Schön, Oliver
author
Meyring, Wilhelm
author
Hanning, Horst
author
Dirks, Wilhelm G.
author
Miller, Konstantin
author
Garritsen, Henk S.P.
author
Lindenmaier, Werner
author
2010-04
Quality of cell products: Authenticity, identity, genomic stability and status of differentiation 2010 (37/2):57-64 Transfusion Medicine and Hemotherapy.
http://hdl.handle.net/10033/118909
Transfusion Medicine and Hemotherapy
Quality of cell products: Authenticity, Identity, Genomic stability and status of differentiation
oai:repository.helmholtz-hzi.de:10033/1207882019-08-30T11:34:22Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Thorey, Fritz
author
Menzel, Henning
author
Lorenz, Corinna
author
Gross, Gerhard
author
Hoffmann, Andrea
author
Windhagen, Henning
author
2011-01
Intramembranous bone formation is essential in uncemented joint replacement to provide a mechanical anchorage of the implant. Since the discovery of bone morphogenic proteins (BMPs) by Urist in 1965, many studies have been conducted to show the influence of growth factors on implant ingrowth. In this study, the influence of bone morphogenetic protein-2 (rhBMP-2) and transforming growth factor β2 (TGF-β2) on implant osseointegration was investigated.
Osseointegration by bone morphogenetic protein-2 and transforming growth factor beta2 coated titanium implants in femora of New Zealand white rabbits. 2011, 45 (1):57-62 Indian J Orthop
1998-3727
21221225
10.4103/0019-5413.73659
http://hdl.handle.net/10033/120788
Indian journal of orthopaedics
Osseointegration by bone morphogenetic protein-2 and transforming growth factor beta2 coated titanium implants in femora of New Zealand white rabbits.
oai:repository.helmholtz-hzi.de:10033/1225252019-08-30T11:27:42Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Koutinas, Michalis
author
Kiparissides, Alexandros
author
Lam, Ming-Chi
author
Silva-Rocha, Rafael
author
Martins dos Santos, Vítor A P
author
Pistikopoulos, Estradios N.
author
Mantalaris, Athanasios
author
2010
Combining genetic circuit and microbial growth kinetic models: A challenge for biological modelling. (2010), 28 (C):301-306 Computer Aided Chemical Engineering
1570-7946
http://hdl.handle.net/10033/122525
European Syposium on Computer Aided Process Engineering
Combining genetic circuit and microbial growth Kinetic models: Achallenge for biological modelling
oai:repository.helmholtz-hzi.de:10033/1427562019-08-30T11:36:32Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
van Duuren, J B J H
author
Brehmer, B
author
Mars, A E
author
Eggink, G
author
Dos Santos, V A P Martins
author
Sanders, J P M
author
2011-06
A limited life cycle assessment (LCA) was performed on a combined biological and chemical process for the production of adipic acid, which was compared to the traditional petrochemical process. The LCA comprises the biological conversion of the aromatic feedstocks benzoic acid, impure aromatics, toluene, or phenol from lignin to cis, cis-muconic acid, which is subsequently converted to adipic acid through hydrogenation. Apart from the impact of usage of petrochemical and biomass-based feedstocks, the environmental impact of the final concentration of cis, cis-muconic acid in the fermentation broth was studied using 1.85% and 4.26% cis, cis-muconic acid. The LCA focused on the cumulative energy demand (CED), cumulative exergy demand (CExD), and the CO(2) equivalent (CO(2) eq) emission, with CO(2) and N(2) O measured separately. The highest calculated reduction potential of CED and CExD were achieved using phenol, which reduced the CED by 29% and 57% with 1.85% and 4.26% cis, cis-muconic acid, respectively. A decrease in the CO(2) eq emission was especially achieved when the N(2) O emission in the combined biological and chemical process was restricted. At 4.26% cis, cis-muconic acid, the different carbon backbone feedstocks contributed to an optimized reduction of CO(2) eq emissions ranging from 14.0 to 17.4 ton CO(2) eq/ton adipic acid. The bulk of the bioprocessing energy intensity is attributed to the hydrogenation reactor, which has a high environmental impact and a direct relationship with the product concentration in the broth.
A limited LCA of bio-adipic acid: manufacturing the nylon-6,6 precursor adipic acid using the benzoic acid degradation pathway from different feedstocks. 2011, 108 (6):1298-306 Biotechnol. Bioeng.
1097-0290
21328320
10.1002/bit.23074
http://hdl.handle.net/10033/142756
Biotechnology and bioengineering
A limited LCA of bio-adipic acid: manufacturing the nylon-6,6 precursor adipic acid using the benzoic acid degradation pathway from different feedstocks.
oai:repository.helmholtz-hzi.de:10033/1242052019-08-30T11:36:32Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
May, Tobias
author
Eccleston, Lee
author
Herrmann, Sabrina
author
Hauser, Hansjörg
author
Goncalves, Jorge
author
Wirth, Dagmar
author
2008
In order to establish cells and organisms with predictable properties, synthetic biology makes use of controllable, synthetic genetic devices. These devices are used to replace or to interfere with natural pathways. Alternatively, they may be interlinked with endogenous pathways to create artificial networks of higher complexity. While these approaches have been already successful in prokaryotes and lower eukaryotes, the implementation of such synthetic cassettes in mammalian systems and even animals is still a major obstacle. This is mainly due to the lack of methods that reliably and efficiently transduce synthetic modules without compromising their regulation properties. To pave the way for implementation of synthetic regulation modules in mammalian systems we utilized lentiviral transduction of synthetic modules. A synthetic positive feedback loop, based on the Tetracycline regulation system was implemented in a lentiviral vector system and stably integrated in mammalian cells. This gene regulation circuit yields a bimodal expression response. Based on experimental data a mathematical model based on stochasticity was developed which matched and described the experimental findings. Modelling predicted a hysteretic expression response which was verified experimentally. Thereby supporting the idea that the system is driven by stochasticity. The results presented here highlight that the combination of three independent tools/methodologies facilitate the reliable installation of synthetic gene circuits with predictable expression characteristics in mammalian cells and organisms.
Bimodal and hysteretic expression in mammalian cells from a synthetic gene circuit. 2008, 3 (6):e2372 PLoS ONE
1932-6203
18523635
10.1371/journal.pone.0002372
http://hdl.handle.net/10033/124205
PloS one
Bimodal and hysteretic expression in mammalian cells from a synthetic gene circuit.
oai:repository.helmholtz-hzi.de:10033/1298362019-08-30T11:26:13Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Gama-Norton, L
author
Herrmann, S
author
Schucht, R
author
Coroadinha, A S
author
Löw, R
author
Alves, P M
author
Bartholomae, C C
author
Schmidt, M
author
Baum, C
author
Schambach, A
author
Hauser, Hansjoerg
author
Wirth, D
author
2010-08
The improvement of safety and titer of retroviral vectors produced in standard retroviral packaging cell lines is hampered because production relies on uncontrollable vector integration events. The influences of chromosomal surroundings make it difficult to dissect the performance of a specific vector from the chromosomal surroundings of the respective integration site. Taking advantage of a technology that relies on the use of packaging cell lines with predefined integration sites, we have systematically evaluated the performance of several retroviral vectors. In two previously established modular packaging cell lines (Flp293A and 293 FLEX) with single, defined chromosomal integration sites, retroviral vectors were integrated by means of Flp-mediated site-specific recombination. Vectors that are distinguished by different long terminal repeat promoters were introduced in either the sense or reverse orientation. The results show that the promoter, viral vector orientation, and integration site are the main determinants of the titer. Furthermore, we exploited the viral production systems to evaluate read-through activity. Read-through is thought to be caused by inefficient termination of vector transcription and is inherent to the nature of retroviral vectors. We assessed the frequency of transduction of sequences flanking the retroviral vectors from both integration sites. The approach presented here provides a platform for systematic design and evaluation of the efficiency and safety of retroviral vectors optimized for a given producer cell line.
Retroviral vector performance in defined chromosomal Loci of modular packaging cell lines. 2010, 21 (8):979-91 Hum. Gene Ther.
1557-7422
20222806
10.1089/hum.2009.089
http://hdl.handle.net/10033/129836
Human gene therapy
Retroviral vector performance in defined chromosomal Loci of modular packaging cell lines.
oai:repository.helmholtz-hzi.de:10033/1411142019-08-30T11:37:44Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Carrondo, Manuel
author
Panet, Amos
author
Wirth, Dagmar
author
Coroadinha, Ana Sofia
author
Cruz, Pedro
author
Falk, Haya
author
Schucht, Roland
author
Dupont, Francis
author
Geny-Fiamma, Cécile
author
Merten, Otto-Wilhelm
author
Hauser, Hansjörg
author
2011-03
The broad application of retroviral vectors for gene delivery is still hampered by the difficulty to reproducibly establish high vector producer cell lines generating sufficient amounts of highly concentrated virus vector preparations of high quality. To enhance the process for producing clinically relevant retroviral vector preparations for therapeutic applications, we have integrated novel and state-of-the-art technologies in a process that allows rapid access to high-efficiency vector-producing cells and consistent production, purification, and storage of retroviral vectors. The process has been designed for various types of retroviral vectors for clinical application and to support a high-throughput process. New modular helper cell lines that permit rapid insertion of DNA encoding the therapeutic vector of interest at predetermined, optimal chromosomal loci were developed to facilitate stable and high vector production levels. Packaging cell lines, cultivation methods, and improved medium composition were coupled with vector purification and storage process strategies that yield maximal vector infectivity and stability. To facilitate GMP-grade vector production, standard of operation protocols were established. These processes were validated by production of retroviral vector lots that drive the expression of type VII collagen (Col7) for the treatment of a skin genetic disease, dystrophic epidermolysis bullosa. The potential efficacy of the Col7-expressing vectors was finally proven with newly developed systems, in particular in target primary keratinocyte cultures and three-dimensional skin tissues in organ culture.
Integrated strategy for the production of therapeutic retroviral vectors. 2011, 22 (3):370-9 Hum. Gene Ther.
1557-7422
21043806
10.1089/hum.2009.165
http://hdl.handle.net/10033/141114
Human gene therapy
Integrated strategy for the production of therapeutic retroviral vectors.
oai:repository.helmholtz-hzi.de:10033/1411152019-08-30T11:37:44Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Sester, Urban
author
Fousse, Mathias
author
Dirks, Jan
author
Mack, Ulrich
author
Prasse, Antje
author
Singh, Mahavir
author
Lalvani, Ajit
author
Sester, Martina
author
2011
T-cell based IFN-γ release assays do not permit distinction of active tuberculosis (TB) from successfully treated disease or latent M. tuberculosis infection. We postulated that IFN-γ and IL-2 cytokine profiles of antigen-specific T cells measured by flow-cytometry ex vivo might correlate with TB disease activity in vivo. Tuberculin (PPD), ESAT-6 and CFP-10 were used as stimuli to determine antigen-specific cytokine profiles in CD4 T cells from 24 patients with active TB and 28 patients with successfully treated TB using flow-cytometry. Moreover, 25 individuals with immunity consistent with latent M. tuberculosis infection and BCG-vaccination, respectively, were recruited. Although the frequency of cytokine secreting PPD reactive CD4 T cells was higher in patients with active TB compared to patients with treated TB (median 0.81% vs. 0.39% of CD4 T cells, p = 0.02), the overlap in frequencies precluded distinction between the groups on an individual basis. When assessing cytokine profiles, PPD specific CD4 T cells secreting both IFN-γ and IL-2 predominated in treated TB, latent infection and BCG-vaccination, whilst in active TB the cytokine profile was shifted towards cells secreting IFN-γ only (p<0.0001). Cytokine profiles of ESAT-6 or CFP-10 reactive CD4 T cells did not differ between the groups. Receiver operator characteristics (ROC) analysis revealed that frequencies of PPD specific IFN-γ/IL-2 dual-positive T cells below 56% were an accurate marker for active TB (specificity 100%, sensitivity 70%) enabling effective discrimination from non-active states. In conclusion, a frequency lower than 56% IFN-γ/IL-2 dual positive PPD-specific circulating CD4 T-cells is strongly indicative of active TB.
Whole-blood flow-cytometric analysis of antigen-specific CD4 T-cell cytokine profiles distinguishes active tuberculosis from non-active states. 2011, 6 (3):e17813 PLoS ONE
1932-6203
21423578
10.1371/journal.pone.0017813
http://hdl.handle.net/10033/141115
PloS one
Whole-blood flow-cytometric analysis of antigen-specific CD4 T-cell cytokine profiles distinguishes active tuberculosis from non-active states.
oai:repository.helmholtz-hzi.de:10033/1427402019-08-30T11:37:44Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Oberhardt, Matthew A
author
Puchałka, Jacek
author
Martins dos Santos, Vítor A P
author
Papin, Jason A
author
2011-03
In the past decade, over 50 genome-scale metabolic reconstructions have been built for a variety of single- and multi- cellular organisms. These reconstructions have enabled a host of computational methods to be leveraged for systems-analysis of metabolism, leading to greater understanding of observed phenotypes. These methods have been sparsely applied to comparisons between multiple organisms, however, due mainly to the existence of differences between reconstructions that are inherited from the respective reconstruction processes of the organisms to be compared. To circumvent this obstacle, we developed a novel process, termed metabolic network reconciliation, whereby non-biological differences are removed from genome-scale reconstructions while keeping the reconstructions as true as possible to the underlying biological data on which they are based. This process was applied to two organisms of great importance to disease and biotechnological applications, Pseudomonas aeruginosa and Pseudomonas putida, respectively. The result is a pair of revised genome-scale reconstructions for these organisms that can be analyzed at a systems level with confidence that differences are indicative of true biological differences (to the degree that is currently known), rather than artifacts of the reconstruction process. The reconstructions were re-validated with various experimental data after reconciliation. With the reconciled and validated reconstructions, we performed a genome-wide comparison of metabolic flexibility between P. aeruginosa and P. putida that generated significant new insight into the underlying biology of these important organisms. Through this work, we provide a novel methodology for reconciling models, present new genome-scale reconstructions of P. aeruginosa and P. putida that can be directly compared at a network level, and perform a network-wide comparison of the two species. These reconstructions provide fresh insights into the metabolic similarities and differences between these important Pseudomonads, and pave the way towards full comparative analysis of genome-scale metabolic reconstructions of multiple species.
Reconciliation of genome-scale metabolic reconstructions for comparative systems analysis. 2011, 7 (3):e1001116 PLoS Comput. Biol.
1553-7358
21483480
10.1371/journal.pcbi.1001116
http://hdl.handle.net/10033/142740
PLoS computational biology
Reconciliation of genome-scale metabolic reconstructions for comparative systems analysis.
oai:repository.helmholtz-hzi.de:10033/1429692019-08-30T11:27:16Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Kugel, Daniela
author
Pulverer, Julia Elisabeth
author
Köster, Mario
author
Hauser, Hansjörg
author
Staeheli, Peter
author
2011-04
We used embryo fibroblasts from Mx2-Luc transgenic mice that express Firefly luciferase under control of the interferon (IFN)-regulated mouse Mx2 promoter to develop simple nonviral bioassays for type I and type III IFN. Since type III IFN is acid-labile, Mx2-Luc fibroblasts detected the presence of type I IFN in acid-treated biological samples with high sensitivity and selectivity. For selective detection of type III IFN, we employed embryo fibroblasts from Mx2-Luc mutant mice that lack functional receptors for type I IFN. The sensitivity of this latter assay remained comparatively low, presumably because type III IFN receptors are not abundantly present on fibroblasts. The main advantages of our novel IFN assays are that they are easy to perform, yield fast results, and can be used in laboratories that are not licensed for work with infectious agents. Further, the type I IFN assay has superior sensitivity than commercially available enzyme-linked immunosorbent assay systems.
Novel nonviral bioassays for mouse type I and type III interferon. 2011, 31 (4):345-9 J. Interferon Cytokine Res.
1557-7465
21138377
10.1089/jir.2010.0079
http://hdl.handle.net/10033/142969
Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research
Novel nonviral bioassays for mouse type I and type III interferon.
oai:repository.helmholtz-hzi.de:10033/1539592019-08-30T11:26:13Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Schucht, Roland
author
Lydford, Simon
author
Andzinski, Lisa
author
Zauers, Jeannette
author
Cooper, James
author
Hauser, Hansjörg
author
Wirth, Dagmar
author
May, Tobias
author
2011-03
The establishment of mammalian cell lines reliably expressing G-protein-coupled receptors (GPCRs) can be a tedious and often time-consuming process. A strategy has been developed to allow the rapid production of such cell lines. The first step of this approach was the generation of a specialized master cell line, characterized by optimized stable expression of a membrane-bound reporter protein. In the second step, this reporter gene was exchanged for that of the GPCR of interest by a DNA recombinase "cut-and-paste" engineering step. It has been demonstrated that the resulting GPCR cell lines inherit the advantages of the master cell line, expressing the GPCR in a homogeneous and stable manner. The case studies presented demonstrate the functionality of the established GPCR cell lines, and most important, because of the highly efficient integration event, these recombinant GPCR-expressing cell lines were generated within a timeframe of 2 to 4 weeks. The advantages of this cut-and-paste approach versus other strategies such as Flp-In or Jump-In are compared.
Rapid establishment of G-protein-coupled receptor-expressing cell lines by site-specific integration. 2011, 16 (3):323-31 J Biomol Screen
1552-454X
21335600
10.1177/1087057110396371
http://hdl.handle.net/10033/153959
Journal of biomolecular screening
Rapid establishment of G-protein-coupled receptor-expressing cell lines by site-specific integration.
oai:repository.helmholtz-hzi.de:10033/1963102019-08-30T11:28:24Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Koutinas, Michalis
author
Kiparissides, Alexandros
author
Silva-Rocha, Rafael
author
Lam, Ming-Chi
author
Martins Dos Santos, Vitor A P
author
de Lorenzo, Victor
author
Pistikopoulos, Efstratios N
author
Mantalaris, Athanasios
author
2011-07
The majority of models describing the kinetic properties of a microorganism for a given substrate are unstructured and empirical. They are formulated in this manner so that the complex mechanism of cell growth is simplified. Herein, a novel approach for modelling microbial growth kinetics is proposed, linking biomass growth and substrate consumption rates to the gene regulatory programmes that control these processes. A dynamic model of the TOL (pWW0) plasmid of Pseudomonas putida mt-2 has been developed, describing the molecular interactions that lead to the transcription of the upper and meta operons, known to produce the enzymes for the oxidative catabolism of m-xylene. The genetic circuit model was combined with a growth kinetic model decoupling biomass growth and substrate consumption rates, which are expressed as independent functions of the rate-limiting enzymes produced by the operons. Estimation of model parameters and validation of the model's predictive capability were successfully performed in batch cultures of mt-2 fed with different concentrations of m-xylene, as confirmed by relative mRNA concentration measurements of the promoters encoded in TOL. The growth formation and substrate utilisation patterns could not be accurately described by traditional Monod-type models for a wide range of conditions, demonstrating the critical importance of gene regulation for the development of advanced models closely predicting complex bioprocesses. In contrast, the proposed strategy, which utilises quantitative information pertaining to upstream molecular events that control the production of rate-limiting enzymes, predicts the catabolism of a substrate and biomass formation and could be of central importance for the design of optimal bioprocesses.
Linking genes to microbial growth kinetics: an integrated biochemical systems engineering approach. 2011, 13 (4):401-13 Metab. Eng.
1096-7184
21315172
10.1016/j.ymben.2011.02.001
http://hdl.handle.net/10033/196310
Metabolic engineering
Linking genes to microbial growth kinetics: an integrated biochemical systems engineering approach.
oai:repository.helmholtz-hzi.de:10033/2100292019-08-30T11:31:49Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Hemmen, Katherina
author
Reinl, Tobias
author
Buttler, Kerstin
author
Behler, Friederike
author
Dieken, Hauke
author
Jänsch, Lothar
author
Wilting, Jörg
author
Weich, Herbert A
author
2011-05
Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis.
High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells. 2011, 14 (2):163-72 Angiogenesis
1573-7209
21234671
10.1007/s10456-011-9200-x
http://hdl.handle.net/10033/210029
Angiogenesis
High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells.
oai:repository.helmholtz-hzi.de:10033/2227372019-08-30T11:30:54Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Nehlsen, Kristina
author
da Gama-Norton, Leonor
author
Schucht, Roland
author
Hauser, Hansjörg
author
Wirth, Dagmar
author
2011-11-22
Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci. 2011, 5 Suppl 8:O6notBMC Proc
1753-6561
22373348
10.1186/1753-6561-5-S8-O6
http://hdl.handle.net/10033/222737
BMC proceedings
Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci.
oai:repository.helmholtz-hzi.de:10033/2276742019-08-30T11:37:23Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Rand, Ulfert
author
Rinas, Melanie
author
Schwerk, Johannes
author
Nöhren, Gesa
author
Linnes, Melanie
author
Kröger, Andrea
author
Flossdorf, Michael
author
Kály-Kullai, Kristóf
author
Hauser, Hansjörg
author
Höfer, Thomas
author
Köster, Mario
author
2012
The cellular recognition of viruses evokes the secretion of type-I interferons (IFNs) that induce an antiviral protective state. By live-cell imaging, we show that key steps of virus-induced signal transduction, IFN-β expression, and induction of IFN-stimulated genes (ISGs) are stochastic events in individual cells. The heterogeneity in IFN production is of cellular-and not viral-origin, and temporal unpredictability of IFN-β expression is largely due to cell-intrinsic noise generated both upstream and downstream of the activation of nuclear factor-κB and IFN regulatory factor transcription factors. Subsequent ISG induction occurs as a stochastic all-or-nothing switch, where the responding cells are protected against virus replication. Mathematical modelling and experimental validation show that reliable antiviral protection in the face of multi-layered cellular stochasticity is achieved by paracrine response amplification. Achieving coherent responses through intercellular communication is likely to be a more widely used strategy by mammalian cells to cope with pervasive stochasticity in signalling and gene expression.
Multi-layered stochasticity and paracrine signal propagation shape the type-I interferon response. 2012, 8:584 Mol. Syst. Biol.
1744-4292
22617958
10.1038/msb.2012.17
http://hdl.handle.net/10033/227674
Molecular systems biology
Multi-layered stochasticity and paracrine signal propagation shape the type-I interferon response.
oai:repository.helmholtz-hzi.de:10033/2304562019-08-30T11:37:00Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Reich, Uta
author
Fadeeva, Elena
author
Warnecke, Athanasia
author
Paasche, Gerrit
author
Müller, Peter
author
Chichkov, Boris
author
Stöver, Timo
author
Lenarz, Thomas
author
Reuter, Günter
author
2012-05
For best hearing sensation, electrodes of auditory prosthesis must have an optimal electrical contact to the respective neuronal cells. To improve the electrode-nerve interface, microstructuring of implant surfaces could guide neuronal cells toward the electrode contact. To this end, femtosecond laser ablation was used to generate linear microgrooves on the two currently relevant cochlear implant materials, silicone elastomer and platinum. Silicone surfaces were structured by two different methods, either directly, by laser ablation or indirectly, by imprinting using laser-microstructured molds. The influence of surface structuring on neurite outgrowth was investigated utilizing a neuronal-like cell line and primary auditory neurons. The pheochromocytoma cell line PC-12 and primary spiral ganglion cells were cultured on microstructured auditory implant materials. The orientation of neurite outgrowth relative to the microgrooves was determined. Both cell types showed a preferred orientation in parallel to the microstructures on both, platinum and on molded silicone elastomer. Interestingly, microstructures generated by direct laser ablation of silicone did not influence the orientation of either cell type. This shows that differences in the manufacturing procedures can affect the ability of microstructured implant surfaces to guide the growth of neurites. This is of particular importance for clinical applications, since the molding technique represents a reproducible, economic, and commercially feasible manufacturing procedure for the microstructured silicone surfaces of medical implants.
Directing neuronal cell growth on implant material surfaces by microstructuring. 2012, 100 (4):940-7 J. Biomed. Mater. Res. Part B Appl. Biomater.
1552-4981
22287482
10.1002/jbm.b.32656
http://hdl.handle.net/10033/230456
Journal of biomedical materials research. Part B, Applied biomaterials
Directing neuronal cell growth on implant material surfaces by microstructuring.
oai:repository.helmholtz-hzi.de:10033/2322312019-08-30T11:30:56Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Behrens, Peter
author
Müller, Peter P.
author
Stieve, Martin
author
Besdo, Silke
author
Ehlert, Nina
author
Lenarz, Thomas
author
2010
Funktionalisierte Mittelohrprothesen 2010, :13 Sonderforschungsbereich 599
978-3-00-032924-1
http://hdl.handle.net/10033/232231
Funktionalisierte Mittelohrprothesen
oai:repository.helmholtz-hzi.de:10033/2322542019-08-30T11:30:52Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Windhagen, Henning
author
Dempwolf, Wiebke
author
Gross, Gerhard
author
2010
Implantatoberflächen 2010, :85 Sonderforschungsbereich 599
978-3-00-032924-1
http://hdl.handle.net/10033/232254
Implantatoberflächen
oai:repository.helmholtz-hzi.de:10033/2449992019-08-30T11:27:16Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Lam, Carolyn M C
author
Suárez Diez, María
author
Godinho, Miguel
author
Martins Dos Santos, Vítor A P
author
2012-07-16
Bacteria have long been used for the synthesis of a wide range of useful proteins and compounds. The developments of new bioprocesses and improvements of existing strategies for syntheses of valuable products in various bacterial cell hosts have their own challenges and limitations. The field of synthetic biology has combined knowledge from different science and engineering disciplines and facilitated the advancement of novel biological components which has inspired the design of targeted biosynthesis. Here we discuss recent advances in synthetic biology with relevance to biosynthesis in bacteria and the applications of computational algorithms and tools for manipulation of cellular components. Continuous improvements are necessary to keep up with increasing demands in terms of complexity, scale, and predictability of biosynthesis products.
Programmable bacterial catalysis - designing cells for biosynthesis of value-added compounds. 2012, 586 (15):2184-90 FEBS Lett.
1873-3468
22710181
10.1016/j.febslet.2012.02.030
http://hdl.handle.net/10033/244999
FEBS letters
Programmable bacterial catalysis - designing cells for biosynthesis of value-added compounds.
oai:repository.helmholtz-hzi.de:10033/2400512019-08-30T11:27:16Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Leprince, Audrey
author
de Lorenzo, Víctor
author
Völler, Petra
author
van Passel, Mark W J
author
Martins dos Santos, Vitor A P
author
2012-06
Cumulative site-directed mutagenesis is of limited suitability for the global analysis of the gene functions in the microbe's cellular network. In order to simplify and stabilize the genome of the soil bacterium Pseudomonas putida, we developed a recyclable three-step excision method based on the combination of customized mini-transposons and the FLP-FRT site-specific recombination system. To demonstrate the powerful potential of these tools, we first established insertion mutant libraries that allow users to study gene functions with respect either to phenotypic characteristics (single insertions) or to their involvement in predicted networks (double insertions). Based on these libraries, we generated as a proof-of-principle, single-deletion mutants lacking ~4.1% of the genome (~3.7% of the gene repertoire). A cyclical application of the method generated four double-deletion mutants of which a maximum of ~7.4% of the chromosome (~6.9% of the gene count) was excised. This procedure demonstrates a new strategy for rapid genome streamlining and gain of new insights into the molecular interactions and regulations.
Random and cyclical deletion of large DNA segments in the genome of Pseudomonas putida. 2012, 14 (6):1444-53 Environ. Microbiol.
1462-2920
22429517
10.1111/j.1462-2920.2012.02730.x
http://hdl.handle.net/10033/240051
Environmental microbiology
Random and cyclical deletion of large DNA segments in the genome of Pseudomonas putida.
oai:repository.helmholtz-hzi.de:10033/2405912019-08-30T11:24:31Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Poblete-Castro, Ignacio
author
Escapa, Isabel F
author
Jäger, Christian
author
Puchalka, Jacek
author
Lam, Carolyn Ming Chi
author
Schomburg, Dietmar
author
Prieto, María Auxiliadora
author
Martins dos Santos, Vítor A P
author
2012
Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures.
The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: highlights from a multi-level omics approach. 2012, 11:34 Microb. Cell Fact.
1475-2859
22433058
10.1186/1475-2859-11-34
http://hdl.handle.net/10033/240591
Microbial cell factories
The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: highlights from a multi-level omics approach.
oai:repository.helmholtz-hzi.de:10033/2459522019-08-30T11:27:46Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Thiele, Wilko
author
Krishnan, Jaya
author
Rothley, Melanie
author
Weih, Debra
author
Plaumann, Diana
author
Kuch, Vanessa
author
Quagliata, Luca
author
Weich, Herbert A
author
Sleeman, Jonathan P
author
2012-08-30
VEGFR-3 is a transmembrane receptor tyrosine kinase that is activated by its ligands VEGF-C and VEGF-D. Although VEGFR-3 has been linked primarily to the regulation of lymphangiogenesis, in the present study, we demonstrate a role for VEGFR-3 in megakaryopoiesis. Using a human erythroleukemia cell line and primary murine BM cells, we show that VEGFR-3 is expressed on megakaryocytic progenitor cells through to the promegakaryoblast stage. Functionally, specific activation of VEGFR-3 impaired the transition to polyploidy of CD41(+) cells in primary BM cultures. Blockade of VEGFR-3 promoted endoreplication consistently. In vivo, long-term activation or blockade of VEGFR-3 did not affect steady-state murine megakaryopoiesis or platelet counts significantly. However, activation of VEGFR-3 in sublethally irradiated mice resulted in significantly elevated numbers of CD41(+) cells in the BM and a significant increase in diploid CD41(+) cells, whereas the number of polyploid CD41(+) cells was reduced significantly. Moreover, activation of VEGFR-3 increased platelet counts in thrombopoietin-treated mice significantly and modulated 5-fluorouracil-induced thrombocytosis strongly, suggesting a regulatory role for VEGFR-3 in megakaryopoiesis.
VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis. 2012, 120 (9):1899-907 Blood
1528-0020
22797697
10.1182/blood-2011-09-376657
http://hdl.handle.net/10033/245952
Blood
VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis.
oai:repository.helmholtz-hzi.de:10033/2465362019-08-30T11:27:46Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Poblete-Castro, Ignacio
author
Becker, Judith
author
Dohnt, Katrin
author
dos Santos, Vitor Martins
author
Wittmann, Christoph
author
2012-03
Since their discovery many decades ago, Pseudomonas putida and related subspecies have been intensively studied with regard to their potential application in industrial biotechnology. Today, these Gram-negative soil bacteria, traditionally known as well-performing xenobiotic degraders, are becoming efficient cell factories for various products of industrial relevance including a full range of unnatural chemicals. This development is strongly driven by systems biotechnology, integrating systems metabolic engineering approaches with novel concepts from bioprocess engineering, including novel reactor designs and renewable feedstocks.
Industrial biotechnology of Pseudomonas putida and related species. 2012, 93 (6):2279-90 Appl. Microbiol. Biotechnol.
1432-0614
22350258
10.1007/s00253-012-3928-0
http://hdl.handle.net/10033/246536
Applied microbiology and biotechnology
Industrial biotechnology of Pseudomonas putida and related species.
oai:repository.helmholtz-hzi.de:10033/2469932019-08-30T11:27:46Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Hogan, Louise
author
Bhuju, Sabin
author
Jones, Des C
author
Laing, Ken
author
Trowsdale, John
author
Butcher, Philip
author
Singh, Mahavir
author
Vordermeier, Martin
author
Allen, Rachel L
author
2012
Leukocyte Immunoglobulin-like receptors (LILR) are innate immune receptors involved in regulating both innate and adaptive immune functions. LILR show more interspecies conservation than the closely related Killer Ig-like receptors, and homologues have been identified in rodents, primates, seals and chickens. The murine equivalents, paired Ig-like receptors (PIR), contain two additional immunoglobulin domains, but show strong sequence and functional similarities to human LILR. The bovine genome was recently sequenced, with preliminary annotations indicating that LILR were present in this species. We therefore sought to identify and characterize novel LILR within the Bos taurus genome, compare these phylogenetically with LILR from other species and determine whether they were expressed in vivo. Twenty six potential bovine LILR were initially identified using BLAST and BLAT software. Phylogenetic analysis constructed using the neighbour-joining method, incorporating pairwise deletion and confidence limits estimated from 1000 replicates using bootstrapping, indicated that 16 of these represent novel bovine LILR. Protein structures defined using protein BLAST predict that the bovine LILR family comprises seven putative inhibitory, four activating and five soluble receptors. Preliminary expression analysis was performed by mapping the predicted sequences with raw data from total transcript sequence generated using Genome Analyzer IIx (Illumina) to provide evidence that all 16 of these receptors are expressed in vivo. The bovine receptor family appears to contain receptors which resemble the six domain rodent PIR as well as the four domain LILR found in other species.
Characterisation of bovine leukocyte Ig-like receptors. 2012, 7 (4):e34291 PLoS ONE
1932-6203
22485161
10.1371/journal.pone.0034291
http://hdl.handle.net/10033/246993
PloS one
Characterisation of bovine leukocyte Ig-like receptors.
oai:repository.helmholtz-hzi.de:10033/2518122019-08-30T11:28:51Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Cudmore, Melissa J
author
Hewett, Peter W
author
Ahmad, Shakil
author
Wang, Ke-Qing
author
Cai, Meng
author
Al-Ani, Bahjat
author
Fujisawa, Takeshi
author
Ma, Bin
author
Sissaoui, Samir
author
Ramma, Wenda
author
Miller, Mark R
author
Newby, David E
author
Gu, Yuchun
author
Barleon, Bernhard
author
Weich, Herbert
author
Ahmed, Asif
author
2012
VEGF-A activity is tightly regulated by ligand and receptor availability. Here we investigate the physiological function of heterodimers between VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (KDR; Flk-1) (VEGFR(1-2)) in endothelial cells with a synthetic ligand that binds specifically to VEGFR(1-2). The dimeric ligand comprises one VEGFR-2-specific monomer (VEGF-E) and a VEGFR-1-specific monomer (PlGF-1). Here we show that VEGFR(1-2) activation mediates VEGFR phosphorylation, endothelial cell migration, sustained in vitro tube formation and vasorelaxation via the nitric oxide pathway. VEGFR(1-2) activation does not mediate proliferation or elicit endothelial tissue factor production, confirming that these functions are controlled by VEGFR-2 homodimers. We further demonstrate that activation of VEGFR(1-2) inhibits VEGF-A-induced prostacyclin release, phosphorylation of ERK1/2 MAP kinase and mobilization of intracellular calcium from primary endothelial cells. These findings indicate that VEGFR-1 subunits modulate VEGF activity predominantly by forming heterodimer receptors with VEGFR-2 subunits and such heterodimers regulate endothelial cell homeostasis.
The role of heterodimerization between VEGFR-1 and VEGFR-2 in the regulation of endothelial cell homeostasis. 2012, 3:972 Nat Commun
2041-1723
22828632
10.1038/ncomms1977
http://hdl.handle.net/10033/251812
Nature communications
The role of heterodimerization between VEGFR-1 and VEGFR-2 in the regulation of endothelial cell homeostasis.
oai:repository.helmholtz-hzi.de:10033/2673322019-08-30T11:28:51Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Mueller, Peter P
author
Arnold, Sylvia
author
Badar, Muhammad
author
Bormann, Dirk
author
Bach, Friedrich-Wilhelm
author
Drynda, Andreas
author
Meyer-Lindenberg, Andrea
author
Hauser, Hansjörg
author
Peuster, Matthias
author
2012-11
A small animal model was established to evaluate the potential of iron as a degradable implant material. After insertion into the tail of mice, the implants gradually degraded over a clinically relevant time period of several months. Histological analysis and gene expression data from whole-genome microarray analyses indicated a limited inflammatory reaction. No evidence of cellular responses to excess iron ions was detected, suggesting that the iron degradation products were metabolically inactive. Iron-rich compounds could be detected in the vicinity of the implant and in individual cells distant from the implantation site. These results demonstrate that the mouse model could be useful for the primary in vivo evaluation of novel implant materials and that iron degradation products can accumulate in diverse organs of the body.
Histological and molecular evaluation of iron as degradable medical implant material in a murine animal model. 2012, 100 (11):2881-9 J Biomed Mater Res A
1552-4965
22623368
10.1002/jbm.a.34223
http://hdl.handle.net/10033/267332
Journal of biomedical materials research. Part A
Histological and molecular evaluation of iron as degradable medical implant material in a murine animal model.
oai:repository.helmholtz-hzi.de:10033/2690122019-08-30T11:33:05Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Wöhl-Bruhn, Stefanie
author
Badar, Muhammad
author
Bertz, Andreas
author
Tiersch, Brigitte
author
Koetz, Joachim
author
Menzel, Henning
author
Mueller, Peter P
author
Bunjes, Heike
author
2012-08-20
Hydrogel systems based on hydroxyethyl starch-polyethylene glycol methacrylate (HES-P(EG)(6)MA) or hydroxyethyl starch methacrylate (HES-MA) were used to assess the protein release behavior. Here, we analyzed the in vitro release of FITC-anti-human antibodies incorporated in either HES-P(EG)(6)MA or HES-MA hydrogel delivery systems in PBS or human serum. In addition, hydrogel disks and microparticles prepared from the two polymers were subcutaneously implanted in BALB/c mice. The in vivo release of FITC-IgG was non-invasively monitored by an in vivo imaging system (IVIS 200) over a time period of up to 3 months. The imaging system allowed to asses individual animals over time, therefore only a small number of animals was required to obtain high quality data. The reduction in fluorescence intensity at the site of administration was compared to in vitro release profiles. These investigations demonstrated a sustained release from HES-MA hydrogel disks compared to rapidly degrading HES-P(EG)(6)MA disks and microparticles. The sustained release from HES-MA disks could be further optimized by using increased polymer concentrations. Human serum as in vitro release medium reflected better the in vivo release from HES-P(EG)(6)MA systems than PBS, suggesting that the presence of organic substances like proteins or lipids may play a significant role for the release kinetics.
Comparison of in vitro and in vivo protein release from hydrogel systems. 2012, 162 (1):127-33 J Control Release
1873-4995
22687287
10.1016/j.jconrel.2012.05.049
http://hdl.handle.net/10033/269012
Journal of controlled release : official journal of the Controlled Release Society
Comparison of in vitro and in vivo protein release from hydrogel systems.
oai:repository.helmholtz-hzi.de:10033/2693132019-08-30T11:33:05Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Lensing, Rebecca
author
Bleich, André
author
Smoczek, Anna
author
Glage, Silke
author
Ehlert, Nina
author
Luessenhop, Tammo
author
Behrens, Peter
author
Müller, Peter Paul
author
Kietzmann, Manfred
author
Stieve, Martin
author
2013-01
Nanoporous silica layers are able to host molecules and release them over a certain period of time. These local drug delivery systems for antibiotics could be a new approach in the treatment of chronic otitis media. The aim of this study was to examine the efficacy of nanoporous silica coatings on middle ear prostheses as a delivery system for antibiotics in vivo. Pseudomonas aeruginosa was inoculated into the middle ear of rabbits to induce an otitis media. The control group received coated Bioverit®II implants without antibiotics. Coated prostheses with loaded ciprofloxacin were implanted into the middle ears of the study group. After 1 week, the rabbits were sacrificed. The clinical examination as well as the microbiological and histological examinations of organs and middle ear irrigation revealed clear differences between the two groups. P. aeruginosa was detected in every middle ear of the control group and was almost completely eliminated in the study group. Organ examinations revealed the presence of P. aeruginosa in the control group and a prevention of a bacterial spread in the study group. The nanoporous silica layer as antibiotic delivery system showed convincing efficacy in induced pseudomonal otitis media in the rabbit.
Efficacy of nanoporous silica coatings on middle ear prostheses as a delivery system for antibiotics: an animal study in rabbits. 2013, 9 (1):4815-25 Acta Biomater
1878-7568
22906623
10.1016/j.actbio.2012.08.016
http://hdl.handle.net/10033/269313
Acta biomaterialia
Efficacy of nanoporous silica coatings on middle ear prostheses as a delivery system for antibiotics: an animal study in rabbits.
oai:repository.helmholtz-hzi.de:10033/2698522019-08-30T11:27:16Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Schniedermann, Judith
author
Rennecke, Moritz
author
Buttler, Kerstin
author
Richter, Georg
author
Städtler, Anna-Maria
author
Norgall, Susanne
author
Badar, Muhammad
author
Barleon, Bernhard
author
May, Tobias
author
Wilting, Jörg
author
Weich, Herbert A
author
2010
Postnatal endothelial progenitor cells (EPCs) have been successfully isolated from whole bone marrow, blood and the walls of conduit vessels. They can, therefore, be classified into circulating and resident progenitor cells. The differentiation capacity of resident lung endothelial progenitor cells from mouse has not been evaluated.
Mouse lung contains endothelial progenitors with high capacity to form blood and lymphatic vessels. 2010, 11:50 BMC Cell Biol.
1471-2121
20594323
10.1186/1471-2121-11-50
http://hdl.handle.net/10033/269852
BMC cell biology
Mouse lung contains endothelial progenitors with high capacity to form blood and lymphatic vessels.
oai:repository.helmholtz-hzi.de:10033/2954872019-08-30T11:34:48Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Badar, Muhammad
author
Lünsdorf, Heinrich
author
Evertz, Florian
author
Rahim, Muhammad Imran
author
Glasmacher, Birgit
author
Hauser, Hansjörg
author
Mueller, Peter P
author
2013-07
Magnesium alloys have been proposed as prospective degradable implant materials. To elucidate the complex interactions between the corroding implants and the tissue, magnesium implants were analyzed in a mouse model and the response was compared to that induced by Ti and by the resorbable polymer polyglactin, respectively. One month after implantation, distinct traces of corrosion were apparent but the magnesium implants were still intact, whereas resorbable polymeric wound suture implants were already fragmented. Analysis of magnesium implants 2weeks after implantation by energy-dispersive X-ray spectroscopy indicated that magnesium, oxygen, calcium and phosphate were present at the implant surface. One month after implantation, the element composition of the outermost layer of the implant was indicative of tissue without detectable levels of magnesium, indicating a protective barrier function of this organic layer. In agreement with this notion, gene expression patterns in the surrounding tissue were highly similar for all implant materials investigated. However, high-resolution imaging using energy-filtered transmission electron microscopy revealed magnesium-containing microparticles in the tissue in the proximity of the implant. The release of such corrosion particles may contribute to the accumulation of calcium phosphate in the nearby tissue and to bone conductive activities of magnesium implants.
The formation of an organic coat and the release of corrosion microparticles from metallic magnesium implants. 2013, 9 (7):7580-9 Acta Biomater
1878-7568
23518475
10.1016/j.actbio.2013.03.012
http://hdl.handle.net/10033/295487
Acta biomaterialia
The formation of an organic coat and the release of corrosion microparticles from metallic magnesium implants.
oai:repository.helmholtz-hzi.de:10033/3019982019-08-30T11:36:32Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Schwerk, Johannes
author
Köster, Mario
author
Hauser, Hansjörg
author
Rohde, Manfred
author
Fulde, Marcus
author
Hornef, Mathias W
author
May, Tobias
author
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.
Generation of mouse small intestinal epithelial cell lines that allow the analysis of specific innate immune functions. 2013, 8 (8):e72700 PLoS ONE
1932-6203
23940817
10.1371/journal.pone.0072700
http://hdl.handle.net/10033/301998
PloS one
Generation of mouse small intestinal epithelial cell lines that allow the analysis of specific innate immune functions.
oai:repository.helmholtz-hzi.de:10033/3048382019-08-30T11:37:24Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Gross, Gerhard
author
Hoffmann, Andrea
author
2013
The repair of tendon injuries still presents a major clinical challenge to orthopedic medicine. Tendons, like some other tissues, are poorly vascularized and heal slowly. In addition, healing often leads to the formation of fibrous tissue and scar tissue which lack flexibility and biomechanical properties. So the treatment of tendon injuries is challenging. We give an overview of the structure and composition of tendons, pathological states of tendon and natural healing, as well as therapeutic options. We focus in particular on biomaterials that have been specifically developed or suggested for the successful repair of tendon injuries. In addition, we also review factor- and cell-dependent strategies to heal tendon and ligament disorders. Although brief, we hope that this review will be helpful, particularly for those readers who are new to the field of tendon tissue engineering.
Therapeutic strategies for tendon healing based on novel biomaterials, factors and cells. 2013, 80 (4):203-10 Pathobiology
1423-0291
23652284
10.1159/000347059
http://hdl.handle.net/10033/304838
Pathobiology : journal of immunopathology, molecular and cellular biology
Therapeutic strategies for tendon healing based on novel biomaterials, factors and cells.
oai:repository.helmholtz-hzi.de:10033/3056592019-08-30T11:36:59Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Roesner, Lennart M
author
Mielke, Christian
author
Fähnrich, Silke
author
Merkhoffer, Yvonne
author
Dittmar, Kurt E J
author
Drexler, Hans G
author
Dirks, Wilhelm G
author
2013-10
The human DNA mismatch repair (MMR) gene family comprises four MutL paralogues capable of forming heterodimeric MutLα (MLH1-PMS2), MutLβ (MLH1-PMS1), and MutLγ (MLH1-MLH3) protein complexes. Human MutL subunits PMS2 and MLH3 contain an evolutionarily conserved amino acid motif DQHA(X)2E(X)4E identified as an endonucleolytic domain capable of incising a defective DNA strand. PMS2 of MutLα is generally accepted to be the sole executor of endonucleolytic activity, but since MLH3 was shown to be able to perform DNA repair at low levels in vitro, our aim was to investigate whether or not MLH3 is activated as a backup under MutLα-deficient conditions. Here, we report stable expression of GFP-tagged MLH3 in the isogenic cell lines 293 and 293T which are functional or defective for MLH1 expression, respectively. As expected, MLH3 formed dimeric complexes with endogenous and recombinant MLH1. MutLγ dimers were recruited to sites of DNA damage induced by UVA micro-irradiation as shown for MutLα. Surprisingly, splicing variant MLH3Δ7 lacking the endonucleolytic motif displayed congruent foci formation, implying that recruitment is not necessarily representing active DNA repair. As an alternative test for repair enzyme activity, we combined alkylation-directed DNA damage with comet formation assays. While recombinant MutLα led to full recovery of DNA damage response in MMR deficient cells, expression of MutLγ or single MLH3 failed to do so. These experiments show recruitment and persistence of MutLγ-heterodimers at UVA-induced DNA lesions. However, we demonstrate that in a MutLα-deficient background no DNA repair-specific function carried out by MutLγ can be detected in living cells.
Stable expression of MutLγ in human cells reveals no specific response to mismatched DNA, but distinct recruitment to damage sites. 2013, 114 (10):2405-14 J. Cell. Biochem.
1097-4644
23696135
10.1002/jcb.24591
http://hdl.handle.net/10033/305659
Journal of cellular biochemistry
Stable expression of MutLγ in human cells reveals no specific response to mismatched DNA, but distinct recruitment to damage sites.
oai:repository.helmholtz-hzi.de:10033/3063812019-08-30T11:25:11Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Reifenrath, Janin
author
Badar, Muhammad
author
Dziuba, Dina
author
Müller, Peter P
author
Heidenblut, Torsten
author
Bondarenko, Alexander
author
Meyer-Lindenberg, Andrea
author
2013
Nowadays, research in magnesium alloys as a biodegradable implant material has increased. The aim of this study was to examine osteoinductive properties and tissue responses to pure magnesium in comparison to conventional permanent (titanium) and to degradable (glyconate) implant materials.
Assessment of cellular reactions to magnesium as implant
material in comparison to titanium and to glyconate using
the mouse tail model. 2013, 11 (2):e89-94 J Appl Biomater Funct Mater
2280-8000
23728545
10.5301/JABFM.5000150
http://hdl.handle.net/10033/306381
Journal of applied biomaterials & functional materials
Assessment of cellular reactions to magnesium as implant
material in comparison to titanium and to glyconate using
the mouse tail model.
oai:repository.helmholtz-hzi.de:10033/3117172019-08-30T11:25:43Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Harari, Daniel
author
Abramovich, Renne
author
Zozulya, Alla
author
Smith, Paul
author
Pouly, Sandrine
author
Köster, Mario
author
Hauser, Hansjörg
author
Schreiber, Gideon
author
2014
We have generated transgenic mice that harbor humanized type I interferon receptors (IFNARs) enabling the study of type I human interferons (Hu-IFN-Is) in mice. These "HyBNAR" (Hybrid IFNAR) mice encode transgenic variants of IFNAR1 and IFNAR2 with the human extracellular domains being fused to transmembrane and cytoplasmic segments of mouse sequence. B16F1 mouse melanoma cells harboring the HyBNAR construct specifically bound Hu-IFN-Is and were rendered sensitive to Hu-IFN-I stimulated anti-proliferation, STAT1 activation and activation of a prototypical IFN-I response gene (MX2). HyBNAR mice were crossed with a transgenic strain expressing the luciferase reporter gene under the control of the IFN-responsive MX2 promoter (MX2-Luciferase). Both the HyBNAR and HyBNAR/MX2-Luciferase mice were responsive to all Hu-IFN-Is tested, inclusive of IFNα2A, IFNβ, and a human superagonist termed YNSα8. The mice displayed dose-dependent pharmacodynamic responses to Hu-IFN-I injection, as assessed by measuring the expression of IFN-responsive genes. Our studies also demonstrated a weak activation of endogenous mouse interferon response, especially after high dose administration of Hu-IFNs. In sharp contrast to data published for humans, our pharmacodynamic readouts demonstrate a very short-lived IFN-I response in mice, which is not enhanced by sub-cutaneous (SC) injections in comparison to other administration routes. With algometric differences between humans and mice taken into account, the HyBNAR mice provides a convenient non-primate pre-clinical model to advance the study of human IFN-Is.
Bridging the species divide: transgenic mice humanized for type-I interferon response. 2014, 9 (1):e84259 PLoS ONE
1932-6203
24416207
10.1371/journal.pone.0084259
http://hdl.handle.net/10033/311717
PloS one
Bridging the species divide: transgenic mice humanized for type-I interferon response.
oai:repository.helmholtz-hzi.de:10033/3167332019-08-30T11:26:42Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Mullen, Lisa
author
Rigby, Anne
author
Sclanders, Michelle
author
Adams, Gill
author
Mittal, Gayatri
author
Colston, Julia
author
Fatah, Rewas
author
Subang, Cristina
author
Foster, Julie
author
Francis-West, Philippa
author
Köster, Mario
author
Hauser, Hansjörg
author
Layward, Lorna
author
Vessillier, Sandrine
author
Annenkov, Alex
author
Al-Izki, Sarah
author
Pryce, Gareth
author
Bolton, Chris
author
Baker, David
author
Gould, David J
author
Chernajovsky, Yuti
author
2014-01
Targeting cytokines to sites of disease has clear advantages because it increases their therapeutic index. We designed fusion proteins of the latent-associated peptide (LAP) derived from TGF-β with various cytokines via a matrix metalloproteinase (MMP) cleavage site. This design confers latency, increased half-life and targeting to sites of inflammation. The aim of this study is to determine whether this approach can be applied to cytokines of different molecular structures and sizes.
Latency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-β. 2014, 11 (1):5-16 Expert Opin Drug Deliv
1744-7593
24073618
10.1517/17425247.2013.839655
http://hdl.handle.net/10033/316733
Expert opinion on drug delivery
Latency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-β.
oai:repository.helmholtz-hzi.de:10033/3241432019-08-30T11:36:05Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Kayser, Hartmut
author
Wray, Victor
author
Nimtz, Manfred
author
2014-05
Biliproteins are present in almost all forms of life, and many of them play vital roles in photobiology. The bilin ligand of a recently characterized 500-kDa biliprotein from an insect has been isolated and its structure elucidated with chemical and spectroscopic techniques (UV-visible, IR, MS, NMR, and CD). This blue pigment, named CV-bilin, represents a unique high molecular mass derivative of biliverdin IXα, with an unusual 10E-configuration and a molecular mass of 852 Da, corresponding to C48H60N4O10. The high mass of this open-chain tetrapyrrole results from the presence of an epoxi-dihydroxyethylfarnesyl substituent at C-18 and a hydroxymethyl substituent at C-13. This substitution pattern exactly reflects that of heme A of mitochondrial cytochrome c oxidases with a hydroxyethylfarnesyl chain and a formyl group at corresponding positions of the cyclic tetrapyrrole. As no other natural product is known to show these structural features (heme O, the precursor of heme A, has a methyl group at C-13), this bilin is presumed to be derived from heme A by cleavage of the α-methine bridge and oxidative modifications at C-13 and the hydroxyethylfarnesyl chain. Possibly, a bilin structurally related to this insect bilin is also produced in other organisms as a result of mitochondrial turnover or degradation. As CV-bilin in complex with a specific protein is accumulated at the end of larval life, stored in the pupa, and finally transferred to the oocytes, a possible role of the free or protein-bound pigment in egg or embryonic development is discussed.
Structure of a novel farnesylated bilin from an insect--formation by α-cleavage of heme A of mitochondrial cytochrome c oxidases? 2014, 281 (10):2366-76 FEBS J.
1742-4658
24655573
10.1111/febs.12789
http://hdl.handle.net/10033/324143
The FEBS journal
Structure of a novel farnesylated bilin from an insect--formation by α-cleavage of heme A of mitochondrial cytochrome c oxidases?
oai:repository.helmholtz-hzi.de:10033/3390342019-08-30T11:36:05Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Charbord, Pierre
author
Livne, Erella
author
Gross, Gerhard
author
Häupl, Thomas
author
Neves, Nuno M
author
Marie, Pierre
author
Bianco, Paolo
author
Jorgensen, Christian
author
2011-03
Genostem (acronym for "Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to the bed side") has been an European consortium of 30 teams working together on human bone marrow Mesenchymal Stem Cell (MSC) biological properties and repair capacity. Part of Genostem activity has been dedicated to the study of basic issues on undifferentiated MSCs properties and on signalling pathways leading to the differentiation into 3 of the connective tissue lineages, osteoblastic, chondrocytic and tenocytic. We have evidenced that native bone marrow MSCs and stromal cells, forming the niche of hematopoietic stem cells, were the same cellular entity located abluminally from marrow sinus endothelial cells. We have also shown that culture-amplified, clonogenic and highly-proliferative MSCs were bona fide stem cells, sharing with other stem cell types the major attributes of self-renewal and of multipotential priming to the lineages to which they can differentiate (osteoblasts, chondrocytes, adipocytes and vascular smooth muscle cells/pericytes). Extensive transcription profiling and in vitro and in vivo assays were applied to identify genes involved in differentiation. Thus we have described novel factors implicated in osteogenesis (FHL2, ITGA5, Fgf18), chondrogenesis (FOXO1A) and tenogenesis (Smad8). Another part of Genostem activity has been devoted to studies of the repair capacity of MSCs in animal models, a prerequisite for future clinical trials. We have developed novel scaffolds (chitosan, pharmacologically active microcarriers) useful for the repair of both bone and cartilage. Finally and most importantly, we have shown that locally implanted MSCs effectively repair bone, cartilage and tendon.
Human bone marrow mesenchymal stem cells: a systematic reappraisal via the genostem experience. 2011, 7 (1):32-42 Stem Cell Rev
1558-6804
20198518
10.1007/s12015-010-9125-6
http://hdl.handle.net/10033/339034
Stem cell reviews
Human bone marrow mesenchymal stem cells: a systematic reappraisal via the genostem experience.
oai:repository.helmholtz-hzi.de:10033/3473152019-08-30T11:25:11Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Jenke, Bok Hee C
author
Fetzer, Christian P
author
Stehle, Isa M
author
Jönsson, Franziska
author
Fackelmayer, Frank O
author
Conradt, Harald
author
Bode, Jürgen
author
Lipps, Hans J
author
2002-04
pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.
An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo. 2002, 3 (4):349-54 EMBO Rep.
1469-221X
11897664
10.1093/embo-reports/kvf070
http://hdl.handle.net/10033/347315
EMBO reports
An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo.
oai:repository.helmholtz-hzi.de:10033/6210112019-08-30T11:31:49Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Martins, José P
author
Santos, Jorge M
author
Almeida, Joana M d
author
Filipe, Mariana A
author
de Almeida, Mariana V T
author
Almeida, Sílvia C C
author
Água-Doce, Ana
author
Varela, Alexandre
author
Gilljam, Mari
author
Stellan, Birgitta
author
Pohl, Susanne
author
Dittmar, Kurt
author
Lindenmaier, Werner
author
Alici, Evren
author
Graça, Luís
author
Cruz, Pedro E
author
Cruz, Helder J
author
Bárcia, Rita N
author
2014-01-17
Abstract Introduction Standardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP). Methods The adaptation focused on different stages of production, from cell isolation steps to cell culturing and cryopreservation. The origin and quality of materials and reagents were considered and steps for avoiding microbiological and endotoxin contamination of the final cell product were implemented. Cell isolation efficiency, MSCs surface markers and genetic profiles, originating from the use of different medium supplements, were compared. The ATMP-compliant UCX® product was also cryopreserved avoiding the use of dimethyl sulfoxide, an added benefit for the use of these cells as an ATMP. Cells were analyzed for expansion capacity and longevity. The final cell product was further characterized by flow cytometry, differentiation potential, and tested for contaminants at various passages. Finally, genetic stability and immune properties were also analyzed. Results The isolation efficiency of UCX® was not affected by the introduction of clinical grade enzymes. Furthermore, isolation efficiencies and phenotype analyses revealed advantages in the use of human serum in cell culture as opposed to human platelet lysate. Initial decontamination of the tissue followed by the use of mycoplasma- and endotoxin-free materials and reagents in cell isolation and subsequent culture, enabled the removal of antibiotics during cell expansion. UCX®-ATMP maintained a significant expansion potential of 2.5 population doublings per week up to passage 15 (P15). They were also efficiently cryopreserved in a DMSO-free cryoprotectant medium with approximately 100% recovery and 98% viability post-thaw. Additionally, UCX®-ATMP were genetically stable upon expansion (up to P15) and maintained their immunomodulatory properties. Conclusions We have successfully adapted a method to consistently isolate, expand and cryopreserve a well-characterized population of human umbilical cord tissue-derived MSCs (UCX®), in order to obtain a cell product that is compliant with cell therapy. Here, we present quality and safety data that support the use of the UCX® as an ATMP, according to existing international guidelines.
Stem Cell Research & Therapy. 2014 Jan 17;5(1):9
http://dx.doi.org/10.1186/scrt398
http://hdl.handle.net/10033/621011
Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data
oai:repository.helmholtz-hzi.de:10033/6210432019-08-30T11:33:30Zcom_10033_6823com_10033_6820col_10033_6891col_10033_6891col_10033_6891col_10033_118931
00925njm 22002777a 4500
dc
Nehlsen, Kristina
author
Schucht, Roland
author
da Gama-Norton, Leonor
author
Krömer, Wolfgang
author
Baer, Alexandra
author
Cayli, Aziz
author
Hauser, Hansjörg
author
Wirth, Dagmar
author
2009-12-14
Abstract Background Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs. Results We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs) were targeted by Flp recombinase mediated cassette exchange (RMCE). The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context. Conclusion RMCE provides a powerful method to specifically design vectors for optimized gene expression with high accuracy. Upon considering the specific requirements of chromosomal sites this method provides a unique tool to exploit such sites for predictable expression of biotechnologically relevant proteins such as antibodies.
BMC Biotechnology. 2009 Dec 14;9(1):100
http://dx.doi.org/10.1186/1472-6750-9-100
http://hdl.handle.net/10033/621043
Recombinant protein expression by targeting pre-selected chromosomal loci
oai:repository.helmholtz-hzi.de:10033/6210332019-08-30T11:32:16Zcom_10033_6823com_10033_6820com_10033_620652col_10033_6891col_10033_620672
00925njm 22002777a 4500
dc
Nehlsen, Kristina
author
da Gama-Norton, Leonor
author
Schucht, Roland
author
Hauser, Hansjörg
author
Wirth, Dagmar
author
2011-11-22
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
Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci
oai:repository.helmholtz-hzi.de:10033/6208412019-08-30T11:32:59Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Hauser, Hansjörg
author
2011-11-22
22nd European Society for Animal Cell Technology (ESACT) Meeting on Cell Based Technologies Vienna, Austria. 15-18 May 2011. Abstracts. 2011, 5 Suppl 8:I1-P134 BMC Proc
1753-6561
22166093
http://hdl.handle.net/10033/620841
BMC proceedings
22nd European Society for Animal Cell Technology (ESACT) Meeting on Cell Based Technologies Vienna, Austria. 15-18 May 2011. Abstracts.
oai:repository.helmholtz-hzi.de:10033/6207912019-08-30T11:26:12Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Quentmeier, Hilmar
author
Eberth, Sonja
author
Romani, Julia
author
Weich, Herbert A
author
Zaborski, Margarete
author
Drexler, Hans G
author
2012-01-17
Abstract Background Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGF-Rs) are important regulators for angiogenesis and lymphangiogenesis. VEGFs and VEGF-Rs are not only expressed on endothelial cells but also on various subtypes of solid tumors and leukemias contributing to the growth of the malignant cells. This study was performed to examine whether VEGF-R2 (KDR) and VEGF-R3 (FLT4) are regulated by DNA methylation. Methods Real-time (RT) PCR analysis was performed to quantify KDR and FLT4 expression in some ninety leukemia/lymphoma cell lines, human umbilical vein endothelial cells (HUVECs) and dermal microvascular endothelial cells (HDMECs). Western blot analyses and flow cytometric analyses confirmed results at the protein level. After bisulfite conversion of DNA we determined the methylation status of KDR and FLT4 by DNA sequencing and by methylation specific PCR (MSP). Western blot analyses were performed to examine the effect of VEGF-C on p42/44 MAPK activation. Results Expression of KDR and FLT4 was observed in cell lines from various leukemic entities, but not in lymphoma cell lines: 16% (10/62) of the leukemia cell lines expressed KDR, 42% (27/65) were FLT4 positive. None of thirty cell lines representing six lymphoma subtypes showed more than marginal expression of KDR or FLT4. Western blot analyses confirmed KDR and FLT4 protein expression in HDMECs, HUVECs and in cell lines with high VEGF-R mRNA levels. Mature VEGF-C induced p42/44 MAPK activation in the KDR- /FLT4+ cell line OCI-AML1 verifying the model character of this cell line for VEGF-C signal transduction studies. Bisulfite sequencing and MSP revealed that GpG islands in the promoter regions of KDR and FLT4 were unmethylated in HUVECs, HDMECs and KDR + and FLT4 + cell lines, whereas methylated cell lines did not express these genes. In hypermethylated cell lines, KDR and FLT4 were re-inducible by treatment with the DNA demethylating agent 5-Aza-2'deoxycytidine, confirming epigenetic regulation of both genes. Conclusions Our data show that VEGF-Rs KDR and FLT4 are silenced by DNA methylation. However, if the promoters are unmethylated, other factors (e.g. transactivation factors) determine the extent of KDR and FLT4 expression.
BMC Cancer. 2012 Jan 17;12(1):19
http://dx.doi.org/10.1186/1471-2407-12-19
http://hdl.handle.net/10033/620791
DNA methylation regulates expression of VEGF-R2 (KDR) and VEGF-R3 (FLT4)
oai:repository.helmholtz-hzi.de:10033/6207942019-08-30T11:33:05Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Poblete-Castro, Ignacio
author
Escapa, Isabel F
author
Jäger, Christian
author
Puchalka, Jacek
author
Chi Lam, Carolyn M
author
Schomburg, Dietmar
author
Prieto, María A
author
Martins dos Santos, Vítor A
author
2012-03-20
Abstract Background Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures. Results We found that 26, 62, and 81% of the cell dry weight consist of PHA under conditions of carbon, dual, and nitrogen limitation, respectively. Under nitrogen limitation a specific PHA production rate of 0.43 (g·(g·h)-1) was obtained. The residual biomass was not constant for dual- and strict nitrogen-limiting growth, showing a different feature in comparison to other P. putida strains. Dual limitation resulted in patterns of gene expression, protein level, and metabolite concentrations that substantially differ from those observed under exclusive carbon or nitrogen limitation. The most pronounced differences were found in the energy metabolism, fatty acid metabolism, as well as stress proteins and enzymes belonging to the transport system. Conclusion This is the first study where the interrelationship between nutrient limitations and PHA synthesis has been investigated under well-controlled conditions using a system level approach. The knowledge generated will be of great assistance for the development of bioprocesses and further metabolic engineering work in this versatile organism to both enhance and diversify the industrial production of PHAs.
Microbial Cell Factories. 2012 Mar 20;11(1):34
http://dx.doi.org/10.1186/1475-2859-11-34
http://hdl.handle.net/10033/620794
The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: Highlights from a multi-level omics approach
oai:repository.helmholtz-hzi.de:10033/5797352019-08-30T11:31:23Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Martins, José Paulo
author
Santos, Jorge Miguel
author
de Almeida, Joana Marto
author
Filipe, Mariana Alves
author
de Almeida, Mariana Vargas Teixeira
author
Almeida, Sílvia Cristina Paiva
author
Água-Doce, Ana
author
Varela, Alexandre
author
Gilljam, Mari
author
Stellan, Birgitta
author
Pohl, Susanne
author
Dittmar, Kurt
author
Lindenmaier, Werner
author
Alici, Evren
author
Graça, Luís
author
Cruz, Pedro Estilita
author
Cruz, Helder Joaquim
author
Bárcia, Rita Nogueira
author
2014
Standardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP).
Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data. 2014, 5 (1):9 Stem Cell Res Ther
1757-6512
24438697
10.1186/scrt398
http://hdl.handle.net/10033/579735
Stem cell research & therapy
Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data.
oai:repository.helmholtz-hzi.de:10033/5933912019-08-30T11:37:24Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Sun, Lijing
author
Hemgård, Gun-Viol
author
Susanto, Sony A
author
Wirth, Manfred
author
2010
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.
Caveolin-1 influences human influenza A virus (H1N1) multiplication in cell culture. 2010, 7:108 Virol. J.
1743-422X
20504340
10.1186/1743-422X-7-108
http://hdl.handle.net/10033/593391
Virology journal
Caveolin-1 influences human influenza A virus (H1N1) multiplication in cell culture.
oai:repository.helmholtz-hzi.de:10033/6208422019-08-30T11:29:17Zcom_10033_6823com_10033_6820col_10033_6891
00925njm 22002777a 4500
dc
Quentmeier, Hilmar
author
Eberth, Sonja
author
Romani, Julia
author
Weich, Herbert A
author
Zaborski, Margarete
author
Drexler, Hans G
author
2012-01-17
Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGF-Rs) are important regulators for angiogenesis and lymphangiogenesis. VEGFs and VEGF-Rs are not only expressed on endothelial cells but also on various subtypes of solid tumors and leukemias contributing to the growth of the malignant cells. This study was performed to examine whether VEGF-R2 (KDR) and VEGF-R3 (FLT4) are regulated by DNA methylation.
DNA methylation regulates expression of VEGF-R2 (KDR) and VEGF-R3 (FLT4). 2012, 12:19 BMC Cancer
1471-2407
22251800
10.1186/1471-2407-12-19
http://hdl.handle.net/10033/620842
BMC cancer
DNA methylation regulates expression of VEGF-R2 (KDR) and VEGF-R3 (FLT4).