Serologische und molekularbiologische Untersuchungen zur Prävalenz von Toxoplasma gondii bei Waschbären (Procyon lotor) in Deutschland

Einleitung: Toxoplasma gondii stellt einen der weltweit häufigsten, ubiquitär vorkommenden, alimentär übertragbaren Krankheitserreger dar. Der Verzehr von nicht ausreichend erhitztem Fleisch und Fleischerzeugnissen, welche infektiöse Stadien des Protozoen enthalten, erwies sich als eine der bedeutendsten Infektionsquellen für den Menschen. Waschbären (Procyon lotor) gelten in Deutschland als invasive Art. Aufgrund der steigenden Jagdstrecke in den letzten Jahrzehnten wird neben der Pelzproduktion auch die Verwendung des Fleisches als Wildbret zunehmend attraktiv. Gegenwärtig gibt es nur wenige Daten zur Erregerlast von T. gondii in für den humanen Konsum relevanten Gewebeteilen bei Waschbären, ferner zur Durchseuchungsrate der Waschbären in Deutschland und assoziierten Risikofaktoren. Ziele der Studie: In dieser Arbeit sollten die Seroprävalenz von T. gondii bei Waschbären in Deutschland sowie damit assoziierte Risikofaktoren ermittelt werden. Die für den menschlichen Verzehr relevanten Fleischteile sollten ergänzend dazu direkt molekularbiologisch untersucht werden, um qualitative und quantitative Ergebnisse zum Vorkommen von T.-gondii-Stadien in ebendiesem Gewebe zu erhalten. Schlussendlich sollten diese Daten als Beitrag zur Risikoabschätzung einer Toxoplasma-gondii-Infektion des Menschen durch den Verzehr des Fleisches von wildlebenden Waschbären aus Deutschland zur Verfügung gestellt werden. Material und Methoden: Im Rahmen dieser Studie wurden Proben von 820 wildlebenden Waschbären, die von Dezember 2017 bis April 2021 erlegt wurden, untersucht. Das Erlegungsdatum, die Herkunftspostleitzahl, Alter, Geschlecht und Gewicht der Tiere wurden jeweils erfasst. Von den Tieren wurden Kopf, Zwerchfell, Vorder- und Hintergliedmaße entnommen, um Fleischsaft durch Auftauen zu generieren. Mittels kommerziellen ELISAs (Enzyme‐Linked Immunosorbent Assay) wurden T.-gondii-spezifische Antikörper im Fleischsaft nachgewiesen und Risikofaktoren, basierend auf den zugrundeliegenden tierassoziierten Daten, ermittelt. Auf Grundlage der serologischen Ergebnisse wurde das Fleisch der Gliedmaßen von 50 negativen, 50 niedrigpositiven und 50 hochpositiven Proben mittels magnetic-capture Polymerase Chain Reaction (PCR) quantitativ untersucht. Zur Untersuchung der Prävalenz-Unterschiede für die einzelnen Risikofaktoren, wurden Chi-Quadrat-Tests durchgeführt. Für das Gewicht wurde eine einfache logistische Regression angewandt. Ergebnisse: Bei 48,5 % (398/820; 95 % Konfidenzintervall KI: 45,1-52,0) der untersuchten Waschbären wurden T.-gondii-spezifische Antikörper nachgewiesen. Weitere 48,5 % der Proben waren negativ und 2,9 % (47/820; 95 % KI: 2,0-4,3) fraglich. Statistisch signifikante Unterschiede wurden für die Faktoren Geschlecht (p = 0,028), Saison (p < 0,0003) und Gewicht (odds ratio: 1,783; 95 % KI: 1,513-2,108; p < 0,0001) festgestellt. Rüden waren häufiger seropositiv als Fähen, im Spätwinter/Frühling erlegte Tiere waren häufiger seropositiv als im Herbst erlegte Tiere und je schwerer die Tiere waren, umso höher war die Chance eines positiven Antikörper-Nachweises. Bei 56 der untersuchten 150 Waschbärenfleischproben wurde T.-gondii-DNA (Deoxyribonucleic Acid) nachgewiesen. Davon gehörten 18 Tiere der serologisch niedrigpositiven Gruppe und 38 Tiere der serologisch hochpositiven Gruppe an. In den Proben der seronegativen Tiere wurde keine T.-gondii-DNA detektiert. In der serologisch hochpositiven Gruppe gab es statistisch signifikant mehr positive Proben (p < 0,0001) und es wurden statistisch signifikant höhere Werte der DNA-Äquivalente (p < 0,0001) pro 100 g Waschbärenfleisch nachgewiesen als bei den serologisch Niedrigpositiven. Schlussfolgerung: Waschbären in Deutschland sind stark mit T. gondii befallen. Bei Handling und Verzehr von Waschbärenfleisch besteht daher grundsätzlich ein potenzielles humanes Gesundheitsrisiko. Das Fleisch sollte vor dem Verzehr ausreichend durcherhitzt werden. Jedoch sind weitere Studien im Rahmen von Bioassay oder Zellkulturen nötig, um eine bessere Aussage über die Infektiosität treffen zu können.:Inhalt Seite 1 Einleitung ............................................................................................................. 1 2 Literaturübersicht ................................................................................................. 3 2.1 Lebenszyklus und Parasitenstadien von Toxoplasma gondii ............................. 3 2.2 Toxoplasmose beim Menschen .......................................................................... 7 2.2.1 Klinische Manifestation ................................................................................... 7 2.2.2 Risikofaktoren ................................................................................................ 8 2.2.3 Seroprävalenz .............................................................................................. 10 2.3 Toxoplasmose beim Tier .................................................................................. 12 2.3.1 Klinische Manifestation ................................................................................. 12 2.3.2 Prävalenz in verschiedenen Tierarten .......................................................... 12 2.3.3 Toxoplasmose bei Waschbären .................................................................... 14 2.4 T. gondii in Lebensmitteln ................................................................................ 16 2.4.1 Vorkommen in Lebensmitteln ....................................................................... 16 2.4.2 Prävention und Inaktivierung von T. gondii ................................................... 17 2.5 Diagnostik ....................................................................................................... 20 2.5.1 Serodiagnostik ............................................................................................. 21 2.5.2 Molekulardiagnostik ..................................................................................... 22 3 Publikationen ..................................................................................................... 25 3.1 Publikation 1 ................................................................................................... 25 3.2 Publikation 2 ................................................................................................... 35 4 Diskussion .......................................................................................................... 43 5 Zusammenfassung.............................................................................................. 50 6 Summary ............................................................................................................ 52 7 Literaturverzeichnis ............................................................................................ 54 8 Danksagung ....................................................................................................... 6

Colorectal Cancers Mimic Structural Organization of Normal Colonic Crypts

Colonic crypts are stereotypical structures with distinct stem cell, proliferating, and differentiating compartments. Colorectal cancers derive from colonic crypt epithelia but, in contrast, form morphologically disarrayed glands. In this study, we investigated to which extent colorectal cancers phenocopy colonic crypt architecture and thus preserve structural organization of the normal intestinal epithelium. A subset of colon cancers showed crypt-like compartments with high WNT activity and nuclear beta-Catenin at the leading tumor edge, adjacent proliferation, and enhanced Cytokeratin 20 expression in most differentiated tumor epithelia of the tumor center. This architecture strongly depended on growth conditions, and was fully reproducible in mouse xenografts of cultured and primary colon cancer cells. Full crypt-like organization was associated with low tumor grade and was an independent prognostic marker of better survival in a collection of 221 colorectal cancers. Our findings suggest that full activation of preserved intestinal morphogenetic programs in colon cancer requires in vivo growth environments. Furthermore, crypt-like architecture was linked with less aggressive tumor biology, and may be useful to improve current colon cancer grading schemes

The Human Blood Transcriptome in a Large Population Cohort and Its Relation to Aging and Health

Background: The blood transcriptome is expected to provide a detailed picture of an organism’s physiological state with potential outcomes for applications in medical diagnostics and molecular and epidemiological research.We here present the analysis of blood specimens of 3,388 adult individuals, together with phenotype characteristics such as disease history, medication status, lifestyle factors, and body mass index (BMI). The size and heterogeneity of this data challenges analytics in terms of dimension reduction, knowledge mining, feature extraction, and data integration. Methods: Self-organizing maps (SOM)-machine learning was applied to study transcriptional states on a population-wide scale. This method permits a detailed description and visualization of the molecular heterogeneity of transcriptomes and of their association with different phenotypic features. Results: The diversity of transcriptomes is described by personalized SOM-portraits, which specify the samples in terms of modules of co-expressed genes of different functional context. We identified two major blood transcriptome types where type 1 was found more in men, the elderly, and overweight people and it upregulated genes associated with inflammation and increased heme metabolism, while type 2 was predominantly found in women, younger, and normal weight participants and it was associated with activated immune responses, transcriptional, ribosomal, mitochondrial, and telomere-maintenance cell-functions. We find a striking overlap of signatures shared by multiple diseases, aging, and obesity driven by an underlying common pattern, which was associated with the immune response and the increase of inflammatory processes. Conclusions: Machine learning applications for large and heterogeneous omics data provide a holistic view on the diversity of the human blood transcriptome. It provides a tool for comparative analyses of transcriptional signatures and of associated phenotypes in population studies and medical applications

A Framework for the Evaluation of the Feasibility of Public – Private Partnership in Local Government in Serbia

The adopton of the New Law for Local Government Financing is currently underway in a Serbian Parliament procedure. The goal of the new law is the creaton of clear government regulatons which will defne the system of fnancing for local government enttes. This will furthermore create an environment of stability and predictability with regard to revenue planning when preparing local government enttes’ budgets, as well as achieving a vertcal balance when distributng revenues amongst various state levels. Additonally, these goals are refected in the establishment of a system to increase the share of public investment in the total expenses of countes and cites, as well as in the vertcal balance with regard to the distributon of revenue and jurisdicton at various state levels. In that sense, it is preferable to understand fnancial models such as public-private partnerships, which have stll not, to an adequate degree, been adopted in Serbia, but one which could potentally contribute to the introducton of additonal sources of local government fnancing. In order to beter perceive the current capacites of this model of fnancing local government in Serbia, a study was conducted during the spring and summer of 2016, taking into account a sample of 150 examinees. The results of the study indicate very low human resource and technical capacites in local government with regard to realizing and comprehending the concept of public-private partnership

SOX2 expression correlates with lymph-node metastases and distant spread in right-sided colon cancer

<p>Abstract</p> <p>Background</p> <p>The transcription factor SOX2, which is involved in the induction of pluripotent stem cells and contributes to colorectal carcinogenesis, is associated with a poor prognosis in colon cancer (CC). Furthermore, SOX2 is a repressor of the transcriptional activity of β-catenin in vitro. Since the majority of CC develop via an activation of the Wnt/β-catenin signalling pathway, indicated by nuclear expression of β-catenin, we wanted to investigate the expression patterns of SOX2 and β-catenin and correlate them with the occurrence of lymph node and distant metastases as indicators of malignant progression.</p> <p>Methods</p> <p>The expression of SOX2 and β-catenin was investigated in a case control study utilizing a matched pair collection (N = 114) of right-sided CCs with either corresponding distant metastases (N = 57) or without distant spread (N = 57) by applying immunohistochemistry.</p> <p>Results</p> <p>Elevated protein expression of SOX2 significantly correlated with the presence of lymph node- (<it>p </it>= 0.006) and distant metastases (<it>p </it>= 0.022). Nuclear β-catenin expression correlated significantly only with distant metastases (<it>p </it>= 0.001). Less than 10% of cases showed a coexpression of high levels of β-catenin and SOX2. The positivity for both markers was also associated with a very high risk for lymph-node metastases (<it>p </it>= 0.007) and distant spread (<it>p </it>= 0.028).</p> <p>Conclusion</p> <p>We demonstrated that increased expression of either SOX2 or nuclear β-catenin are associated with distant metastases in right-sided CC. Additionally, SOX2 is also associated with lymph-node metastases. These data underline the importance of stemness-associated markers for the identification of CC with high risk for distant spread.</p

Subcellular distribution of human RDM1 protein isoforms and their nucleolar accumulation in response to heat shock and proteotoxic stress

The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by alternative pre-mRNA splicing and differential usage of two translational start sites, resulting in proteins with long or short N-terminus and a great diversity in the exonic composition of their C-terminus. By using tagged proteins and fluorescent microscopy, we examined the subcellular distribution of full-length RDM1 (renamed RDM1α), and other RDM1 isoforms. We show that RDM1α undergoes subcellular redistribution and nucleolar accumulation in response to proteotoxic stress and mild heat shock. In unstressed cells, the long N-terminal isoforms displayed distinct subcellular distribution patterns, ranging from a predominantly cytoplasmic to almost exclusive nuclear localization, suggesting functional differences among the RDM1 proteins. However, all isoforms underwent stress-induced nucleolar accumulation. We identified nuclear and nucleolar localization determinants as well as domains conferring cytoplasmic retention to the RDM1 proteins. Finally, RDM1 null chicken DT40 cells displayed an increased sensitivity to heat shock, compared to wild-type (wt) cells, suggesting a function for RDM1 in the heat-shock response