Aufhebung der peripheren Immuntoleranz durch Kopplung eines Melanomantigens mit immunogenen Fremdproteinen

Die Dissertationsarbeit mit dem Titel "Aufhebung der peripheren Immuntoleranz durch Kopplung eines Melanomantigens mit immunogenen Fremdproteinen" befasst sich mit der Weiterentwicklung einer effektiven Melanomvakzine im tierexperimentellen Modell im Hinblick auf die spätere klinische Anwendung im Patienten. Als Antigen wurde das in Melanomzellen und in Melanozyten natürlicherweise exprimierte Protein TRP2 verwendet. Dieses Antigen kann bei Mäusen und Menschen von zellulären Komponenten des Immunsystems erkannt werden. Durch die Fusion des wenig immunogenen Selbstantigens mTRP2 mit dem immunogenen Fusionspartner EGFP gelang es bei C57BL/6 Mäusen, die körpereigene Toleranz gegen Selbstantigene zu umgehen und so eine antigenspezifische Immunantwort zu induzieren. In den mit der cDNA von EGFPmTRP2aa30-519 immunisierten Mäusen konnte eine vitiligoartige Felldepigmentierung nachgewiesen werden, die nach Immunisierung mit der cDNA von mTRP2 nicht beobachtet wurde. In diesen Tieren ließen sich auch im Elispot TRP2-spezifische T-Zellen nachweisen. Tiere, die mit Ad-EGFPmTRP2aa30-519 immunisiert wurden waren gegen das Wachstum von transplantierten Melanomzellen geschützt. Eine Immunantwort und ein Tumorschutz konnte jedoch nur induziert werden, wenn eine direkte Fusion zwischen dem Selbstantigen TRP2 und dem immunogenen Fusionspartner EGFP besteht. In Versuchen mit knockout Mäusen und Depletionsexperimenten konnte gezeigt werden, das CD4+ T-Zellen eine entscheidende Rolle in der Primingphase CD8+T-Zellen von die Induktion von antigenspezifischen CD8+T-Zellen spielen. In weiteren Experimenten nach Immunisierung mit dem Fusionskonstrukten EGFPmTRP2aa30-188, EGFPmTRP2aa30-179 und EGFPmTRP2aa180-188 konnte gezeigt werden, dass das Peptidepitop mTRP2 aa180-188 für die Induktion einer Felldepigmentierung und für eine effektive Abwehr von transplantierten Melanomzellen vorhanden sein muss. Um den Einfluss von kompetitiven MHC-Klasse I bindenden Peptidepitopen auf die Induktion einer Immunantwort zu untersuchen wurden Mäuse mit dem Fusionskonstrukt ß-Galaktosidase-mTRP2aa180-188 immunisiert. In diesen Mäusen konnte eine vtiligoartige Felldepigmentierung der beschossenen Bauchhaut und im Elispot-Test gleichzeitig TRP2 und ß-gal spezifische T-Zellen nachgewiesen werden. Im Gegensatz zu Arbeiten anderer Arbeitsgruppen scheint in diesem System keine kompetitive Hemmung zwischen dem ß-gal und dem TRP2 Peptid zu bestehen. Um den Einfluss der Kopplung auf die intrazelluläre Lokalisation zu charakterisieren wurden Fusionskonstrukte bestehend aus EGFP und mTRP2 mit unterschiedlichen Lokalisationssequenzen generiert, DCEK-Zellen stabil transfiziert und die intrazelluläre Lokalisation mit konfokaler Mikroskopie bestimmt. Nach Immunsisierung von Mäusen mit diesen Fusionskonstrukten konnte gezeigt werde, das die intrazelluläre Lokalisation keinen Einfluss auf die Entstehung einer Immunantwort hat. Für die spätere Anwendung der Kopplung in klinischen Studien wurde ein Fusionskonstrukt bestehend aus mTRP2 und dem gut charakterisiertem Antigen des humanen Cytomegalovirus pp65 generiert. Nach Gene-Gun Immunisierung von C57BL/6 Mäusen mit pp65mTRP2 zeigte sich ähnlich wie bei EGFPmTRP2 eine vitiligoartige Felldepigmentierung. Im Elispot-Test ließen sich TRP2 spezifische T-Zellen nachweisen. Das Ergebnis bestätigt die Annahme, dass auch pp65 zur Verstärkung einer antigenspezifischen Immunantwort gegen ein wenig immunogenes Selbstantigen in der Lage ist

Arginase 1+ IL-10+ polymorphonuclear myeloid-derived suppressor cells are elevated in patients with active pemphigus and correlate with an increased Th2/Th1 response

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells, which are characterized by their capability to suppress T-cell responses. While MDSCs have been traditionally associated with cancer diseases, their role as regulators of autoimmune diseases is emerging. Pemphigus is a chronic autoimmune blistering skin disease characterized by dysregulated T-cell responses and autoantibody production. The role of MDSCs in pemphigus disease has not been defined yet. The aim of this study was to characterize MDSCs in pemphigus patients and to dissect their relationship with CD4(+) T-cell subsets and clinical disease assessments. For this purpose, we performed a cross-sectional analysis of 20 patients with pemphigus. Our results indicate that a population of CD66b(+)CD11b(+) polymorphonuclear-like MDSCs (PMN-MDSCs) is expanded in the peripheral blood mononuclear cell fraction of pemphigus patients compared to age-matched healthy donors. These PMN-MDSCs have the capability of suppressing allogeneic T-cell proliferation in vitro and show increased expression of characteristic effector molecules such as arginase I and interleukin-10. We further demonstrate that PMN-MDSCs are especially expanded in patients with active pemphigus, but not in patients in remission. Moreover, MDSC frequencies correlate with an increased Th2/Th1 cell ratio. In conclusion, the identification of a functional PMN-MDSC population suggests a possible role of these cells as regulators of Th cell responses in pemphigus

DNA methylation-based classification of central nervous system tumours.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology

Thermography and thermoregulation of the face

BACKGROUND: Although clinical diagnosis of thermoregulation is gaining in importance there is no consistent evidence on the value of thermography of the facial region. In particular there are no reference values established with standardised methods. METHODS: Skin temperatures were measured in the facial area at 32 fixed measuring sites in 26 health subjects (7–72 years) with the aid of a contact thermograph (Eidatherm). A total of 6 measurements were performed separately for the two sides of the face at intervals of equal lengths (4 hours) over a period of 24 hours. Thermoregulation was triggered by application of a cold stimulus in the region of the ipsilateral ear lobe. RESULTS: Comparison of the sides revealed significant asymmetry of face temperature. The left side of the face showed a temperature that was on the average 0.1°C lower than on the right. No increase in temperature was found following application of the cold stimulus. However, a significant circadian rhythm with mean temperature differences of 0.7°C was observed. CONCLUSION: The results obtained should be seen as an initial basis for compiling an exact thermoprofile of the surface temperature of the facial region that takes into account the circadian rhythm, thus closing gaps in studies on physiological changes in the temperature of the skin of the face

Intracerebral Human Regulatory T Cells: Analysis of CD4+CD25+FOXP3+ T Cells in Brain Lesions and Cerebrospinal Fluid of Multiple Sclerosis Patients

Impaired suppressive capacity of CD4+CD25+FOXP3+ regulatory T cells (Treg) from peripheral blood of patients with multiple sclerosis (MS) has been reported by multiple laboratories. It is, however, currently unresolved whether Treg dysfunction in MS patients is limited to reduced control of peripheral T cell activation since most studies analyzed peripheral blood samples only. Here, we assessed early active MS lesions in brain biopsies obtained from 16 patients with MS by FOXP3 immunohistochemistry. In addition, we used six-color flow cytometry to determine numbers of Treg by analysis of FOXP3/CD127 expression in peripheral blood and cerebrospinal fluid (CSF) of 17 treatment-naïve MS patients as well as quantities of apoptosis sensitive CD45ROhiCD95hi cells in circulating and CSF Treg subsets. Absolute numbers of FOXP3+ and CD4+ cells were rather low in MS brain lesions and Treg were not detectable in 30% of MS biopsies despite the presence of CD4+ cell infiltrates. In contrast, Treg were detectable in all CSF samples and Treg with a CD45ROhiCD95hi phenotype previously shown to be highly apoptosis sensitive were found to be enriched in the CSF compared to peripheral blood of MS patients. We suggest a hypothetical model of intracerebral elimination of Treg by CD95L-mediated apoptosis within the MS lesion

"Inner voices" : the cerebral representation of emotional voice cues described in literary texts

While non-verbal affective voice cues are generally recognized as a crucial behavioral guide in any day-to-day conversation their role as a powerful source of information may extend well beyond close-up personal interactions and include other modes of communication such as written discourse or literature as well. Building on the assumption that similarities between the different ‘modes’ of voice cues may not only be limited to their functional role but may also include cerebral mechanisms engaged in the decoding process, the present functional magnetic resonance imaging study aimed at exploring brain responses associated with processing emotional voice signals described in literary texts. Emphasis was placed on evaluating ‘voice’ sensitive as well as task- and emotion-related modulations of brain activation frequently associated with the decoding of acoustic vocal cues. Obtained findings suggest that several similarities emerge with respect to the perception of acoustic voice signals: results identify the superior temporal, lateral and medial frontal cortex as well as the posterior cingulate cortex and cerebellum to contribute to the decoding process, with similarities to acoustic voice perception reflected in a ‘voice’-cue preference of temporal voice areas as well as an emotion-related modulation of the medial frontal cortex and a task-modulated response of the lateral frontal cortex

Hydrogen Embrittlement Mechanism in Fatigue Behaviour of Austenitic and Martensitic Stainless Steels

In the present study, the influence of hydrogen on the fatigue behaviour of the high strength martensitic stainless steel X3CrNiMo13-4 and the metastable austenitic stainless steels X2Crni19-11 with various nickel contents was examined in the low and high cycle fatigue regime. The focus of the investigations was the changes in the mechanisms of short crack propagation. The aim of the ongoing investigation is to determine and quantitatively describe the predominant processes of hydrogen embrittlement and their influence on the short fatigue crack morphology and crack growth rate. In addition, simulations were carried out on the short fatigue crack growth, in order to develop a detailed insight into the hydrogen embrittlement mechanisms relevant for cyclic loading conditions