Regulatory T cells (Tregs) play a non-redundant role in maintenance of immune homeostasis. This is achieved by suppressing both, priming of naïve cells and effector cell functions. Although Tregs have been implicated in modulating allergic immune responses, their influence on distinct phases of development of allergies remains unclear. In this study, by using bacterial artificial chromosome (BAC)-transgenic Foxp3-DTR (DEREG) mice we demonstrate that the absence of Foxp3+ Tregs during the allergen challenge surprisingly does not exacerbate allergic airway inflammation in BALB/c mice. As genetic disposition due to strain specificity may contribute significantly to development of allergies, we performed similar experiment in C57BL/6 mice, which are less susceptible to allergy in the model of sensitization used in this study. We report that the genetic background does not influence the consequence of this depletion regimen. These results signify the temporal regulation exerted by Foxp3+ Tregs in limiting allergic airway inflammation and may influence their application as potential therapeutics.

Documentation of experiments is essential for best research practice and ensures scientific transparency and data integrity. Traditionally, the paper lab notebook (pLN) has been employed for documentation of experimental procedures, but over the course of the last decades, the introduction of electronic tools has changed the research landscape and the way that work is performed. Nowadays, almost all data acquisition, analysis, presentation and archiving are done with electronic tools. The use of electronic tools provides many new possibilities, as well as challenges, particularly with respect to documentation and data quality. One of the biggest hurdles is the management of data on different devices with a

Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.

The transmigration of African trypanosomes across the human blood-brain barrier (BBB) is the critical step during the course of human African trypanosomiasis. The parasites Trypanosoma brucei gambiense and T. b. rhodesiense are transmitted to humans during the bite of tsetse flies. Trypanosomes multiply within the bloodstream and finally invade the central nervous system (CNS), which leads to the death of untreated patients. This project focused on the mechanisms of trypanosomal traversal across the BBB. In order to establish a suitable in vitro BBB model for parasite transmigration, different human cell lines were used, including ECV304, HBMEC and HUVEC, as well as C6 rat astrocytes. Validation of the BBB models with Escherichia coli HB101 and E. coli K1 revealed that a combination of ECV304 cells seeded on Matrigel as a semisynthetic basement membrane and C6 astrocytes resulted in an optimal BBB model system. The BBB model showed selective permeability for the pathogenic E. coli K1 strain, and African trypanosomes were able to traverse the optimized ECV304-C6 BBB efficiently. Furthermore, coincubation indicated that paracellular macrophage transmigration does not facilitate trypanosomal BBB traversal. An inverse assembly of the BBB model demonstrated that trypanosomes were also able to transmigrate the optimized ECV304-C6 BBB backwards, indicating the relevance of the CNS as a possible reservoir of a relapsing parasitaemia. INTRODUCTIONHuman African trypanosomiasis is a vector-borne parasitic disease, which currently causes about 10 000 deaths each year. At the end of the last century the number of lethal cases was estimated as up to 300 000, according to WHO data. The disease threatens over 60 million people of 36 African nations, reaching lethality of 100 % without treatment (WHO, 2010). The parasites, called trypanosomes, are transmitted during a blood meal of tsetse flies of the Glossina genus and can infect humans as well as cattle. Trypanosoma brucei brucei is one of the causative agents of Nagana, a severe cattle disease, which hampers intensive cattle farming in endemic areas (Steverding, 2008). T. b. gambiense and T. b. rhodesiense are human pathogens and multiply within the blood circulation system. Therein, the parasites evade the host immune system by different strategies, for instance by switching their surface-coat antigens (Dubois et al., 2005). As the disease progresses, the parasites infect the central nervous system (CNS), leading to the severe outcome of the disease. Once inside the CNS, parasites are hardly reached by drugs or by the immune system. Depending on the trypanosome subspecies the parasites transmigrate through the human blood-brain barrier (BBB) within a few weeks (T. b. rhodesiense), some months or even years (T. b. gambiense). So far, the invasion into the CNS of African trypanosomes is poorly understood (Grab & Kennedy, 2008). It has been shown that secreted proteases (Nikolskaia et al., 2006(Nikolskaia et al., , 2008, in the case of T. b. rhodesiense, and the composition of ...