Generation of anti-TLR2 intrabody mediating inhibition of macrophage surface TLR2 expression and TLR2-driven cell activation

<p>Abstract</p> <p>Background</p> <p>Toll-like receptor (TLR) 2 is a component of the innate immune system and senses specific pathogen associated molecular patterns (PAMPs) of both microbial and viral origin. Cell activation via TLR2 and other pattern recognition receptors (PRRs) contributes to sepsis pathology and chronic inflammation both relying on overamplification of an immune response. Intracellular antibodies expressed and retained inside the endoplasmatic reticulum (ER-intrabodies) are applied to block translocation of secreted and cell surface molecules from the ER to the cell surface resulting in functional inhibition of the target protein. Here we describe generation and application of a functional anti-TLR2 ER intrabody (αT2ib) which was generated from an antagonistic monoclonal antibody (mAb) towards human and murine TLR2 (T2.5) to inhibit the function of TLR2. αT2ib is a scFv fragment comprising the variable domain of the heavy chain and the variable domain of the light chain of mAb T2.5 linked together by a synthetic (Gly₄Ser)₃amino acid sequence.</p> <p>Results</p> <p>Coexpression of αT2ib and mouse TLR2 in HEK293 cells led to efficient retention and accumulation of TLR2 inside the ER compartment. Co-immunoprecipitation of human TLR2 with αT2ib indicated interaction of αT2ib with its cognate antigen within cells. αT2ib inhibited NF-κB driven reporter gene activation via TLR2 but not through TLR3, TLR4, or TLR9 if coexpressed in HEK293 cells. Co-transfection of human TLR2 with increasing amounts of the expression plasmid encoding αT2ib into HEK293 cells demonstrated high efficiency of the TLR2-αT2ib interaction. The αT2ib open reading frame was integrated into an adenoviral cosmid vector for production of recombinant adenovirus (AdV)-αT2ib. Transduction with AdVαT2ib specifically inhibited TLR2 surface expression of murine RAW264.7 and primary macrophages derived from bone marrow (BMM). Furthermore, TLR2 activation dependent TNFα mRNA accumulation, as well as TNFα translation and release by macrophages were largely abrogated upon transduction of αT2ib. αT2ib was expressed in BMM and splenocytes over 6 days upon systemic infection with AdVαT2ib. Systemic transduction applying AdVαT2ib rendered immune cells largely non-responsive to tripalmitoyl-peptide challenge. Our results show persistent paralysis of TLR2 activity and thus inhibition of immune activation.</p> <p>Conclusion</p> <p>The generated anti-TLR2 scFv intrabody inhibits specifically and very efficiently TLR2 ligand-driven cell activation <it>in vitro </it>and <it>ex vivo</it>. This indicates a therapeutic potential of αT2ib in microbial or viral infections.</p

High efficient adenoviral-mediated VEGF and Ang-1 gene delivery into osteogenically differentiated human mesenchymal stem cells.

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

Use of Autostitch for automatic stitching of microscope images.

Image stitching is the process of combining multiple images to produce a panorama or larger image. In many biomedical studies, including those of cancer and infection, the use of this approach is highly desirable in order to acquire large areas of certain structures or whole sections, while retaining microscopic resolution. In this study, we describe the application of Autostitch, viz. software that is normally used for the generation of panoramas in photography, in the seamless stitching of microscope images. First, we tested this software on image sets manually acquired by normal light microscopy and compared the performance with a manual stitching approach performed with Paint Shop Pro. Secondly, this software was applied to an image stack acquired by an automatic microscope. The stitching results were then compared with that generated by a self-programmed rectangular tiling macro integrated in Image J. Thirdly, this program was applied in the image stitching of images from electron microscopy. Thus, the automatic stitching program described here may find applications in convenient image stitching and virtual microscopy in the biomedical research

Six-color segmentation of multicolor images in the infection studies of Listeria monocytogenes.

Multiple immunofluorescent staining is a powerful strategy for visualizing the spatial and temporal relationship between antigens, cell populations, and tissue components in histological sections. To segment different cell populations from the multicolor image generated by immunostaining based on color addition theory, a systems approach is proposed for automatic segmentation of six colors. After image acquisition and processing, images are automatically segmented with the proposed approach and six-pseudo channels for individual or colocalized fluorescent dye are generated to distinguish different cell types. The principle of this approach is the classification of each pixel into one of six colors (red, green, blue, yellow, magenta, and cyan) by choosing the minimal angular deviation between the RGB vector of the given pixel and six classically defined edge vectors. In the present infection studies of Listeria monocytogenes, the new multicolor staining methods based on the color addition were applied and the proposed color segmentation was performed for multicolor analysis. Multicolor analysis was accomplished to study the migration and interaction of Listeria and different cell subpopulations such as CD4CD25 double positive T regulatory cells; we also visualized simultaneously the B cells, T cells, dendritic cells, macrophages, and Listeria in another experiment. After Listeria infection, ERTR9 macrophages and dendritic cells formed cluster with Listeria in the infection loci. The principle of color addition and the systems approach for segmentation may be widely applicable in infection and immunity studies requiring multicolor imaging and analysis. This approach can also be applied for image analysis in the multicolor in vivo imaging, multicolor FISH or karyotyping or other studies requiring multicolor analysis

Molecular Diagnostics in Transfusion Medicine: In Capillary, on a Chip, in Silico, or in Flight?

Serology, defined as antibody-based diagnostics, has been regarded as the diagnostic gold standard in transfusion medicine. Nowadays however the impact of molecular diagnostics in transfusion medicine is rapidly growing. Molecular diagnostics can improve tissue typing (HLA typing), increase safety of blood products (NAT testing of infectious diseases), and enable blood group typing in difficult situations (after transfusion of blood products or prenatal non-invasive RhD typing). Most of the molecular testing involves the determination of the presence of single nucleotide polymorphisms (SNPs). Antigens (e.g. blood group antigens) mostly result from single nucleotide differences in critical positions. However, most blood group systems cannot be determined by looking at a single SNP. To identify members of a blood group system a number of critical SNPs have to be taken into account. The platforms which are currently used to perform molecular diagnostics are mostly gel-based, requiring time-consuming multiple manual steps. To implement molecular methods in transfusion medicine in the future the development of higher-throughput SNP genotyping non-gel-based platforms which allow a rapid, cost-effective screening are essential. Because of its potential for automation, high throughput and cost effectiveness the special focus of this paper is a relative new technique: SNP genotyping by MALDI-TOF MS analysis

Quality of Cell Products: Authenticity, Identity, Genomic Stability and Status of Differentiation

Cellular therapies that either use modifications of a patient's own cells or allogeneic cell lines are becoming in vogue. Besides the technical issues of optimal isolation, cultivation and modification, quality control of the generated cellular products are increasingly being considered to be more important. This is not only relevant for the cell's therapeutic application but also for cell science in general. Recent changes in editorial policies of respected journals, which now require proof of authenticity when cell lines are used, demonstrate that the subject of the present paper is not a virtual problem at all. In this article we provide 2 examples of contaminated cell lines followed by a review of the recent developments used to verify cell lines, stem cells and modifications of autologous cells. With relative simple techniques one can now prove the authenticity and the quality of the cellular material of interest and therefore improve the scientific basis for the development of cells for therapeutic applications. The future of advanced cellular therapies will require production and characterization of cells under GMP and GLP conditions, which include proof of identity, safety and functionality and absence of contamination

Reduction of circulating soluble Flt-1 alleviates preeclampsia-like symptoms in a mouse model

Preeclampsia (PE) is characterized by widespread endothelial damage with hypertension, proteinuria, glomeruloendotheliosis and elevated soluble Flt-1 (sFlt-1), a natural occurring antagonist of vascular endothelial growth factor (VEGF). Cancer patients receiving anti-VEGF therapy exhibit similar symptoms. We suggested that a decrease in circulating sFlt-1 would alleviate the symptoms associated with PE. Adenoviral (Adv) overexpression of sFlt-1 induced proteinuria, caused glomerular damage and increase in blood pressure in female Balb/c mice. Circulating level of sFlt-1 above 50 ng/ml plasma induced severe vascular damage and glomerular endotheliosis. Albumin concentration in urine was elevated up to 30-fold, compared to control AdvGFP-treated animals. The threshold of kidney damage was in the range of 20-30 ng/ml sFlt-1 in plasma (8-15 ng/ml in urine). Co-administration of AdvsFlt-1 with AdvVEGF to neutralize circulating sFlt-1 resulted in more than a 70% reduction in free sFlt-1 in plasma, more than 80% reduction in urine and rescued the damaging effect of sFlt-1 on the kidneys. This demonstrates that below a critical threshold sFlt-1 fails to elicit damage to the fenestrated endothelium and that co-expression of VEGF is able to rescue effects mediated by sFlt-1 overexpression