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

Dietary Enterococcus faecium NCIMB 10415 and Zinc Oxide Stimulate Immune Reactions to Trivalent Influenza Vaccination in Pigs but Do Not Affect Virological Response upon Challenge Infection

Swine influenza viruses (SIV) regularly cause significant disease in pigs worldwide. Since there is no causative treatment of SIV, we tested if probiotic Enterococcus (E.) faecium NCIMB 10415 or zinc (Zn) oxide as feed supplements provide beneficial effects upon SIV infection in piglets. Seventy- two weaned piglets were fed three different diets containing either E. faecium or different levels of Zn (2500 ppm, Zn(high); 50 ppm, Zn(low)). Half of the piglets were vaccinated intramuscularly (VAC) twice with an inactivated trivalent SIV vaccine, while all piglets were then infected intranasally with H3N2 SIV. Significantly higher weekly weight gains were observed in the E. faecium group before virus infection, and piglets in Zn(high) and E. faecium groups gained weight after infection while those in the control group (Zn(low)) lost weight. Using ELISA, we found significantly higher H3N2-specific antibody levels in the E. faecium+VAC group 2 days before and at the day of challenge infection as well as at 4 and 6 days after challenge infection. Higher hemagglutination inhibition (HI) titers were also observed in the Zn(high)+VAC and E. faecium+VAC groups at 0, 1 and 4 days after infection. However, there were no significant differences in virus shedding and lung lesions between the dietary groups. Using flow cytometry analysis significantly higher activated T helper cells and cytotoxic T lymphocyte percentages in the PBMCs were detected in the Zn(high) and E. faecium groups at single time points after infection compared to the Zn(low) control group, but no prolonged effect was found. In the BAL cells no influence of dietary supplementation on immune cell percentages could be detected. Our results suggest that feeding high doses of zinc oxide and particularly E. faecium could beneficially influence humoral immune responses after vaccination and recovery from SIV infection, but not affect virus shedding and lung pathology

A Novel Approach to Space Systems Engineering Education through the Construction of High Altitude Balloons

UC CubeCats is a student organization from the University of Cincinnati dedicated to the education of its members through the development of CubeSats. For every university CubeSat program, there are certain challenges that exist. One of the largest challenges for university CubeSat programs is new member recruitment and retention. New members are often intimidated by the knowledge and experience of more senior members because they have no experience in space systems engineering. In order to mitigate these issues, UC CubeCats has developed a high altitude balloon (HAB) educational program known as the CubeCats Applied Training in Space Exploration (CATiSE) program. By building a HAB, members of the CATiSE program can complete a project using a well-documented space mission engineering process that is similar to the process used for CubeSats. However, unlike CubeSat missions, HAB missions tend to have a shorter lifecycle and lower cost, allowing new members to experiment and learn in a low-risk environment. This CATiSE program includes multiple design reviews, an integration and verification plan, design drawings, and designs of the mission and system architecture. The program was designed to last a full school year, starting with concept exploration in the fall and launch in early April. This is UC CubeCats second HAB launch and the first time the CATiSE program has been implemented. The project to be launched this April, code named project TOYGER, will travel 30km into the stratosphere while the payload measures both radiation energy and light wavelengths. The payload will also take 360-degree images throughout the flight of the balloon. This data will be stored on an external storage device and recovered along with the payload. In order to track the payload, GPS data will be transmitted to the automatic packet reporting system (APRS) as well as a ground station constructed by the members of the CATiSE program. At the end of this 8-month program, new members of UC CubeCats will have a well-founded understanding of the space mission and systems engineering process and will have increased their engineering ability to develop and launch a system that must operate in the harsh environment of space

Exploring the genetics of irritable bowel syndrome: A GWA study in the general population and replication in multinational case-control cohorts

OBJECTIVE: IBS shows genetic predisposition, but adequately powered gene-hunting efforts have been scarce so far. We sought to identify true IBS genetic risk factors by means of genome-wide association (GWA) and independent replication studies. DESIGN: We conducted a GWA study (GWAS) of IBS in a general population sample of 11\u2005326 Swedish twins. IBS cases (N=534) and asymptomatic controls (N=4932) were identified based on questionnaire data. Suggestive association signals were followed-up in 3511 individuals from six case-control cohorts. We sought genotype-gene expression correlations through single nucleotide polymorphism (SNP)-expression quantitative trait loci interactions testing, and performed in silico prediction of gene function. We compared candidate gene expression by real-time qPCR in rectal mucosal biopsies of patients with IBS and controls. RESULTS: One locus at 7p22.1, which includes the genes KDELR2 (KDEL endoplasmic reticulum protein retention receptor 2) and GRID2IP (glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein), showed consistent IBS risk effects in the index GWAS and all replication cohorts and reached p=9.31 710(-6) in a meta-analysis of all datasets. Several SNPs in this region are associated with cis effects on KDELR2 expression, and a trend for increased mucosal KDLER2 mRNA expression was observed in IBS cases compared with controls. CONCLUSIONS: Our results demonstrate that general population-based studies combined with analyses of patient cohorts provide good opportunities for gene discovery in IBS. The 7p22.1 and other risk signals detected in this study constitute a good starting platform for hypothesis testing in future functional investigations. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions

Evidence that breast cancer risk at the 2q35 locus is mediated through IGFBP5 regulation.

GWAS have identified a breast cancer susceptibility locus on 2q35. Here we report the fine mapping of this locus using data from 101,943 subjects from 50 case-control studies. We genotype 276 SNPs using the 'iCOGS' genotyping array and impute genotypes for a further 1,284 using 1000 Genomes Project data. All but two, strongly correlated SNPs (rs4442975 G/T and rs6721996 G/A) are excluded as candidate causal variants at odds against >100:1. The best functional candidate, rs4442975, is associated with oestrogen receptor positive (ER+) disease with an odds ratio (OR) in Europeans of 0.85 (95% confidence interval=0.84-0.87; P=1.7 × 10(-43)) per t-allele. This SNP flanks a transcriptional enhancer that physically interacts with the promoter of IGFBP5 (encoding insulin-like growth factor-binding protein 5) and displays allele-specific gene expression, FOXA1 binding and chromatin looping. Evidence suggests that the g-allele confers increased breast cancer susceptibility through relative downregulation of IGFBP5, a gene with known roles in breast cell biology

A prognostic CpG score derived from epigenome-wide profiling of tumor tissue was independently associated with colorectal cancer survival

Background: Results of previous studies on the association of the CpG island methylator phenotype (CIMP) with colorectal cancer (CRC) prognosis were inconsistent and mostly based on different CIMP definitions. The current study aimed to comprehensively investigate the associations between DNA methylation on genes previously used to define CIMP status with CRC survival. Results: Patients with CRC followed up for a median of 5.2 years were divided into a study cohort (n = 568) and a validation cohort (n = 308). DNA methylation was measured in tumor tissue using the Illumina Infinium HumanMethylation450 BeadChip and restricted to 43 genes used to define CIMP status in previous studies. Cox proportional hazard regression models were used to estimate adjusted hazard ratios (HR) and 95% confidence intervals (CI) of survival after CRC, including adjustment for tumor stage, microsatellite instability, and BRAF mutation status. In the study cohort, ten CpG sites were identified to be associated with CRC survival. Seven of these ten CpG sites were also associated with CRC survival in the validation cohort and were used to construct a prognostic score. CRC patients with a prognostic score of the lowest methylation level showed poorer disease-specific survival compared with patients with the highest methylation level in both the study cohort and the validation cohort (HR = 3.11 and 95% CI = 1.97–4.91, and HR = 3.06 and 95% CI = 1.71–5.45, respectively). Conclusions: A CpG panel consisting of seven CpG sites was found to be strongly associated with CRC survival, independent from important clinical factors and mutations associated with CIMP. Further studies are required to confirm these findings

BRCA2 polymorphic stop codon K3326X and the risk of breast, prostate, and ovarian cancers

Background: The K3326X variant in BRCA2 (BRCA2*c.9976A>T; p.Lys3326*; rs11571833) has been found to be associated with small increased risks of breast cancer. However, it is not clear to what extent linkage disequilibrium with fully pathogenic mutations might account for this association. There is scant information about the effect of K3326X in other hormone-related cancers. Methods: Using weighted logistic regression, we analyzed data from the large iCOGS study including 76 637 cancer case patients and 83 796 control patients to estimate odds ratios (ORw) and 95% confidence intervals (CIs) for K3326X variant carriers in relation to breast, ovarian, and prostate cancer risks, with weights defined as probability of not having a pathogenic BRCA2 variant. Using Cox proportional hazards modeling, we also examined the associations of K3326X with breast and ovarian cancer risks among 7183 BRCA1 variant carriers. All statistical tests were two-sided. Results: The K3326X variant was associated with breast (ORw = 1.28, 95% CI = 1.17 to 1.40, P = 5.9x10- 6) and invasive ovarian cancer (ORw = 1.26, 95% CI = 1.10 to 1.43, P = 3.8x10-3). These associations were stronger for serous ovarian cancer and for estrogen receptor–negative breast cancer (ORw = 1.46, 95% CI = 1.2 to 1.70, P = 3.4x10-5 and ORw = 1.50, 95% CI = 1.28 to 1.76, P = 4.1x10-5, respectively). For BRCA1 mutation carriers, there was a statistically significant inverse association of the K3326X variant with risk of ovarian cancer (HR = 0.43, 95% CI = 0.22 to 0.84, P = .013) but no association with breast cancer. No association with prostate cancer was observed. Conclusions: Our study provides evidence that the K3326X variant is associated with risk of developing breast and ovarian cancers independent of other pathogenic variants in BRCA2. Further studies are needed to determine the biological mechanism of action responsible for these associations