Asthma is a chronic respiratory disease characterized by reversible airway obstruction, persistent airway hyperresponsiveness, chronic airway inflammation, and chronic airway remodeling. Most adult asthmatics have neutrophilic airway inflammation that correlates to increasing disease severity, and fail to respond to corticosteroid therapies that mitigate other asthma endotypes. Accordingly, there is a need to investigate the molecular mechanisms responsible for neutrophilic asthma. Pasture heaves, a respiratory disease affecting horses housed on pasture in conditions of high heat and humidity, shares the aforementioned characteristics of human asthma, including neutrophilic inflammation. The cause is undetermined, but genetic propensities for reactivity to seasonally inhaled, pasture-associated, aeroallergens are presumed. Complexities of diseases like asthma and pasture heaves, that include temporal interactions between environmental and genetic factors, lend themselves to exploration using -omics technologies. An emergent paradigm in disease pathogenesis views disease as the result of imbalances in a biological system of thousands of proteins that maintain eukaryotic homeostasis. Consistent with this paradigm, this dissertation describes systematic efforts to identify groups of proteins in the bronchoalveolar lavage fluids of horses with pasture heaves that are altered in a manner that influences neutrophilic airway inflammation, and are similarly changed in human asthma. This is the first use of -omics technologies to investigate pasture heaves. This was accomplished first by improving functional annotation of the equine genome by providing functional annotation for an equine oligoarray, thereby facilitating future functional modeling of equine gene products. Next, through comparative modeling of protein functions in normal bronchoalveolar lavage fluid (BALF) proteomes from horse, human, and mouse, we demonstrated conservation of protein functions in lung fluids across these species. Finally, comparative modeling of pasture heaves-affected and non-diseased BALF proteomes demonstrated that proteins in diseased BALF favor airway neutrophilic inflammation by increasing neutrophil migration, chemotaxis, adhesion, detachment, transmigration, and degranulation, while reducing activation, cell spreading, infiltration, phagocytosis, respiratory burst, apoptosis, and clearance. Collectively, these molecular events contribute to airway neutrophilic inflammation in pasture heaves, and are conserved in human asthma. This method further validates pasture heaves as a robust model for human neutrophilic asthma, and highlights proteins of potential clinical and therapeutic relevance.

Gonorrhea is the second most common sexually transmitted infection worldwide and is caused by Gram-negative, human-specific diplococcus Neisseria gonorrhoeae. It colonizes the mucosal surface of the female reproductive tract and the male urethra. A rapid increase in antibiotic resistance makes gonorrhea a serious threat to public health worldwide. Since N. gonorrhoeae is a human-specific pathogen, animal infection models are not able to recapitulate all the features of infection. Therefore, a realistic in vitro cell culture model is urgently required for studying the gonorrhea infection. In this study, we established and characterized three independent 3D tissue models based on the porcine small intestinal submucosa (SIS) scaffold by co-culturing human dermal fibroblasts with human colorectal carcinoma, endometrial epithelial, and male uroepithelial cells. The histological, immunohistochemical, and ultra-structural analysis showed that the 3D SIS scaffold-based models closely mimic the main characteristics of the site of gonococcal infection in the human host including the formation of epithelial monolayer, underlying connective tissue, mucus production, tight junction (TJ), and microvilli. In addition, functional analysis such as transepithelial electrical resistance (TEER) and barrier permeability indicated high barrier integrity of the cell layer. We infected the established 3D tissue models with different N. gonorrhoeae strains and derivatives presenting various phenotypes regarding adhesion and invasion. The results showed disruption of TJs and growing the interleukins production in response to the infection, which depends on the type of strain and cell. In addition, the 3D tissue models supported bacterial survival, which provided an appropriate in vitro model for long-term infection study. This could be mainly because of the high resilience of the 3D tissue models based on the SIS scaffold to the infection in terms of alteration in permeability, cell destruction, and bacterial transmigration. During gonorrhea infection, a high level of neutrophils migrates to the site of infection. The studies also showed that N. gonorrhoeae can survive or even replicate inside the neutrophils. Therefore, studying the interaction between neutrophils and N. gonorrhoeae is substantially under scrutiny. For this purpose, we generated a 3D tissue model by triple co-culturing of human primary fibroblast cells, human colorectal carcinoma cells, and human umbilical vein endothelial cells. The tissue model was subsequently infected by N. gonorrhoeae. A perfusion-based bioreactor system was employed to recreate blood flow in the side of endothelial cells and consequently study human neutrophils transmigration to the site of infection. We observed neutrophils activation upon the infection. Furthermore, we demonstrated the uptake of N. gonorrhoeae by human neutrophils and reverse transmigration of neutrophils to the basal side carrying N. gonorrhoeae. In summary, the introduced 3D tissue models in this research represent a promising tool to investigate N. gonorrhoeae infections under close-to-natural conditions. / Tripper ist die zweithäufigste sexuell übertragbare Krankheit weltweit und wird durch Gram negative, humanspezifische Diplokokken Neisseria gonorrhoeae verursacht. Das human Pathogen besiedelt die Schleimhautoberfläche des weiblichen Fortpflanzungstraktes und der männlichen Harnröhre. Die rasante Zunahme der Antibiotikaresistenzen macht Gonorrhö zu einer ernsthaften Bedrohung für die öffentliche Gesundheit weltweit. Da N. gonorrhoeae ein humanspezifischer Erreger ist, ist es nicht möglich alle Merkmale einer Infektion in Tiermodellen nachzustellen, daher ist ein realistisches In-vitro-Zellkulturmodell für die Untersuchung der Gonorrhö-Infektion dringend erforderlich. In dieser Studie haben wir drei unabhängige 3D- Gewebemodelle etabliert und charakterisiert, die auf dem Gerüst der Schweine-Submukosa (SIS) basieren, indem wir menschliche dermale Fibroblasten mit menschlichen Darmkrebs-, Endometrialepithel- und männlichen Uroepithelzellen kultivieren. Die histologischen, immunhistochemischen und ultrastrukturellen Analysen zeigten, dass die 3D SIS-Gerüstmodelle die Hauptmerkmale der Stelle der Gonokokken Infektion im menschlichen Wirt genau nachahmen, indem sich Epithelien Monoschichten ausbildeten, unter denen sich Bindegewebe erstrechte. Zudem wiesen die Zellen enge Zell-Zell Kontakte auf und es kam zur Produktion von einer Mukosaschicht sowie Mikrovilli in den Modellen. Darüber hinaus zeigten Funktionsanalysen wie die Messung des transepithelialen elektrischen Widerstands (TEER) und die der Barriere Durchlässigkeit eine hohe Barriere Integrität der Zellschicht. Wir haben die etablierten 3D- Gewebemodelle mit verschiedenen N. gonorrhoeae-Stämmen und Derivaten infiziert, die verschiedene Phänotypen bezüglich der Adhäsion und der Invasion aufwiesen. Die Ergebnisse zeigten eine Unterbrechung der engen Zellverbindungen und eine Zunahme der Interleukin Produktion als Reaktion auf die Infektion, dessen Ausprägung allerdings stark von der Art des Stammes und des verwendeten Zelltyps abhängig ist. Darüber hinaus unterstützten die 3D- Gewebemodelle das bakterielle Überleben, was ein geeignetes In-vitro-Modell für Langzeit- Infektionsstudien liefert. Dies könnte vor allem auf die hohe Widerstandsfähigkeit der SIS- Gerüstmodelle gegen Infektionen in Bezug auf Veränderung der Permeabilität, Zellzerstörung und Bakterienwanderung zurückzuführen sein. Während der Gonorrhoe-Infektion wandert ein hoher Anteil an Neutrophilen an den Ort der Infektion. Die Studie zeigte auch, dass N. gonorrhoeae in den Neutrophilen überleben konnten oder sich sogar in diesen vermehren konnten. Deshalb wurde besonderes die Interaktion zwischen Neutrophilen und N. gonorrhoeae näher betrachtet. Zu diesem Zweck haben wir ein 3D-Gewebemodell mit Hilfe einer dreifache Co-Kultivierung von menschlichen primären Fibroblasten Zellen, menschlichen kolorektalen Karzinomzellen und menschlichen Nabelvenenendothelzellen erstellt. Das Gewebemodell wurde anschließend mit N. gonorrhoeae infiziert. Ein perfusionsbasiertes Bioreaktorsystem wurde eingesetzt, um den Blutfluss auf der Seite der Endothelzellen nachzuahmen und somit konnte die Auswanderung menschlicher Neutrophile zur Infektionsstelle untersucht werden. Darüber hinaus konnte mit diesem Modell die Aufnahme von N. gonorrhoeae in menschliche Neutrophilen und deren Rückwanderung in den Blutfluss beladen mit N. gonorrhoeae nachgewiesen werden. Zusammenfassend lässt sich sagen, dass das in dieser Forschung vorgestellte 3D-Gewebemodell ein vielversprechendes Instrument zur Untersuchung von N. gonorrhoeae-Infektionen unter naturnahen Bedingungen darstellt.

No description available.

Runx1/AML1 transcription factor expression in hematopoietic cell lineages is differentially regulated via usage of two distinct promoters. The 5' UTR and a 19 amino acid encoding sequence transcribed from the distal promoter is inserted via alternative splicing into the 5' end of the mRNA transcript, replacing the 5' UTR and a 5 amino acid encoding sequence usually transcribed from the proximal promoter. Expression of proximal Runx1 in 32Dcl.3 cells delays G-CSF induced neutrophil terminal differentiation by increasing viability compared to distal Runx1. We utilized Runx1 Nterminal deletion and point mutants of three evolutionarily conserved residues to describe dual N-terminal isoform motifs that promote two distinct differentiation phenotypes as regulatory elements in hematopoietic cell differentiation. Runx1 isoforms were evaluated in established hematopoietic in vitro and ex vivo differentiation systems. Deletion of amino acids 3’-14’ (Δ3-14) or 3’-19’ (Δ3-19) of the distal Runx1 N-terminus delayed terminal differentiation of the 32Dcl.3 myeloid cell line, indicating a regulatory motif in distal Runx1 abrogates the delay of terminal differentiation induced by proximal Runx1. Deletion of amino acids 3’-8’ (Δ3-8) or mutation of amino acids serine 3’, serine 5’ and phenylalanine 7’ of the distal Runx1 N-terminus reduce Runx1 expression in the 32Dcl.3 cell line. The N-terminus motif, runt domain and nuclear matrix-targeting sequence of Runx1 modulated Ets1 activity on the KIR3DL1 bidirectional promoter element. The transcription factor YY1 promotes both forward and reverse activation of the KIR3DL1 bidirectional promoter element dominantly in the presence of Runx1, and additively with Ets1. Distinct Runx1 proximal and distal N-termini induced phenotypes were observed in myeloid and thymocyte differentiation, but not with the KIR3DL1 luciferase assay system. This work identifies a previously unknown N-terminal regulatory motif that acts with spatio-temporal and gene target specificity to add another level of control over Runx1 activity during hematopoiesis.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.