Using tissue culture to model early events in Francisella tularensis pathogenesis

Francisella tularensis (Ft) is a highly infectious bacterium that causes tularemia, which manifests in multiple presentations such as ulceroglandular, pneumonic, or typhoidal forms. Pneumonic tularemia, a more severe manifestation, can result from inhalation of as few as 10 colony forming units of Ft. The low infectious dose, its potential for aerosolization, and severity of disease has resulted in Ft classification as a Select Agent by the CDC for bioweapon potential. The severe clinical and financial burden a bioweapon attack utilizing Ft would impose on the U.S. makes development of therapeutics and vaccines an important contribution to protecting public health. Determination of vaccine targets requires knowledge of the early pathogenesis of Ft in the lungs. Ft infects a wide variety of cells, including lung macrophages and lung epithelial cells, likely involved in initial infection from aerosol; few have compared the permissivity of infection between these cell types to infection by Ft. We utilized an in vitro infection assay with murine macrophages (J774) and human alveolar epithelial cells (A549), and developed an ex vivo infection assay for 3D-cultured human primary bronchial epithelium (HBE), intended to mimic lung architecture. Early cellular events within SCHU S4 infected rabbit tissue, a model which exhibits clinical disease similar to humans, was assessed for cellular infiltration, changes in lung architecture, and apoptosis. I found that different strains of Ft (SCHU S4, LVS, and U112), grow at similar rates in A549 as J774 after initial infection. Moreover, I have demonstrated that Ft can infect HBE in the 3D culture system. These data suggest that it takes Ft longer to infect the HBE cells than the A549 or J774 cells. This is the first infection assay performed within a 3D HBE culture, to our knowledge. Over the course of the first five days post-exposure there is an increasing amount of inflammation, hemorrhaging and apoptosis in rabbit lung. When taken altogether, these data suggest lung epithelial cells have an underappreciated role in Ft early pathogenesis and dissemination

Characterization of the scrapie-infection in CD40L-deficient mice

Titel und Inhaltsverzeichnis Einleitung Zielsetzung der Arbeit Eigene Untersuchungen Ergebnisse Diskussion Zusammenfassung Summary Literaturverzeichnis Anhang Danksagung SelbständigkeitserklärungIn dieser Arbeit wurde die Bedeutung des CD40-CD40L-Systems in transmissiblen Spongiformen Enzephalopathien (TSE) untersucht. In einem Mausmodell der Alzheimer Krankheit bewirkte die Hemmung der CD40-CD40L-Signalübertragung eine verringerte Amyloid-Ablagerung und eine verminderte Neuroinflammation und wurde deshalb als therapeutische Strategie vorgeschlagen. Andererseits sind aber auch neuroprotektive Funktionen eines intakten CD40-CD40L-Systems entdeckt worden: CD40 defiziente Neuronen zeigten sich empfindlicher gegenüber Serum- und NGF-β-Entzug und CD40-defiziente Mäuse entwickelten im Alter eine ausgeprägte Neurodegeneration. Anhand des Vergleichs der Scrapie-Infektion von Wildtyp-Mäusen und CD40L-defizienten Mäusen (CD40L-/--Mäusen) wurde in dieser Arbeit untersucht, ob die Aussschaltung CD40L-Gens in diesem TSE-Modell günstige oder schädliche Effekte hat. CD40L-/--Mäuse starben im Durchschnitt 40 Tage früher als Wildtyp-Mäuse. Sie wiesen eine stärkere Aktivierung von Mikroglia, eine stärkere Vakuolisierung des Neuropils und einen erhöhten Verlust an GABAergen Neuronen auf. Hinsichtlich der Ablagerung von fehlgefaltetem PrPSc und der Astrozytenaktivierung wurden keine Unterschiede festgestellt. Das experimentelle Modell zeigt, dass eine Defizienz für CD40L hochgradig schädlich bei Prionerkrankungen ist und stützt die Annahme neuroprotektiver Funktionen eines intakten CD40-CD40L-Systems. Die Stimulierung neuroprotektiver Signalübertragungswege könnte eine Möglichkeit bieten, den Beginn und Verlauf der TSE Erkrankung im zentralen Nervensystem zu verzögern.The aim of this study was to clarify the role of CD40-CD40L-interactions in transmissible spongiform encephalopathies (TSEs).In a mouse model of Alzheimer s disease (AD) the inhibition of CD40L mediated signaling led to a reduced amyloid deposition and neuroinflammation and therefore was suggested as a therapeutic strategy for the treatment of AD. On the other hand, neuroprotective properties of intact CD40-CD40L-interactions were reported, as CD40-deficient neurons proved to be more vulnerable to stress associated with ageing as well as nerve growth factor-beta and serum withdrawal, respectively.We studied the scrapie infection of CD40L deficient (CD40L-/-) mice to see whether ablation of the CD40L gene would be beneficial or detrimental in this model of a neurodegenerative amyloidosis. CD40L-/- mice died on average 40 days earlier than wild type controls and exhibited a more pronounced vacuolization of the neuropil, an increased microglia activation, and a higher loss of GABAergic neurons. No differences were observed concerning the deposition of misfolded PrPSc-amyloid and the activation of astrocytes. The experimental model shows that a deficiency for CD40L is highly detrimental in prion diseases and reinforces the neuroprotective function of intact CD40-CD40L interactions. The stimulation of neuroprotective pathways may represent a possibility to delay the disease onset in prion infections of the central nervous system therapeutically

Allelotyping of pooled DNA with 250 K SNP microarrays

BACKGROUND: Genotyping technologies for whole genome association studies are now available. To perform such studies to an affordable price, pooled DNA can be used. Recent studies have shown that GeneChip Human Mapping 10 K and 50 K arrays are suitable for the estimation of the allele frequency in pooled DNA. In the present study, we tested the accuracy of the 250 K Nsp array, which is part of the 500 K array set representing 500,568 SNPs. Furthermore, we compared different algorithms to estimate allele frequencies of pooled DNA. RESULTS: We could confirm that the polynomial based probe specific correction (PPC) was the most accurate method for allele frequency estimation. However, a simple k-correction, using the relative allele signal (RAS) of heterozygous individuals, performed only slightly worse and provided results for more SNPs. Using four replicates of the 250 K array and the k-correction using heterozygous RAS values, we obtained results for 104.141 SNPs. The correlation between estimated and real allele frequency was 0.983 and the average error was 0.046, which was comparable to the results obtained with the 10 K array. Furthermore, we could show how the estimation accuracy depended on the SNP type (average error for A/T SNPs: 0.043 and for G/C SNPs: 0.052). CONCLUSION: The combination of DNA pooling and analysis of single nucleotide polymorphisms (SNPs) on high density microarrays is a promising tool for whole genome association studies

Elevated dietary zinc oxide levels do not have a substantial effect on porcine reproductive and respiratory syndrome virus (PPRSV) vaccination and infection

Background Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important infectious agents for the swine industry worldwide. Zinc (Zn) salts, which are widely used as a dietary supplement in swine nutrition, have shown antiviral effects in vitro as well as in vivo. The purpose of this study was to determine the influence of dietary zinc oxide supplementation on vaccination and challenge infection with PRRSV. Findings The clinical course of PRRS and the success of vaccination with an experimental inactivated vaccine were compared between animals receiving a conventional diet (50 ppm Zn, control group) and diets supplemented with Zn oxide (ZnO) at final Zn concentrations of 150 or 2,500 ppm. Pigs receiving higher dietary Zn levels showed a tendency towards higher neutralizing antibody levels after infection, while dietary Zn levels did not substantially influence the number of antiviral IFN-gamma secreting cells (IFN-gamma-SC) or percentages of blood immune cell subsets after infection. Finally, feeding higher dietary Zn levels reduced neither clinical symptoms nor viral loads. Conclusions Our results suggest that higher levels of dietary ZnO do not have the potential to stimulate or modulate systemic immune responses after vaccination and heterologous PRRSV infection to an extent that could improve the clinical and virological outcome

High-dose dietary zinc oxide mitigates infection with transmissible gastroenteritis virus in piglets

Zinc (Zn) supplementation has been shown to reduce the incidence of diarrhea and to protect animals from intestinal diseases, but the mechanisms of this protective effect against virus infection in vivo have not yet been elucidated. Transmissible gastroenteritis virus (TGEV) causes diarrhea in piglets with an age-dependent decrease of severity. RESULTS: We used 60 weaned piglets that were divided into three groups to evaluate the effect of different Zn levels added to a conventional diet (50 mg Zn/kg diet, Znlow, control group). The other groups received the diet supplemented with ZnO at final concentrations of 150 mg Zn/kg diet (Znmed), or 2,500 mg/kg diet (Znhigh). Oral challenge infection with TGEV was performed when the pigs had been fed for 1 week with the respective diet. Half of the piglets of each group were sacrificed at day 1 and 18 after challenge infection. Fecal consistency was improved and body weights increased in the Znhigh group when compared to the other groups, but no direct effect of Zn concentrations in the diet on fecal TGEV shedding and mucosal immune responses was detectable. However, in the Znhigh group, we found a prevention of villus atrophy and decreased caspase-3-mediated apoptosis of jejunal epithelium. Furthermore, pigs receiving high Zn diet showed a down-regulation of interferon (IFN)-α, oligoadenylate synthetase (OAS), Zn transporter SLC39A4 (ZIP4), but up- regulation of metallothionein-1 (MT1), as well as the Zn transporters SLC30A1 (ZnT1) and SLC30A5 (ZnT5). In addition, forskolin-induced chloride secretion and epithelial resistance were controlled at a physiological level in the Znhigh but not the other groups. Finally, in the Znhigh group, we documented an earlier and higher systemic TGEV-specific serum antibody response. CONCLUSIONS: These results suggest that high dietary Zn could provide enhanced protection in the intestinal tract and stimulate the systemic humoral immune response against TGEV infection

Decision Support for Intoxication Prediction Using Graph Convolutional Networks

Every day, poison control centers (PCC) are called for immediate classification and treatment recommendations if an acute intoxication is suspected. Due to the time-sensitive nature of these cases, doctors are required to propose a correct diagnosis and intervention within a minimal time frame. Usually the toxin is known and recommendations can be made accordingly. However, in challenging cases only symptoms are mentioned and doctors have to rely on their clinical experience. Medical experts and our analyses of a regional dataset of intoxication records provide evidence that this is challenging, since occurring symptoms may not always match the textbook description due to regional distinctions, inter-rater variance, and institutional workflow. Computer-aided diagnosis (CADx) can provide decision support, but approaches so far do not consider additional information of the reported cases like age or gender, despite their potential value towards a correct diagnosis. In this work, we propose a new machine learning based CADx method which fuses symptoms and meta information of the patients using graph convolutional networks. We further propose a novel symptom matching method that allows the effective incorporation of prior knowledge into the learning process and evidently stabilizes the poison prediction. We validate our method against 10 medical doctors with different experience diagnosing intoxication cases for 10 different toxins from the PCC in Munich and show our method's superiority in performance for poison prediction.Comment: 10 pages, 3 figure