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In NMR spectroscopy, residual dipolar couplings (RDCs) have emerged as one of the most exquisite probes of biological structure and dynamics. The measurement of RDCs relies on the partial alignment of the molecule of interest, for example by using a liquid crystal as a solvent. Here, we establish bacterial type 1 pili as an alternative liquid-crystalline alignment medium for the measurement of RDCs. To achieve alignment at pilus concentrations that allow for efficient NMR sample preparation, we elongated wild-type pili by recombinant overproduction of the main structural pilus subunit. Building on the extraordinary stability of type 1 pili against spontaneous dissociation and unfolding, we show that the medium is compatible with challenging experimental conditions such as high temperature, the presence of detergents, organic solvents or very acidic pH, setting it apart from most established alignment media. Using human ubiquitin, HIV-1 TAR RNA and camphor as spectroscopic probes, we demonstrate the applicability of the medium for the determination of RDCs of proteins, nucleic acids and small molecules. Our results show that type 1 pili represent a very useful alternative to existing alignment media and may readily assist the characterization of molecular structure and dynamics by NMR., Angewandte Chemie. International Edition, ISSN:1433-7851, ISSN:1521-3773, ISSN:0570-0833

SUMMARYAdhesive type 1 pili from uropathogenicEscherichia colistrains are filamentous, supramolecular protein complexes consisting of a short tip fibrillum and a long, helical rod formed by up to several thousand copies of the major pilus subunit FimA. Here, we reconstituted the entire type 1 pilus rod assembly reactionin vitro, using all constituent protein subunits in the presence of the assembly platform FimD, and identified the so-far uncharacterized subunit FimI as an irreversible assembly terminator. We provide a complete, quantitative model of pilus rod assembly kinetics based on the measured rate constants of FimD-catalyzed subunit incorporation. The model reliably predicts the length distribution of assembled pilus rods as a function of the ratio between FimI and the main pilus subunit FimA and is fully consistent with the length distribution of membrane-anchored pili assembledin vivo. The results show that the natural length distribution of adhesive pili formed via the chaperone-usher pathway results from a stochastic chain termination reaction.

We study the dynamics of charged, spherically symmetric dust shells in the presence of a positive cosmological constant. We find generalizations of the well-known solutions in asymptotically flat spacetime, in particular orbits into "parallel universes", but also new solutions corresponding to a "bounce" of the shell before an event horizon has formed. We also discuss "bubble" solutions, in which a charged shell and an oppositely charged singularity are spontaneously created and annihilated., Comment: 16 page2; v2 final version

A hallmark of N-linked glycosylation in the secretory compartments of eukaryotic cells is the sequential remodeling of an initially uniform oligosaccharide to a site-specific, heterogeneous ensemble of glycostructures on mature proteins. To understand site-specific processing, we used protein disulfide isomerase (PDI), a model protein with five glycosylation sites, for molecular dynamics (MD) simulations and compared the result to a biochemical in vitro analysis with four different glycan processing enzymes. As predicted by an analysis of the accessibility of the N-glycans for their processing enzymes derived from the MD simulations, N-glycans at different glycosylation sites showed different kinetic properties for the processing enzymes. In addition, altering the tertiary structure of the glycoprotein PDI affected its N-glycan remodeling in a site-specific way. We propose that the observed differential N-glycan reactivities depend on the surrounding protein tertiary structure and lead to different glycan structures in the same protein through kinetically controlled processing pathways.

A hallmark of N-linked glycosylation in the secretory compartments of eukaryotic cells is the sequential remodeling of an initially uniform oligosaccharide to a site-specific, heterogeneous ensemble of glycostructures on mature proteins. To understand site-specific processing, we used protein disulfide isomerase (PDI), a model protein with five glycosylation sites, for molecular dynamics (MD) simulations and compared the result to a biochemical in vitro analysis with four different glycan processing enzymes. As predicted by an analysis of the accessibility of the N-glycans for their processing enzymes derived from the MD simulations, N-glycans at different glycosylation sites showed different kinetic properties for the processing enzymes. In addition, altering the tertiary structure of the glycoprotein PDI affected its N-glycan remodeling in a site-specific way. We propose that the observed differential N-glycan reactivities depend on the surrounding protein tertiary structure and lead to different glycan structures in the same protein through kinetically controlled processing pathways., Atomistic glycoprotein simulations reveal a site-specific availability of glycan substrates in time-resolved mass spectrometry of maturating enzyme kinetics.

Conventional Bioreactor systems for cultivating cells in Life Science have been widely used for decades. An in vitro cell cultivation bioreactor should reliably and reproducibly mimic the in vivo microenvironment of the cultured cells. Normally, mammalian cell cultures are performed in conventional bioreactor devices such as culture flasks and culture-dishes. However, these tools have fundamental limitations due to being inappropriate for high throughput screening and consume a considerable amount of resources and time [1]. Therefore, there is a trend towards miniaturization, disposables and even micro platforms that fulfill increasing demands strongly aiming for production and testing of novel pharmaceutical products. Here we present the development and manufacture of a disposable miniaturized flow-through bioreactor system that can be produced in large numbers at low costs. nanoporous hollow fibers are located at the fluidic sources and drains of the miniaturized bioreactors and retain cells. The necessary mixture of oxygen and carbon dioxide is provided via diffusion through a semi-permeable membrane. Fluidic connections allow the continuous feeding of the cells adding nutrient solution at constant rates at the inlet of the micro bioreactor and removing the solution at the same rate at the outlet. This medium can be collected and used for subsequent analysis. Different designs and concepts for such bioreactors were carried out with varying numbers of plates, and integrated or joined miniaturized reactor chambers. First tests show full technical and biological functionality, cells could successfully be cultivated at high viability rates for some days.

The immunogenicity of protein aggregates has been investigated in numerous studies. Nevertheless, it is still unknown which kind of protein aggregates enhance immunogenicity the most. The ability of the currently used in vitro and in vivo systems regarding their predictability of immunogenicity in humans is often questionable, and results are partially contradictive. In this study, we used a 2D in vitro assay and a complex 3D human artificial lymph node model to predict the immunogenicity of protein aggregates of bevacizumab and adalimumab. The monoclonal antibodies were exposed to different stress conditions such as light, heat, and mechanical stress to trigger the formation of protein aggregates and particles, and samples were analyzed thoroughly. Cells and culture supernatants were harvested and analyzed for dendritic cell marker and cytokines. Our study in the artificial lymph node model revealed that bevacizumab after exposure to heat triggered a TH1- and proinflammatory immune response, whereas no trend of immune responses was seen for adalimumab after exposure to different stress conditions. The human artificial lymph node model represents a new test model for testing the immunogenicity of protein aggregates combining the relevance of a 3D human system with the rather easy handling of an in vitro setup.

Cdc48 is a AAA+ ATPase that plays an essential role for many cellular processes in eukaryotic cells. An archaeal homologue of this highly conserved enzyme was shown to directly interact with the 20S proteasome. Here, we analyze the occurrence and phylogeny of a Cdc48 homologue in Actinobacteria and assess its cellular function and possible interaction with the bacterial proteasome. Our data demonstrate that Cdc48-like protein of actinobacteria (Cpa) forms hexameric rings and that the oligomeric state correlates directly with the ATPase activity. Furthermore, we show that the assembled Cpa rings can physically interact with the 20S core particle. Comparison of the Mycobacterium smegmatis wild-type with a cpa knockout strain under carbon starvation uncovers significant changes in the levels of around 500 proteins. Pathway mapping of the observed pattern of changes identifies ribosomal proteins as a particular hotspot, pointing amongst others toward a role of Cpa in ribosome adaptation during starvation., eLife, 7, ISSN:2050-084X

Background Insect-bite hypersensitivity is the most common allergic dermatitis in horses. Excoriated skin lesions are typical symptoms of this seasonal and refractory chronic disease. On a cellular level, the skin lesions are characterized by massive eosinophil infiltration caused by an underlying allergic response. Objective To target these cells and treat disease, we developed a therapeutic vaccine against equine IL-5 (eIL-5), the master regulator of eosinophils. Methods The vaccine consisted of eIL-5 covalently linked to a virus-like particle derived from cucumber mosaic virus containing the tetanus toxoid universal T-cell epitope tt830-843 (CMVTT). Thirty-four Icelandic horses were recruited and immunized with 400 μg of eIL-5–CMVTT formulated in PBS without adjuvant (19 horses) or PBS alone (15 horses). Results The vaccine was well tolerated and did not reveal any safety concerns but was able to induce anti–eIL-5 autoantibody titers in 17 of 19 horses. This resulted in a statistically significant reduction in clinical lesion scores when compared with previous season levels, as well as levels in placebo-treated horses. Protection required a minimal threshold of anti–eIL-5 antibodies. Clinical improvement by disease scoring showed that 47% and 21% of vaccinated horses reached 50% and 75% improvement, respectively. In the placebo group no horse reached 75% improvement, and only 13% reached 50% improvement. Conclusion Our therapeutic vaccine inducing autoantibodies against self IL-5 brings biologics to horses, is the first successful immunotherapeutic approach targeting a chronic disease in horses, and might facilitate development of a similar vaccine against IL-5 in human subjects., Journal of Allergy and Clinical Immunology, 142 (4), ISSN:0091-6749, ISSN:1097-6825

Subunit vaccines often require adjuvants to elicit sustained immune activity. Here, a method is described to evaluate the efficacy of single vaccine candidates in the preclinical stage based on cytokine and gene expression analysis. As a model, the recombinant human respiratory syncytial virus (RSV) fusion protein (RSV-F) was produced in CHO cells. For comparison, wild-type and glycoengineered, afucosylated RSV-F were established. Both glycoprotein vaccines were tested in a commercial Human Artificial Lymph Node in vitro model (HuALN®). The analysis of six key cytokines in cell culture supernatants showed well-balanced immune responses for the afucosylated RSV-F, while immune response of wild-type RSV-F was more Th1 accentuated. In particular, stronger and specific secretion of interleukin-4 after each round of re-stimulation underlined higher potency and efficacy of the afucosylated vaccine candidate. Comprehensive gene expression analysis by nCounter gene expression assay confirmed the stronger onset of the immunologic reaction in stimulation experiments with the afucosylated vaccine in comparison to wild-type RSV-F and particularly revealed prominent activation of Th17 related genes, innate immunity, and comprehensive activation of humoral immunity. We, therefore, show that our method is suited to distinguish the potency of two vaccine candidates with minor structural differences.

Xylose is a general component of O-glycans in mammals. Core-xylosylation of N-glycans is only found in plants and helminth. Consequently, xylosylated N-glycans cause immunological response in humans. We have used the F-protein of the human respiratory syncytial virus (RSV), one of the main causes of respiratory tract infection in infants and elderly, as a model protein for vaccination. The RSV-F protein was expressed in CHO-DG44 cells, which were further modified by co-expression of β1,2-xylosyltransferase from Nicotiana tabacum. Xylosylation of RSV-F N-glycans was shown by monosaccharide analysis and MALDI-TOF mass spectrometry. In immunogenic studies with a human artificial lymph node model, the engineered RSV-F protein revealed improved vaccination efficacy.

It has been widely recognised that the phylogenetic distance between laboratory animals and humans limits the former's predictive value for immunogenicity testing of biopharmaceuticals and nanostructure-based drug delivery and adjuvant systems. 2D in vitro assays have been established in conventional culture plates with little success so far. Here, we detail the status of various 3D approaches to emulate innate immunity in non-lymphoid organs and adaptive immune response in human professional lymphoid immune organs in vitro. We stress the tight relationship between the necessarily changing architecture of professional lymphoid organs at rest and when activated by pathogens, and match it with the immunity identified in vitro. Recommendations for further improvements of lymphoid tissue architecture relevant to the development of a sustainable adaptive immune response in vitro are summarized. In the end, we sketch a forecast of translational innovations in the field to model systemic innate and adaptive immunity in vitro.

Quantitative real-time PCR (qPCR) is commonly usedforgeneexpressionanalyseswithdefineddocumentation guidelines to compare published results. To minimize the impactof variancesfromqPCRperformance,samplehandling and processing reference genes are used. Their selection pro- cess cannot be completely aligned due to variations in exper- imental conditions. Furthermore, the named sources of error are alsopresent when determiningthestabilityofthereference genes themselves. Even software applications that are used to identify the best reference genes rarely coincide on their rankings and can be misleading under certain conditions. In previous experiments, peripheral blood mononuclear cells (PBMC) were analyzed to identify the most stable reference gene(s). Twelve of the 13 investigated genes showed sample type specific differences in the expression. Direct mRNA measurement was performed in the form of a NanoString analysis, a multiplexed absolute quantification method. The external validation showed a high concordance of the refer- ence gene expression levels. However, it identified the same sample type specific expression pattern for only some of the tested reference genes. By comparing various combinations of reference genes with both methods we are able to suggest a set of well-performing reference genes.

Type 1 pili are extracellular, supramolecular protein complexes required for the attachment of pathogenic E. coli strains to host cells. Hundreds to thousands of protein subunits are assembled within minutes in vivo and form filaments with unique kinetic stability against dissociation. Here, we review recent work on the structure, assembly mechanism and potential technical applications derived from this exciting biological system.

Decades of costly failures in translating drug candidates from preclinical disease models to human therapeutic use warrant reconsideration of the priority placed on animal models in biomedical research. Following an international workshop attended by experts from academia, government institutions, research funding bodies, and the corporate and non-governmental organisation (NGO) sectors, in this consensus report, we analyse, as case studies, five disease areas with major unmet needs for new treatments. In view of the scientifically driven transition towards a human pathways-based paradigm in toxicology, a similar paradigm shift appears to be justified in biomedical research. There is a pressing need for an approach that strategically implements advanced, human biology-based models and tools to understand disease pathways at multiple biological scales. We present recommendations to help achieve this.

Mesenchymal stromal cells (MSC), known for their high immune modulatory capacity are promising tools for several cell-based therapies. To better mimic the in vivo situation of MSC interactions with immune cells, we applied an artificial lymph node (ALN)-bioreactor culture system combining a miniaturized perfusion bioreactor with a 3D matrix-based cell culture of immune competent cells forming micro-organoids.Rat lymph node cells and allogeneic bone marrow-derived MSCs were seeded in a 20:1 ratio within the agarose matrix of the ALN-reactor. Lymphocytes were pre-incubated with Concanavalin A (ConA) and then co-cultured with MSC in the matrix with additional ConA in the perfusing medium. Live/dead staining showed survival of the co-cultures during the 8-day ALN-reactor run. Paraffin sections of bioreactor matrices were analyzed by proliferating cell nuclear antigen (PCNA)-specific stai-ning to determine MSC proliferation. Immune modulatory capacity was defined by daily analysis of cytokine secretion profiles (TNFa, IFNy, IL-1a, IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-12p40/p70, GM-CSF).Cytokine peak secretion at day 2 was significantly inhibited by MSCs for TNFa (96.8 ± 4.8%) and IFNy (88.7 ± 12.0%) in 3D co-cultures. In contrast, other cytokines (IL-1, IL-6, IL-12) were induced. Furthermore, we detected a significantly higher (58.8%) fraction of proliferating MSCs in the presence of immune cells compared to control bioreactors loaded with MSCs only.In the future, this system might be an excellent tool to investigate the mechanisms of MSC-mediated immune modulation during simulated in vivo conditions.

The immune system is regulated by the complex interaction of multiple cytokines, which are secreted signaling molecules affecting other cells. In this work, we studied the cytokine response to several well-known stimulants, such as OKT-3, Con A, PWM, and SEB. Healthy donor cells (PBMCs) were cultivated for up to 72 h and the mRNA levels and cytokine release of four key cytokines (IL-2, IL-4, IFN-γ, and TNF-α) were analyzed by RT-PCR and bead-based multiplex analyses. The generated cytokine profiles showed characteristic expression patterns and secretion kinetics for each cytokine and substance. PWM/SEB and OKT-3 led to a very fast and long-lasting immune response, whereas Con A induced the slowest cytokine production. Cytokine concentrations also differed greatly. The highest IFN-γ concentration was 1000 times higher than the respective IL-4 concentration. Gene expression and cytokine concentration profiles were strongly correlated during the time course. The chronological response of the donors' cytokine profiles coincided, but showed individual characteristics regarding the strength of the cytokine release. The comparison of stimulation experiments using freshly isolated and cryopreserved PBMCs showed that, for the observation of an immunological response at early points in time, gene expression experiments are more reliable than the measurement of cytokines in the cell culture supernatant. However, the freezing of cells influences the response significantly. The measurement of secreted proteins is the superior method at later points in time.

All IgG-type antibodies are N-glycosylated in their Fc part at Asn-297. Typically, a fucose residue is attached to the first N-acetylglucosamine of these complex-type N-glycans. Antibodies lacking core fucosylation show a significantly enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and an increased efficacy of anti-tumor activity. In cases where the clinical efficacy of an antibody is to some extent mediated by its ADCC effector function, afucosylated N-glycans could help to reduce dose requirement and save manufacturing costs. Using Chinese hamster ovary (CHO) cells as a model, we demonstrate here that heterologous expression of the prokaryotic enzyme GDP-6-deoxy-d-lyxo-4-hexulose reductase within the cytosol can efficiently deflect the fucose de novo pathway. Antibody-producing CHO cells that were modified in this way secrete antibodies lacking core fucose as demonstrated by MALDI-TOF mass spectrometry and HPAEC-PAD monosaccharide analysis. Engineering of the fucose de novo pathway has led to the construction of IgGs with a strongly enhanced ADCC effector function. The method described here should have broad practical applicability for the development of next-generation therapeutic antibodies.

Nonproteinogenic amino acids that either occur naturally or are synthesized chemically are becoming important tools in modern drug discovery. In this context, fluorinated amino acids have great potential in the development of novel pharmaceuticals and drugs. To assess whether different fluorinated aromatic amino acid analogues of phenylalanine, tyrosine, and tryptophan are potentially interesting as therapeutic drugs, we examined their cytostatic and cytotoxic effects on the growth of the human breast cancer cell line MCF-7. Of all the tested analogues L-4-fluorotryptophan, L-6-fluorotryptophan and L-p-fluorophenylalanine effectively and irreversibly inhibited cell growth with IC(50) values in the low micromolar range (3-15 microM). Additionally, using L-4-[14C]fluorotryptophan, and L-6-[14C]fluorotryptophan, we discovered that the cellular uptake of these fluorinated amino acids occurs through active transport with a 70-fold excess of intracellular over extracellular concentrations. We identified system L as the responsible amino acid transporter. Our findings fully support the idea that fluorinated aromatic amino acid analogues are promising chemotherapeutics with the potential for use in combination with classical cancer therapy, and as new cytotoxic drugs for certain tumor types such as melanoma.

We describe the manufacture of highly stable and elastic alginate membranes with good cell adhesivity and adjustable permeability. Clinical grade, ultra-high viscosity alginate is gelled by diffusion of Ba2+ followed by use of the "crystal gun" [Zimmermann H. et al., Fabrication of homogeneously cross-linked, functional alginate microcapsules validated by NMR-, CLSM- and AFM-imaging. Biomaterials 2003;24:2083-96]. Burst pressure of well-hydrated membranes is between 34 and 325kPa depending on manufacture and storage details. Water flows induced by sorbitol and raffinose (probably diffusional) are lower than those caused by PEG 6000, which may be related to a Hagen-Poiseuille flow. Hydraulic conductivity, L(p), from PEG-induced flows ranges between 2.4x10(-12) and 6.5x10(-12) m Pa(-1)s(-1). Hydraulic conductivity measured with hydrostatic pressure up to 6 kPa is 2-3 orders of magnitude higher and decreases with increasing pressure to about 3x10(-10) m Pa(-1)s(-1) at 4kPa. Mechanical introduction of 200 microm-diameter pores increases hydraulic conductivity dramatically without loss of mechanical stability or flexibility. NMR imaging with Cu2+ as contrast agent shows a layered structure in membranes cross-linked for 2h. Phase contrast and atomic force microscopy in liquid environment reveals surface protrusions and cavities correlating with steps of the production process. Murine L929 cells adhere strongly to the rough surface of crystal-bombarded membranes. NaCl-mediated membrane swelling can be prevented by partial replacement of salt with sorbitol allowing cell culture on the membranes.

Extracorporeal human lymphatic organs are expected to be excellent tools in the study of human molecular and cellular bases of the immunologic balance and tissue harmony. A rational approach and process to design a device and a procedure to recreate the human lymph node environment in vitro is described with emphasis on T-cell activation. Based on this approach, a bioreactor and a process supporting self-assembly of human lymphatic tissues due to proper emulation of human architecture and homeostasis could be developed.

Die Messung von Zytokinen in Stimulationsexperimenten zur Bestimmung von Stärke und Umfang einer Immunreaktion werden in der Praxis häufig an kryokonserviertem Zellmaterial durchgeführt. Bisherige Untersuchungen zum Einfluss der Kryokonservierung auf die Zytokinexpression sind widersprüchlich. In den hier durchgeführten Experimenten wurden die Genexpression und/ oder Sekretion der Zytokine IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, GM-CSF, IFN-γ und TNF-α in frisch isolierten und kryokonservierten Immunzellen aus Blut (PBMC) eines gesunden Spenders bestimmt. Diese wurden mit den Immunsystem-aktivierenden Stoffen OKT-3 und Concanavalin A (Con A) stimuliert und für 4 bzw. 24 h kultiviert. Ein Vergleich der frisch-isolierten und kryokonservierten PBMC in Stimulationsexperimenten zeigt, (1) dass die Messung der Genexpression genauere Einblicke zu Beginn der einsetzenden Immunreaktion verschafft, als die Messung der ausgeschütteten Zytokine, (2) dass durch Einfrieren die Immunreaktion insbesondere zu diesem Zeitpunkt beeinflusst wird und (3) dass zu späteren Zeitpunkten die Konzentrationsbestimmung der Zytokine im Zellkulturüberstand das Mittel der Wahl ist. The measurement of cytokines in stimulation experiments with the aim to determine the strength and the complexitiy of an immune reaction is commonly carried out on cryopreserved cells. Previous studies, investigating the impact of cryopreservation on the cytokine expression, are inconsistent in their findings. Experiments conducted in this study determine the gene expression and/or secretion of IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, GM-CSF, IFN-γ and TNF-α in freshly isolated and cryopreserved PBMC of a healthy donor. The cells were stimulated with the substances OKT-3 and Concanavalin A (Con A) and cultured for 4h and 24h. The comparison of freshly isolated and cryopreserved cells in stimulation experiments showed (1) that the measurement of the gene expression offers a more accurate insight into the immune-reaction at early timepoints than the measurement of the secreted cytokines; (2) that freezing of the cells affect the immune response at this early stage and (3) that at later points the measurement of secreted proteins is the method of choice.

An alternative to tagging: The thermodynamically most stable protein ligand complex known to date (K D=1.5×10 20M) consists of the two components FimGt and DsF portions of two different subunits of the type 1 pilus protein complex. The first technical applications of this new complex are the single step purification and identification of multiprotein complexes from cell extracts. Copyright © 2012 WILEY VCH Verlag GmbH Co. KGaA Weinheim.

The TanDEM-X satellite is essentially a rebuild of the TerraSAR-X satellite operated successfully since 2007. The TanDEM-X mission became a vision during the development of the TerraSAR-X satellite. Therefore, necessary design extensions have already been implemented on TerraSAR-X to support future synchronised operation of both radars. The bus design of TanDEM-X has been extended with respect to TerraSAR-X to allow formation flight of both satellites. The software changes relevant for the Tandem mission have been verified during the TanDEM-X on-ground tests and then also uplinked to the TerraSAR-X satellite in preparation of the formation flight. This paper describes the TanDEM-X design and In-Orbit verification results concentrating on the differences to TerraSAR-X and the aspects of bistatic operation

Die simultane Bestimmung mehrerer Analyten und die Erstellung komplexer Parameterprofi e erlangt immer größere Bedeutung in der heutigen Labordiagnostik. Die Bead-basierte Multiplexanalytik bietet hier eine flexible, schnelle und einfache Methode zur Erstellung individueller Analysen. Aufgrund der Vielzahl von Anwendungsmöglichkeiten, die diese moderne Nachweismethode im Bereich molekularbiologischer Fragestellungen bietet, ist die Etablierung der Beadbasierten Multiplexanalytik im Labor für Molekularbiologie und funktionelle Genomik der Technischen Hochschule Wildau von großem Nutzen. Zur Einarbeitung in das Testsystem wurden die Konzentrationen der Zytokine IL-2, IL-4, IFN-γ und TNF-α in Zellkulturüberständen mit kommerziellen Fertigsystemen gemessen und mit mRNA-Expressionsraten der gleichen Proben verglichen. Des Weiteren wurde ein Testsystem zum Nachweis von humanen Antikörpern der Klassen IgG und IgM sowie deren antigen-spezifischer Anteil in Zellkulturüberständen entwickelt. Außerdem konnte durch die erfolgreiche Detektion von DNA-gekoppelten Beads mittels markierter Oligonukleotidsequenz die Kopplung und die Anwendbarkeit der Methode auf Bindungsexperimente mit Nukleotidsequenzen gezeigt werden The simultaneous determination of multiple analytes and the generation of complex parameter profi les gains increasing importance in today’s laboratory diagnostics. The bead-based multiplex assay is offering a flexible, rapid and easy to handle method for the creation of individual analyses. Because of the multitude of applications this modern detection system offers, the establishment of the bead-based multiplex technique is of great benefit for the Laboratory for Molecular Biology and Functional Genomics of the Technical University of Applied Sciences Wildau. To familiarize with the testing system the concentrations of the cytokines IL-2, IL-4, IFN-γ and TNF-α in cell culture supernatant were analysed with commercially available assays and compared with mRNA expression ratios of the same samples. Furthermore a custom testing system was developed for the detection of human IgG and IgM antibodies and also antigen specific antibodies in cell culture supernatants. The assignability of the method to binding experiments with nucleotide sequences could be shown by the successful detection of DNA-modified beads by conjugated oligonucleotides

Pharmaceutical drugs and compounds used for consumer products may bear the risk of unexpected immuno-toxicological side effects, such as sensitization, allergy, anaphylaxis or immunogenicity. Modern biopharmaceuticals with high potency and target specificity, like antibodies and cytokines need to be tested for their therapeutical doses, their exposition regimens and their immune functionality prior to first-in-man applications. For the latter, existing in vitro tests and animal models do not sufficiently reflect the complexity and specificity of the human immune system. Even novel humanised animal models have limitations in their systemic reactions. Monolayer or suspended cell culture possesses neither tissue functionality nor organ physiology, and also cannot be used for long term culture and experiments. In contrast, solid tissue biopsies, e.g. tonsil preparations of tonsillitis patients typically show inflammatory artefacts and degrade in long term culture due to preparation-induced damage. The construction of tissue-like structures in vitro, so-called "micro-organoids", can overcome these limitations. Key structures of secondary lymphatic organs, e.g. lymph nodes or the spleen are the primary lymphatic follicles and germinal centres, in particular during the "activated state" of an inflammation or infection. To remodel lymphatic follicles, functional and structural cells, e.g. lymphoid cells derived from peripheral blood mononuclear cells (PBMCs) and stromal cells need to be combined with biogenic or artificial matrices and scaffolds to produce a suitable 3D tissue-mimicking environment. Therefore, a unique human lymph node model (HuALN) was designed to operate over several weeks, and allow long term and repeated drug exposure to induce and monitor both cellular and humoral immune responses. Cellular immunity is monitored, for example, by cytokine release patterns; humoral immunity is analysed, for example, by B cell activation, plasma cell formation and antibody secretion profiles (IgM and IgG). Moreover, cellular composition and micro-organoid formation are analysed by flow cytometry, histology and in situ imaging.

Bioreactor systems play an important role in tissue engineering, as they enable reproducible and controlled changes to be made in specifi c environmental factors. They can also provide the technical means to perform controlled studies aimed at understanding specifi c biological, chemical, or physical effects. Furthermore, bioreactors allow for a safe and reproducible production of tissue constructs. For later clinical applications, the bioreactor system should be an advantageous method in terms of low contamination risk, ease of handling, and scalability. With respect to drug screening, the main challenge is the effi cient, reproducible handling of a large quantity of tissue constructs in parallel (high-throughput screening). To date, the goals and expectations of bioreactor development have been fulfi lled only to some extent, as bioreactor design in tissue engineering is very complex and still at an early stage of development, especially for use in drug screening. In this chapter, important aspects of bioreactor design are summarized, and an overview of existing concepts is provided. An artifi cial immunosystem will be used as an example to demonstrate how an increased fundamental understanding of biological, biochemical, and engineering aspects can signifi cantly improve the properties of 3D tissue constructs.

Bei einer adaquaten Aussaat der zu klonierenden bzw. zu charakterisierenden Zellen ist es moglich, die Syntheseleistungen der Zelle durch Einsatz spezifischer Fluoreszenzmarker zu visualisieren. Ziel dieser Arbeit ist die qualitative Erfassung dieser Visualisierung, so dass eine Aussage uber die spezifische Syntheseleistung der jeweiligen Einzelzelle moglich ist. Dabei findet das Konzept der unscharfen Klassifikation Anwendung. Nach der Identifikation der „Hochproduzenten“ unter den Zellen ist mit dem Visualisierungs- und Analyse verfahren ein Entscheidungssystem zu entwickeln, dass die Moglichkeiten der Ansteuerung und Gewinnung dieser Zellen mittels Mikromanipulation auch im Kultursystem schafft.

Adhesive chaperone-usher pili are long, supramolecular protein fibers displayed on the surface of many bacterial pathogens. The type 1 and P pili of uropathogenic Escherichia coli (UPEC) play important roles during urinary tract colonization, mediating attachment to the bladder and kidney, respectively. The biomechanical properties of the helical pilus rods allow them to reversibly uncoil in response to flow-induced forces, allowing UPEC to retain a foothold in the unique and hostile environment of the urinary tract. Here we provide the 4.2-Å resolution cryo-EM structure of the type 1 pilus rod, which together with the previous P pilus rod structure rationalizes the remarkable “spring-like” properties of chaperone-usher pili. The cryo-EM structure of the type 1 pilus rod differs in its helical parameters from the structure determined previously by a hybrid approach. We provide evidence that these structural differences originate from different quaternary structures of pili assembled in vivo and in vitro., Structure, 25 (12), ISSN:0969-2126, ISSN:1878-4186