Quantitative Analysis of T Cell Stimulation and Costimulation using Cellular Microsystems

Die Aktivierung der T-Zellen des Immunsystems erfolgt durch Interaktionen der T-Zell-Rezeptoren (TCR) mit spezifischen Komplexen aus MHC-Molekülen und antigenen Peptiden auf Ziel-Zellen, wodurch eine Kaskade von durch Kinasen vermittelten Protein-Phosphorylierungen ausgelöst wird. Die entstehenden Phospho-Tyrosin-Motive fungieren in der Rekrutierung und Aktivierung weiterer Enzyme. Ein Beispiel hierfür ist ist die Phosphorylierung des TCR/CD3-Komplex durch die Kinase LCK, gefolgt von der Rekrutierung der ZAP-70 Kinase. Im Zusammenspiel des TCR mit Costimuli wie z.B. CD28 werden zelluläre Antworten wie Genexpression und zum Kontakt-stabilisierenden Rearrangement des Zytoskeletts ausgelöst. Wegen der Bedeutung extrazellulärer Stimuli für die Immunantwort besteht ein großes Interesse an der Entwicklung analytischer Techniken, die die quantitative Analyse zellulärer Interaktionen und unterliegender molekularer Prozesse ermöglichen und so die Integration dieser Ereignisse in Signal-Netzwerke ermöglichen. Da die an der T-Zell-Aktivierung beteiligten Komponenten weitgehend bekannt sind, ist der nächste Schritt, die zur Aktivierung der T-Zelle führenden Ereignisse unter Berücksichtigung zeitlicher Abläufe und Stimulus-Intensitäten zu analysieren. Im ersten Teil dieser Arbeit wurden die Signalwege der T-Zell-Aktivierung auf der morphologischen Ebene untersucht. Zum Einen wurde die Empfindlichkeit eines Kontakts und zwischen einer T-Zelle und einer mit anti-CD3-Antikörpern beschichteten Oberfläche gegenüber einem zu unterschiedlichen Zeitpunkten zugegebenen LCK-Inhibitor untersucht. Weiterhin wurde der Einfluss von Inhibitoren verschiedener Signalproteine auf die Zell-Anheftung und Aktivierung des Transkriptionsfaktors NFAT von mit anti-CD3 und anti-CD28 in unterschiedlichen Kombinationen stimulierten Zellen in einem zellulären Mikroarray quantifiziert. Weiterhin dienten mikrostrukturierte Oberflächen zur Analyse der Einflüsse lokaler Stimulation auf die Rekrutierung von Signalproteinen. Auf der biochemischen Ebene wurde mit Hilfe von aus Protein-Protein Interaktionsmotiven abgeleiteten Peptidmikroarrays die stimulationsabhängige Ausbildung von Multiprotein-Komplexen analysiert.The activation of T cells in the immune system occurs via interactions of the T cell-receptors (TCR) with specfic complexes formed by MHC molecules and antigenic peptides on the surface of target cells, initiating a cascade of kinase-mediated protein phosphorylations. The resulting phosphotyrosine motifs enable the recruitment and activation of further enzymes.One example for this is the phosphorylation of the TCR/CD3-complex by the LCK kinase, followed by the recruitment of the ZAP-70 kinase. In concert of the TCR with costimuli such as CD28, cellular responses such as gene expression and contact-stabilizing rearrangements of the cytoskeleton are initiated. Because of the importance of extracellular stimuli for the immune response there is a great interest in the development of analytical techniques enabling the quantitative analysis of cellular interactions and the underlying molecular processes and thus the integration of these events into signalling networks. Since the components involved in T cell activation are widely known, the next step would be the analysis of these events considering time dependence and signal intensities. In the first part of this thesis, T cell signalling pathways were analyzed on the morphological scale. Firstly, the sensitivity of a contact between a T cell and an anti-CD3 functionalized surface to an LCK inhibitor was analyzed at different time points. In the next step, the influence of inhibitors of different signalling proteins on cell adhesion and the activation of the transcription factor NFAT induced by stimulation was quantified in a microarray, in which cells were stimulated by different immobilization concentrations of anti-CD3 and anti-CD28 antibodies. Moreover, microstructured surfaces were employed to analyze the influence of local stimulation on the recruitment of signalling proteins. On the biological scale, peptide microarrays derived from protein-protein interaction motifs were used to analyze the stimulation-dependent formation of multiprotein complexes

Simultanes Emulgieren und Mischen

Emulgieren und Mischen sind etablierte Grundoperationen der Verfahrenstechnik. In dieser Arbeit wird dargestellt, wie in einer Zerkleinerungseinheit diese Grundoperationen kombiniert werden können und somit Produkte wie Milch und Pickering Emulsionen effizienter hergestellt werden können. Mit der neuen Simultanen Emulgier und Misch-Technik wird es damit möglich, sowohl neue Prozesse, die effizienter und einfacher sind, als auch neue Produkte wie emulgatorfreie Suspensionen zu entwickeln

Dynamics of Natural Killer cell receptor revealed by quantitative analysis of photoswitchable protein

Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies which rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report a novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. Thus, following a localized switching event, the movement of the photoswitched molecules can be temporally and spatially resolved by monitoring fluorescence in two regions of interest. By comparing images with mathematical models, we evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 +- 0.06 micron^2/s) and assessed how synapse formation affects receptor dynamics. Our data conclude that the inhibitory NK cell receptor KIR2DL1 is continually trafficked into the synapse and remains surprisingly stable there. Unexpectedly however, in NK cells forming synapses with multiple target cells simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus, our results reveal a previously undetected inter-synaptic exchange of protein.Comment: 25 pages, 5 figure

EMF Model Refactoring based on Graph Transformation Concepts

The Eclipse Modeling Framework (EMF) provides a modeling and code generation framework for Eclipse applications based on structured data models. Within model driven software development based on EMF, refactoring of EMF models become a key activity. In this paper, we present an approach to define EMF model refactoring methods as transformation rules being applied in place on EMF models. Performing an EMF model refactoring, EMF transformation rules are applied and can be translated to corresponding graph transformation rules, as in the graph transformation environment AGG. If the resulting EMF model is consistent, the corresponding result graph is equivalent and can be used for validating EMF model refactoring. Results on conflicts and dependencies of refactorings for example, can help the developer to decide which refactoring is most suitable for a given model and why

Morphological characterization of the human corneal epithelium by in vivo confocal laser scanning microscopy

Background: Regarding the growing interest and importance of understanding the cellular changes of the cornea in diseases, a quantitative cellular characterization of the epithelium is becoming increasingly important. Towards this, the latest research offers considerable improvements in imaging of the cornea by confocal laser scanning microscopy (CLSM). This study presents a pipeline to generate normative morphological data of epithelial cell layers of healthy human corneas. Methods: 3D in vivo CLSM was performed on the eyes of volunteers (n=25) with a Heidelberg Retina Tomograph II equipped with an in-house modified version of the Rostock Cornea Module implementing two dedicated piezo actuators and a concave contact cap. Image data were acquired with nearly isotropic voxel resolution. After image registration, stacks of en-face sections were used to generate full-thickness volume data sets of the epithelium. Beyond that, an image analysis algorithm quantified en-face sections of epithelial cells regarding the depth-dependent mean of cell density, area, diameter, aggregation (Clark and Evans index of aggregation), neighbor count and polygonality. Results: Imaging and cell segmentation were successfully performed in all subjects. Thereby intermediated cells were efficiently recognized by the segmentation algorithm while efficiency for superficial and basal cells was reduced. Morphological parameters showed an increased mean cell density, decreased mean cell area and mean diameter from anterior to posterior (5,197.02 to 8,190.39 cells/mm²; 160.51 to 90.29 µm²; 15.9 to 12.3 µm respectively). Aggregation gradually increased from anterior to posterior ranging from 1.45 to 1.53. Average neighbor count increased from 5.50 to a maximum of 5.66 followed by a gradual decrease to 5.45 within the normalized depth from anterior to posterior. Polygonality gradually decreased ranging from 4.93 to 4.64 sides of cells. The neighbor count and polygonality parameters exhibited profound depth-dependent changes. Conclusions: This in vivo study demonstrates the successful implementation of a CLSM-based imaging pipeline for cellular characterization of the human corneal epithelium. The dedicated hardware in combination with an adapted image registration method to correct the remaining motion-induced image distortions followed by a dedicated algorithm to calculate characteristic quantities of different epithelial cell layers enabled the generation of normative data. Further significant effort is necessary to improve the algorithm for superficial and basal cell segmentation

Corneal Subbasal Nerve Plexus Changes in Severe Diabetic Charcot Foot Deformity: A Pilot Study in Search for a DNOAP Biomarker

Introduction. Diabetic neuroosteoarthropathy (DNOAP) early symptoms are unspecific, mimicking general infectious symptoms and rendering a diagnosis challenging. Consequently, unfavourable outcomes occur frequently, with recurrent foot ulceration, infectious complications, and eventually amputation. Corneal confocal microscopy (CCM) of the subbasal nerve plexus (SNP) is used to detect early peripheral neuropathy in diabetic patients without diabetic retinopathy. This pilot study was designed to determine if specific SNP changes manifest in severe DNOAP in comparison to a healthy control group. Methods. This pilot study utilized a matched-pair analysis to investigate SNP changes by in vivo CCM for 26 patients (mean patient age 63.7 years, range 27 to 78) with severe DNOAP defined by condition after the need for reconstructive foot surgery (n=13) and a healthy control group (n=13). Corneal nerve fibre length (CNFL), nerve fibre density (CNFD), nerve branch density (CNBD), average weighted corneal nerve fibre thickness (CNFTh), nerve connecting points (CNCP), and average weighted corneal nerve fibre tortuosity (CNFTo) were assessed as well as the general clinical status, diabetic status, and ophthalmologic basic criteria. Results. In vivo CCM revealed significantly reduced SNP parameters in the DNOAP group for CNFL (p=0.010), CNFD (p=0.037), CNBD (p=0.049), and CNCP (p=0.012) when compared to the healthy control group. Six patients (46%) of the DNOAP group suffered from diabetic retinopathy and none of the control group. Conclusions. This pilot study revealed a rarefication of SNP in all measured parameters in patients with severe DNOAP. We see a potential value of CCM providing a SNP-based biomarker for early stages of DNOAP prior to the development of any foot deformities that needs to be evaluated in further studies. This trial is registered with German Clinical Trials Register (DKRS) DRKS00007537

3D confocal laser-scanning microscopy for large-area imaging of the corneal subbasal nerve plexus

The capability of corneal confocal microscopy (CCM) to acquire high-resolution in vivo images of the densely innervated human cornea has gained considerable interest in using this non-invasive technique as an objective diagnostic tool for staging peripheral neuropathies. Morphological alterations of the corneal subbasal nerve plexus (SNP) assessed by CCM have been shown to correlate well with the progression of neuropathic diseases and even predict future-incident neuropathy. Since the field of view of single CCM images is insufficient for reliable characterisation of nerve morphology, several image mosaicking techniques have been developed to facilitate the assessment of the SNP in large-area visualisations. Due to the limited depth of field of confocal microscopy, these approaches are highly sensitive to small deviations of the focus plane from the SNP layer. Our contribution proposes a new automated solution, combining guided eye movements for rapid expansion of the acquired SNP area and axial focus plane oscillations to guarantee complete imaging of the SNP. We present results of a feasibility study using the proposed setup to evaluate different oscillation settings. By comparing different image selection approaches, we show that automatic tissue classification algorithms are essential to create high-quality mosaic images from the acquired 3D dataset

Real-time large-area imaging of the corneal subbasal nerve plexus

The morphometric assessment of the corneal subbasal nerve plexus (SNP) by confocal microscopy holds great potential as a sensitive biomarker for various ocular and systemic conditions and diseases. Automated wide-field montages (or large-area mosaic images) of the SNP provide an opportunity to overcome the limited field of view of the available imaging systems without the need for manual, subjective image selection for morphometric characterization. However, current wide-field montaging solutions usually calculate the mosaic image after the examination session, without a reliable means for the clinician to predict or estimate the resulting mosaic image quality during the examination. This contribution describes a novel approach for a real-time creation and visualization of a mosaic image of the SNP that facilitates an informed evaluation of the quality of the acquired image data immediately at the time of recording. In cases of insufficient data quality, the examination can be aborted and repeated immediately, while the patient is still at the microscope. Online mosaicking also offers the chance to identify an overlap of the imaged tissue region with previous SNP mosaic images, which can be particularly advantageous for follow-up examinations

In vivo monitoring of corneal dendritic cells in the subbasal nerve plexus during Trastuzumab and Paclitaxel breast cancer therapy - a one-year follow-up

Paclitaxel and trastuzumab have been associated with adverse effects including chemotherapy-induced peripheral neuropathy (CIPN) or ocular complications. In vivo confocal laser scanning microscopy (CLSM) of the cornea could be suitable for assessing side effects since the cornea is susceptible to, i.e., neurotoxic stimuli. The study represents a one-year follow-up of a breast cancer patient including large-area in vivo CLSM of the subbasal nerve plexus (SNP), nerve function testing, and questionnaires during paclitaxel and trastuzumab therapy. Six monitoring sessions (one baseline, four during, and one after therapy) over 58 weeks were carried out. Large-area mosaics of the SNP were generated, and identical regions within all sessions were assigned. While corneal nerve morphology did not cause alterations, the number of dendritic cells (DCs) showed dynamic changes with a local burst at 11 weeks after baseline. Simultaneously, paclitaxel treatment was terminated due to side effects, which, together with DCs, returned to normal levels as the therapy progressed. Longitudinal in vivo CLSM of the SNP could complement routine examinations and be helpful to generate a comprehensive clinical picture. The applied techniques, with corneal structures acting as biomarkers could represent a diagnostic tool for the objective assessment of the severity of adverse events and the outcome

Volumetric measurements of paranasal sinuses and examination of sinonasal communication in healthy Shetland ponies: anatomical and morphometric characteristics using computed tomography.

BACKGROUND: Despite clinical importance and frequent occurrence of sinus disease, little is known about the size of paranasal sinuses and their communication in ponies and small horses. To examine the shape and volume of the paranasal sinuses and evaluate the sinonasal communication, three-dimensional (3D) reconstructions of computed tomography (CT) datasets of 12 healthy adult Shetland ponies were performed and analysed. Linear measurements of head length and width were taken. Using semi-automatic segmentation, 3D-models of all sinus compartments were created. Volumetric measurement of the seven sinus compartments were conducted and statistical analysis was performed. Sinus volumes were compared between the left and right sinuses and the relation to age and head size was evaluated. RESULTS: Structure and shape of the paranasal sinus system in Shetland ponies was similar to that of large horses. All seven sinus compartments on each side of the head were identified (rostral maxillary sinus, ventral conchal sinus, caudal maxillary sinus, dorsal conchal sinus, middle conchal sinus, frontal sinus, sphenopalatine sinus). The existence of a bilateral cranial and a caudal system formed by a maxillary septum was visible in all 12 individuals. The volumetric sizes of the left and right sinuses did not differ significantly (p > 0.05). A positive correlation between the size of the paranasal sinuses and the head length was shown. A relation between sinus volumes and age could not be proved in adult ponies aged > six years. Communication between single sinus compartments was identified. Furthermore, communication with the nasal cavity over the nasomaxillary aperture (Apertura nasomaxillaris) and a common sinonasal channel (Canalis sinunasalis communis) as well as its splitting up into a rostral and a caudolateral channel could be seen. Examination of the sinonasal communication was challenging and only a descriptive evaluation was possible. CONCLUSIONS: Our findings concerning the size, shape and volumetric dimensions of Shetland pony CT images could help improve CT interpretation of abnormal clinical cases as well as aiding clinicians to develop and select appropriate instruments for medical inspection and treatments