genomewide characterization of host-pathogen interactions by ...
genomewide characterization of host-pathogen interactions by ... genomewide characterization of host-pathogen interactions by ...
Maren Depke Introduction mixture of different cell types which might even feature contrary reactions. Therefore, in vitro models were additionally analyzed in which only one defined host cell type was studied. Macrophages are an example for an immune cell type involved in the first steps of the encounter between the host and a pathogen. A model to study reactions of macrophages is the preparation of bone marrow stem cells and the in vitro differentiation of the stem cells into so-called bonemarrow derived macrophages (BMM). The advantage of this approach is that these macrophages have never been under any immunological influence which might result from the immune status of the animal even under standardized laboratory conditions. Thus, the third part of this thesis focuses on the reaction of BMM. BMM of different mouse strains were treated with IFN-γ, a modulator of macrophage function which is one of the first signals during initiation of the immune response in vivo. But not only immune cells or specially phagocytes get in touch with pathogens, but also cells responsible for functional and structural integrity of host organs and tissue, like epithelial and endothelial cells. Such cells are actually part of the first line of recognition and reaction to a pathogenic invasion into the host. The bronchial epithelial cell line S9 was used as an in vitro model system for the infection with staphylococci. The fourth chapter in this thesis includes host gene expression signatures of S9 cell after in vitro infection with S. aureus RN1HG. Finally, the following chapter addresses the pathogen expression profile which was first recorded from agitated, aerobic S. aureus RN1HG cultures in different growth phases as a starting and reference point. Afterwards, the already described S9 cell in vitro infection model was used to extract staphylococci after an internalization phase inside the host cell. Internalized bacteria were analyzed at two time points in comparison to different control samples by tiling array gene expression analysis. 38
Maren Depke M A T E R I A L A N D M E T H O D S LIVER GENE EXPRESSION PATTERN IN A MOUSE PSYCHOLOGICAL STRESS MODEL Animal experiments [performed by Cornelia Kiank] Female BALB/c mice aged 6 to 8 weeks were randomly grouped into the experimental and control groups starting at least 4 weeks before being used in experiments. The group size in different experiments differed from 6 to 12 mice per cage. Animals stayed in their group until the end of the experiments and were not mixed up to avoid social stress. All animals were maintained with sterilized food (ssniff R−Z; ssniff Spezialdiäten GmbH, Soest, Germany) and tap water ad libitum for adaptation under minimal stress conditions. Influences of irregularities of the estrous cycles of unisexually grouped female mice were not analyzed selectively and may cause higher SD values in the statistical analyses. Animal rooms had a 12 h light, 12 h dark cycle and were maintained at a constant environment before the experiment. To avoid any additional effect, e. g. acoustic or olfactory effects, the handling of mice during the adaptation period and during the experiments was restricted to one investigator. All animal procedures were performed as approved by the Ethics Committee for Animal Care of Mecklenburg-Vorpommern, Germany. Repeated stress model [performed by Cornelia Kiank] Mice were exposed to combined acoustic and restraint stress on 4 successive days, for 2 h twice a day during the physiological recovery phase of rodents (0800–1000 and 1600–1800 h). On day 5, only one stress session was performed in the morning. For immobilization mice were placed in 50 ml conical centrifuge tubes with multiple ventilation holes without penning the tail. Acoustic stress was induced by a randomized ultrasound emission device between 19 kHz and 25 kHz with 0 dB to 35 dB waves in attacks (patent no. 109977; SiXiS, Taipei, Taiwan), allowing the mice no adaptation to the stressor. Between the stress sessions, mice stayed in their home cages and had free access to food and tap water. Control mice were kept isolated from stressed animals during the 4.5 days stress exposure to avoid any acoustic or olfactory communication between the groups. Therefore, the nonstressed group stayed in the incubator where the animals were adapted. The stressed mice remained outside the incubator in the same animal laboratory during the whole period of the stress model. All successive experiments and analyses were performed starting at 1000 h after the ninth stress exposure. Different in vivo analyses were performed with 6 to 12 mice per group in at least two experiments according to the experimental protocol to ensure reproducibility. For array analysis two independent stress experiments were performed with nine mice per group (first experiment) and eight mice per group (second experiment). 39
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Maren Depke<br />
Introduction<br />
mixture <strong>of</strong> different cell types which might even feature contrary reactions. Therefore, in vitro<br />
models were additionally analyzed in which only one defined <strong>host</strong> cell type was studied.<br />
Macrophages are an example for an immune cell type involved in the first steps <strong>of</strong> the encounter<br />
between the <strong>host</strong> and a <strong>pathogen</strong>. A model to study reactions <strong>of</strong> macrophages is the preparation<br />
<strong>of</strong> bone marrow stem cells and the in vitro differentiation <strong>of</strong> the stem cells into so-called bonemarrow<br />
derived macrophages (BMM). The advantage <strong>of</strong> this approach is that these macrophages<br />
have never been under any immunological influence which might result from the immune status<br />
<strong>of</strong> the animal even under standardized laboratory conditions. Thus, the third part <strong>of</strong> this thesis<br />
focuses on the reaction <strong>of</strong> BMM. BMM <strong>of</strong> different mouse strains were treated with IFN-γ, a<br />
modulator <strong>of</strong> macrophage function which is one <strong>of</strong> the first signals during initiation <strong>of</strong> the<br />
immune response in vivo. But not only immune cells or specially phagocytes get in touch with<br />
<strong>pathogen</strong>s, but also cells responsible for functional and structural integrity <strong>of</strong> <strong>host</strong> organs and<br />
tissue, like epithelial and endothelial cells. Such cells are actually part <strong>of</strong> the first line <strong>of</strong><br />
recognition and reaction to a <strong>pathogen</strong>ic invasion into the <strong>host</strong>. The bronchial epithelial cell line<br />
S9 was used as an in vitro model system for the infection with staphylococci. The fourth chapter<br />
in this thesis includes <strong>host</strong> gene expression signatures <strong>of</strong> S9 cell after in vitro infection with<br />
S. aureus RN1HG. Finally, the following chapter addresses the <strong>pathogen</strong> expression pr<strong>of</strong>ile which<br />
was first recorded from agitated, aerobic S. aureus RN1HG cultures in different growth phases as<br />
a starting and reference point. Afterwards, the already described S9 cell in vitro infection model<br />
was used to extract staphylococci after an internalization phase inside the <strong>host</strong> cell. Internalized<br />
bacteria were analyzed at two time points in comparison to different control samples <strong>by</strong> tiling<br />
array gene expression analysis.<br />
38
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