Patterns of Early Gut Colonization Shape Future Immune Responses of the Host

The most important trigger for immune system development is the exposure to microbial components immediately after birth. Moreover, targeted manipulation of the microbiota can be used to change host susceptibility to immune-mediated diseases. Our aim was to analyze how differences in early gut colonization patterns change the composition of the resident microbiota and future immune system reactivity. Germ-free (GF) mice were either inoculated by single oral gavage of caecal content or let colonized by co-housing with specific pathogen-free (SPF) mice at different time points in the postnatal period. The microbiota composition was analyzed by denaturing gradient gel electrophoresis for 16S rRNA gene followed by principal component analysis. Furthermore, immune functions and cytokine concentrations were analyzed using flow cytometry, ELISA or multiplex bead assay. We found that a single oral inoculation of GF mice at three weeks of age permanently changed the gut microbiota composition, which was not possible to achieve at one week of age. Interestingly, the ex-GF mice inoculated at three weeks of age were also the only mice with an increased pro-inflammatory immune response. In contrast, the composition of the gut microbiota of ex-GF mice that were co-housed with SPF mice at different time points was similar to the gut microbiota in the barrier maintained SPF mice. The existence of a short GF postnatal period permanently changed levels of systemic regulatory T cells, NK and NKT cells, and cytokine production. In conclusion, a time window exists that enables the artificial colonization of GF mice by a single oral dose of caecal content, which may modify the future immune phenotype of the host. Moreover, delayed microbial colonization of the gut causes permanent changes in the immune system

A predictive divergence compensation approach for the fabrication of three-dimensional microstructures using focused ion beam machining

In this paper, the major factors which cause fabrication divergence in the focused ion beam (FIB) milling process are discussed. A divergence compensation approach is outlined which calculates the corrected dwell time in order to allow for the divergence caused by the FIB milling process; this is due to overlap effects and the angular-dependent sputter yield. A multi-pass scanning method is used to reduce the fabrication divergence precipitated by atom redeposition. Microstructures, such as parabolic, hemispherical and sinusoidal shapes and a nano hemispherical structure have been produced during the FIB milling experiments using conventional bitmap milling and proposed divergence compensation approaches. A flat top/bottom surface is obtained in convex/concave structures when using the conventional bitmap FIB milling approach. Further research shows that the reasons for this phenomenon are mainly related to both the aspect ratio of the structures and the existence of an oxide layer on the substrate surface. The flat top/bottom phenomenon can be removed by using the combination of a novel divergence compensation approach and the removal of the oxide layer from the substrate surface prior to FIB machining. The experimental results show that the surface form accuracy has been greatly improved by using this method and the overlap effect can be suppressed by carefully choosing the normalized pixel spacing