The role of the stem cell in Rauscher murine leukaemia

The essential problem of malignancy is associated with the nature of the disturbance which leads to the purposeless multiplication and spread of cells in the body of the afflicted individual. Before overt manifestations of malignancy - cancer or leukaemia - are discernable, a whole series of events takes place some of which can be investigated either clinically or experimentally. The most interesting objects of current research are the identification of a causative agent and the kinetics of the proliferation of the transformed cells at the expense of the normal cells of the body. The knowledge gathered from these investigations will hopefully provide a basis for the institution of a therapy which results in lasting remissions while avoiding the damage of normal cells such as occurs with the present forms of treatment

Identification and characterization of transformed cells in jaagsiekte, a contagious lung tumour of sheep

Jaagsiekte is a contagious lung tumour of sheep in which two types of secretory epithelial cell in the lower respiratory tract are transformed. These cells are type II pneumocytes in the alveoli, and the cells of Clara in the terminal bronchioles.An efficient and reproducible technique is described for the isolation of transformed SPA cells. It includes three basic steps: Prolonged trypsinisation to kill fibroblasts, magnetic removal of macrophages, and adherence to remove the rapidly adherent cells. The resultant preparations of lung cells were enriched to 96.6% type II pneumocytes.Four cell lines were initiated from lungs of sheep with pulmonary adenomatosis and two of the cell lines have been continuously propagated in vitro for more than 140 passages. The morphologic and cytogenetic characteristics of these lines have been examined. The cells are epithelial and demonstrate ultrastructural features consistent with the tumour of origin. The lines are aneuploid and contain a spectrum of cytogenetic marker chromosomesSPARV P25 was detected in all cell lines in early passes, but was undetectable after 11th passage.After 16 or 20 in vitro passages, three cell lines grew in soft agar with low cloning efficiency. However, the efficiency increased with higher passage numbers.The effect of bromhexine HC1 and prednisolone on the morphology and ultrastructure of SPA cell lines ( JS7 and JS8 ) was studied. Cells treated with prednisolone lost their squamous epithelial shape and assumed a fusiform swirling appearance. Coincident with this change in morphology, the JS7 cells also lost the characteristic cytoplasmic lamellar bodies but not apical microvilli nor desmosomes.Cells treated with bromhexine HC1 showed an increase in the number and size of lamellar bodies, and retained their squamous epithelial appearance.When the cell lines were transplanted subcutaneously in athymic nude mice, they induced palpable fluid-filled, cyst-like tumours at the site of injection. Tumour cells had a morphology similar to that of the parent cells and the original adenomatosis lung tumour cells of jaagsiekte, even though they had been through many in vitro passages. Ultrastructural and cytogenetic analysis of cell cultures derived from the nude mice tumours showed these to possess only the sheep karyotype.SPA was successfully reproduced in neonatal lambs by intratracheal inoculation of passaged cells. Macroscopic lesions of SPA were observed in lambs and confirmed by histopathology and electron microscopy. Cell cultures were established from the lungs of these lambs. Replication of a retrovirus in these cultures was demonstrated by the detection in the supernate of SPARV P25. The presence of the retrovirus in the lung fluids from these lambs was also demonstrated.Thus for the first time permanent cell lines that contain the SPARV are available and should considerably facilitate further studies on this virus and the tumour it causes

The development of the sporozoite of Plasmodium gallinaceum (Apicomplexa Haemosporina)

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Studies in the ecology and biocontrol of Burkholderia pseudomallei

Burkholderia pseudomallei is a saprophytic bacterium that causes melioidosis, the most common cause of fatal bacterial community acquired pneumonia in the tropics. The disease is endemic in North Queensland and has been studied extensively, yet the ecology of the bacterium remains uncertain. The organism must persist and multiply in the environment to maintain a clinically significant reservoir, This study examined the questions of whether B. pseudomallei was maintained in plants in an endemic region, whether local and imported rice species could be infected in the laboratory, and if so what effect the bacterium had on the growth of the plants and whether biocontrol agents could decrease bacterial loads of B. pseudomallei in a plant infection model. Two rice models of infection with B. pseudomallei were developed, a domestic rice grown in Australia which was then examined by qPCR and fluorescent antibody microscopy and a native (wild) rice found in the local endemic region, also examined using qPCR. Rice cultivar differences in susceptibility to B. pseudomallei and near-neighbor species were identified with another cultivar of rice identified as resistant to any growth inhibition. Two possible agents, a bacteriocin-like compound derived from a Papua New Guinea strain of Burkholderia ubonensis and a bacteriophage cocktail were developed in two different rice species models of infection (one native and one introduced rice species) and a biofilm model. The first biocontrol candidate, a previously reported bacteriocin-like compound extracted from B. ubonensis, was partially purified and chacterised. The second biocontrol agent was a bacteriophage cocktail developed from previously isolated bacteriophages at James Cook University, which were screened for activity against B. pseudomallei isolates of interest, amplified, measured by transmission electron microscopy, quantified and combined. Individual phage samples were all partially effective against the bacterial isolate of interest. However, on combination they completely cleared growth on bacterial lawns. Both the bacteriocin-like compound and the bacteriophage cocktail were equally able to significantly inhibit biofilm formation in a 96 well plate model over 24 hr. The bacteriocin was less effective by 48 hr, while the bacteriophage cocktail, due to self replication activity, maintained its ability to keep the biofilm from growing. As the bacteriocin lost activity over time, it was not used directly in the rice models and B. ubonensis was used instead to identify whether interaction with B. pseudomallei would result in less inhibition of growth. B. ubonensis did not act as an effective biocontrol agent, probably because it was also somewhat inhibitory at the load applied. The bacteriophage cocktail was effective, with significant increases in growth in treatment groups relative to infected groups. The domestic rice model of infection produced partial recovery of infection load while the wild rice model of infection produced complete recovery and a two log drop in bacterial load. The wild rice species likely has a long exposure to B. pseudomallei and has natural defences which, in combination with phage, are able to control the high B. pseudomallei infection. A spatial / botantical analysis of Castle hill in the dry season (a previously described melioidosis-endemic site in Townsville, Queensland) identified particular plant species most commonly found near B. pseudomallei positive soil, however a wet season analysis failed to find B. pseudomallei in roots, although it was found in low levels around the roots and at increased numbers at 10 and 30 cm below ground level. Low exposure and natural resistance of native species to B. pseudomallei, or simply infection below detection limits are probable explanations and it is likely that at this endemic site, plants may play less of a role in survival of B. pseudomallei than does the physiochemical attributes of soil. This study has developed rice models of B. pseudomallei infection and used these to examine the viability of a biocontrol approach with two different types of biocontrol agents. This approach has shown promise in the laboratory. In addition, this study examined the role played by plants in the ecology of melioidosis in an endemic region and how those plants related to B. pseudomallei presence in the soil

53rd National Meeting of the Italian Society of Biochemistryand Molecular Biology (SIB)andNational Meeting of Chemistry of Biological Systems – Italian Chemical Society (SCI - Section CSB)

The 53rd National Congress of the Italian Society of Biochemistry and Molecular Biology (SIB), which will be held in Riccione from 23 to 26 September, is characterised by the elevated scientific level and interdisciplinary interest of the numerous sessions in which it is organised. The Scientific Programme comprises three joint Symposia of the SIB and the Chemistry of Biological Systems section of the Italian Chemistry Society (SCI) on Molecular Systems Biology, Chemistry of Nucleic Acids, Protein and Drug Structure, and Environmental Biotechnology. These Symposia address groundbreaking arguments, making the joint interest of the two societies particularly fascinating; the joint organisation of these events in fact signals the shared intention to proceed along the path of scientific exchange. The topics of the other sessions have been chosen by the Scientific Committee on the basis of their scientific relevance and topicality, with particular attention paid to the selection of the speakers. The SIB sessions will range from Signal Transduction and Biomolecular Targets, Protein Misfolding and its Relationship with Disease, Emerging Techniques in Biochemistry, Gene Silencing, Redox Signalling and Oxidative Stress, Lipids in Cell Communication and Signal Transduction, Mitochondrial Function and Dysfunction

Mechanisms of Chemically-Induced Hepatocarcinogenesis in Western Mosquitofish (Gambusia Affinis).

A need exists for fish sentinel species for monitoring environmental changes. These studies were performed to examine the suitability of western mosquitofish (Gambusia affinis) as a sentinel species and to explore basic mechanisms of hepatocarcinogenesis. Neoplastic lesions were induced in laboratory-reared mosquitofish fry and adults using single pulse exposures to 1 or 10 ppm methylazoxymethanol acetate. Early changes in the liver of exposed fish included severe cytotoxicity, megalocytosis, and widespread apoptosis. Neoplastic changes were found in the liver in approximately 20% of the exposed fish by 8 weeks after exposure and increased to approximately 70% by 41 weeks. These included foci of cellular alteration, hepatocellular carcinomas and cholangiocellular carcinomas. There was also significant incidence of a spindle cell tumor, believed to be of hepatobiliary stem cell origin. Tumors were found in less than 1% of controls. Immunohistochemistry was performed on histologic sections of the fish from the histopathology study. Specimens were stained for proliferating cell nuclear antigen (PCNA, PC10). Significantly increased cell proliferation detected in liver cell populations of the high dose groups at early time points was attributed primarily to regenerative hyperplasia, and at later time points mainly to preneoplastic and/or neoplastic cell proliferation. Additional sections of the same fish specimens were stained for p53 protein. Overexpression of p53 was detected as early as 48 hours after exposure and in approximately 38% of the tumors. However, at the early sampling points, p53 immunohistochemistry did not appear to have good predictive value of later tumor formation. Levels of the DNA adducts O\sp6-methylguanine and 7-methylguanine were measured in the liver in additional alkylating agent exposures using stable isotope dilution-GC/mass spectrometry. Adapted for small fish species, this assay gave detection limits of approximately 1.6 femtomoles per microgram of DNA, or about 1 adduct per 2 million DNA bases. Levels of O\sp6-methylguanine ranging from approximately 55-185 picograms per microgram DNA, measured in the first 72 hours after exposure, were correlated with a 33% liver tumor incidence after 25 weeks in parallel histopathology studies. The carcinogen sensitivity, ease of laboratory culture, biology, and wide range of Gambusia affinis appear to indicate excellent potential for its use as a warmwater sentinel species

Phenolic Compounds

Phenolics are commonly available compounds in foods, beverages, and spices. They have great importance in all aspects of daily life including industry, health, and research. As such, this book presents a comprehensive overview of phenolic compounds and their potential applications in industry, environment, and public health. Chapters cover such topics as the production of these compounds and their uses in environmental sustainability, climate change, green industry, and treatment of human disease

Charakterisierung der Virulenzfaktoren von Corynebacterium diphtheriae und Corynebacterium ulcerans

Corynebacterium diphtheriae is the causative agent of respiratory diphtheria, a major cause of infant mortality until the 1930s (Hadfield et al., 2000; Burkovski, 2014). Although the incidence of diphtheria has declined due to vaccination with diphtheria toxoid, the disease has not been completely eradicated. Moreover, the occurrence of non-toxigenic strains which are associated with invasive infections have been increasingly reported. Recently, diphtheria-like infections caused by the emerging pathogen Corynebacterium ulcerans have become more numerous in the Western Europe. The zoonotic transmission of C. ulcerans has become a cause of concern. Despite the medical and veterinary importance of these pathogenic microorganisms, only a few virulence factors have been characterized in detail including pili structures and a few other adhesion factors which play a pivotal role in the intracellular communication and host-pathogen interactions. In the frame of my dissertation, important findings were obtained which contributed to the elucidation of virulence properties of pathogenic corynebacteria: In studies from Peixoto et al., an invasive, non-toxigenic C. diphtheriae strain was characterized with respect to its ability to cause colonization and killing of Caenorhabditis elegans, adhesion to and invasion of epithelial cells, survival within human macrophages as well as induction of osteomyelitis in mice (Peixoto et al., 2016). Furthermore, the DIP0733 protein a multifunctional pathogenicity determinant of C. diphtheriae, was studied in respect to its role in the interaction with biotic surfaces, nematodes, human epithelial and macrophage cell lines. Bioinformatic analyses revealed the presence of several short linear motifs and structural elements of DIP0733. Among those motifs, we were interested in the C-terminal coiled-coil domain of DIP0733 as it was only identified in pathogenic corynebacteria. By demonstrating the binding properties to extracellular matrix proteins, adhesion to and invasion of epithelial cells, detrimental effects on epithelial cells and killing of nematodes, we were able to show that the mutants lacking the coiled-coil domain were less virulent, while the fusion of the coiled-coil domain to DIP0733 of a non-pathogenic strain enhanced its pathogenicity. Therefore, this study indicated the crucial role of the coiled-coil domain of DIP0733 in virulence of corynebacteria (Weerasekera et al., 2018). Pathogenicity of corynebacteria seems to be a multifactorial process and sometimes corynebacteria acquire new virulence factors by horizontal gene transfer. The recent identification of the putative ribosome-binding protein of C. ulcerans 809, which showed a striking structural similarity to the A chain of Shiga-like toxins from Escherichia coli (Trost et al., 2011), indicated a higher pathogenic risk of these species. In this study, the first functional characterization of this putative toxin in C. ulcerans strain 809 was analyzed together with a homologous protein identified in C. diphtheriae strain HC04 which has not been reported before. We were able to show that the overexpression of the corresponding rbp genes led to severe detrimental effects in invertebrate model systems as well as in epithelial and macrophage cell lines, while the rbp disrupted mutant showed a reduced cytotoxic effect (Weerasekera et al., 2019b). Further, FACS analyses revealed that both C. ulcerans 809 and C. diphtheriae HC04 caused necrosis in human THP-1 cells (Weerasekera et al., in preparation). Since the interaction of corynebacteria with macrophages is not well understood, we explored the activation of pattern recognition receptors expressed on different immune cells upon C. diphtheriae contact by utilizing murine and human phagocytes. Our study revealed that the absence of Mincle resulted in reduced G-CSF production while the uptake of the bacteria was not influenced. In contrast, both the uptake of the bacteria and the production of cytokines were blocked when Myd88 was absent. Hence, we propose that the phagocytosis of corynebacteria occur only when TLR/Myd88 pathway is functional. In addition, strain specific differences were observed in the internalization of the bacteria, indicating although all Corynebacterium strains in our study were recognized by TLR2, some strains had unknown mechanisms to avoid uptake by the host cells irrespective of the presence of the diphtheria toxin (Weerasekera et al., 2019a).Corynebacterium diphtheriae ist der Erreger der respiratorischen Diphtherie, eine der Hauptursachen für eine hohe Kindersterblichkeit bis in die 1930er Jahre (Hadfield et al., 2000; Burkovski, 2014). Obwohl die Inzidenz von Diphtherie aufgrund der Impfung mit Diphtherie-Toxoid zurückgegangen ist, wurde die Krankheit nicht vollständig ausgerottet. Darüber hinaus wurde zunehmend über das Auftreten nicht-toxigener Stämme berichtet, die mit invasiven Infektionen in Verbindung gebracht werden. Zudem haben Diphtherie-ähnliche Infektionen,die durch den Erreger Corynebacterium ulcerans verursacht werden, in Westeuropa zugenommen. Die zoonotische Übertragung von C. ulcerans gibt Anlass zur Sorge. Trotz der medizinischen und veterinärmedizinischen Bedeutung dieser pathogenen Mikroorganismen wurden nur wenige Virulenzfaktoren im Detail charakterisiert, darunter Pili-Strukturen und einige andere Adhäsionsfaktoren, die eine zentrale Rolle bei der intrazellulären Kommunikation und den Wechselwirkungen zwischen Wirt und Pathogen spielen. Im Rahmen meiner Dissertation wurden wichtige Erkenntnisse erzielt, die zur Aufklärung der Virulenz pathogener Corynebakterien beitrugen: In einer Studie von Peixoto et al. wurde ein invasiver, nicht-toxigener C. diphtheriae-Stamm in Bezug auf seine Fähigkeit charakterisiert, eine Besiedlung und Abtötung von Caenorhabditis elegans, die Adhäsion an und Invasion von Epithelzellen, das Überleben in menschlichen Makrophagen sowie die Induktion von Osteomyelitis bei Mäusen zu bewirken (Peixoto et al., 2016). Darüber hinaus wurde das DIP0733-Protein, ein multifunktionaler Pathogenitätsfaktor von C. diphtheriae, hinsichtlich seiner Rolle bei der Wechselwirkung mit biotischen Oberflächen, Nematoden, humanen Epithel- und Makrophagen-Zelllinien untersucht. Bioinformatische Analysen zeigten das Vorhandensein mehrerer kurzer linearer Motive und Strukturelemente von DIP0733. Von besonderem Interesse war dabei eine C-terminalen coiled-coil-Domäne von DIP0733, da diese nur in pathogenen Corynebakterien identifiziert wurde. Durch den Nachweis der Bindung an extrazelluläre Matrixproteine, der Adhäsion an und Invasion in 4 Epithelzellen, der schädlichen Auswirkungen auf Epithelzellen und der Abtötung von Nematoden konnten wir zeigen, dass die Mutanten ohne coiled-coil-Domäne weniger virulent waren, während die Fusion der coiled-coil-Domäne mit DIP0733 eines nicht pathogenen Stammes, desen Pathogenität erhöhte. Damit wurde in dieser Studie die entscheidende Rolle der coiled-coil-Domäne von DIP0733 bei der Virulenz von Corynebakterien aufgeklärt (Weerasekera et al., 2018). Die Pathogenität von Corynebakterien ist ein multifaktorieller Prozess, wobei Corynebakterien auch neue Virulenzfaktoren durch horizontalen Gentransfer erwerben können. Die kürzlich erfolgte Identifizierung des putativen ribosomenbindenden Proteins (Rbp) von C. ulcerans 809, zeigte eine auffällige strukturelle Ähnlichkeit mit der A-Kette von Shiga-ähnlichen Toxinen aus Escherichia coli (Trost et al., 2011) und stellt damit ein höheres Virulenzpotential dieser Spezies dar. In einer Studie erfolgte die erste funktionelle Charakterisierung dieses putativen Toxins im C. ulcerans-Stamm 809 zusammen mit einem homologen Protein, das im zuvor nicht beschriebenen C. diphtheriae-Stamm HC04 identifiziert wurde. Es konnte gezeigt werden, dass die Überexpression der entsprechenden rbp-Gene in C. ulcerans bzw. C. diphtheriae in Infektionsexperimenten mit verschiedenen Modellsystemen zu starken Schädigungen führte, während die rbp-Mutante eine verringerte zytotoxische Wirkung aufwies (Weerasekera et al., 2019b). Ferner zeigten FACS-Analysen, dass sowohl C. ulcerans 809 als auch C. diphtheriae HC04 Nekrose in menschlichen THP-1-Zellen verursachten (Weerasekera et al., in Vorbereitung). Da die Wechselwirkung von Corynebakterien mit Makrophagen nicht gut verstanden ist,untersuchten wir die Aktivierung von Mustererkennungsrezeptoren, die bei C. diphtheriae-Kontakt auf verschiedenen Immunzellen exprimiert wurden, unter Verwendung von murinen und humanen Phagozyten. Die Studie ergab, dass die Abwesenheit von Mincle zu einer verringerten G-CSF-Produktion führte, während die Aufnahme der Bakterien nicht beeinflusst wurde. Im Gegensatz dazu wurden sowohl die Aufnahme der Bakterien als auch die Produktion von Zytokinen blockiert, wenn Myd88 fehlte. Daraus schließen wir, dass die Phagozytose von Corynebakterien nur auftritt, wenn der TLR/Myd88-Signalweg funktionsfähig 5 ist. Zusätzlich wurden stammspezifische Unterschiede bei der Internalisierung der Bakterien beobachtet, was darauf hinweist, dass obwohl alle Corynebacterium-Stämme in unserer Studie durch TLR2 erkannt wurden, einige Stämme durch nicht charakterisierte Mechanismen in der Lage waren, die Aufnahme durch die Wirtszellen, unabhängig vom Vorhandensein des Diphtherietoxins, zu verhindern (Weerasekera et al., 2019a)

New strategies for oral immunization : targeting soluble antigens or delivery of particles to the intestinal mucosa

Ondanks de huidige kennis en knowhow bestaan er tegen vele ziekten nog steeds geen doeltreffende vaccins. Deze besmettelijke ziekten worden zeer dikwijls veroorzaakt door pathogenen die de mucosale oppervlakten koloniseren en/of binnendringen. Vaccinatie ter hoogte van de mucosa kan leiden tot een beschermende mucosale immuniteit maar de ontwikkeling van mucosale vaccins is moeilijk omdat voldoende antigenen de mucosale epitheellaag moet passeren zonder ziekte of tolerantie te veroorzaken. De F4ac fimbriae van enterotoxigene Escherichia coli (ETEC) behoren tot een beperkte groep van unieke moleculen die in staat zijn om een immuunrespons te induceren na orale immunisatie. Recent identificeerde onze onderzoeksgroep het varkens aminopeptidase N (pAPN) als nieuwe receptor voor F4ac+ ETEC. In dit proefschrift evalueerden we het gebruik van pAPN als doelwit voor de aflevering van orale vaccins. Bovendien werden twee bijkomende benaderingen gebruikt om biggen oraal te vaccineren, namelijk het gebruik van het maltose-bindend eiwit (MBP) als antigeendrager en poreuze pellets geladen met F4ac

Molecular analysis of the Friend virus complex

This work was undertaken to molecularly clone the Nirand strain of the polycythaemia inducing Friend spleen focus forming virus (F-SFFVp) together with its replication competent helper virus. Friend murine leukaemia virus (F-MuLV). To this aim viral extrachromosomal DNA molecules (of both linear and circular nature corresponding to both of these viruses) which could be induced in Friend cell lines were molecularly characterised with respect to their quantitative increase during differentiation and subcellular location. One cell line, F4-6 in which considerable amounts of these extrachromosomal DNAs could be detected was utilised for the large scale production and purification of both SFFVp and F-MuLV extrachromosomal DNA molecules. Restriction enzyme analysis of such molecules in combination with Southern blotting enabled the construction of primary restriction enzyme maps which allowed the selection of a molecular cloning strategy. Both of these viruses were subsequently molecularly cloned and were subsequently shown to be biologically active after transfection into recipient cells. A further aspect of this work was to construct a biologically active SFFVp containing a dominant selectable marker gene. A selectable construct was generated by joining different regions of the genome of the myeloproliferative sarcoma virus (MPSV) and SFFVp. A construct with the U3 region from the long terminal repeat (LTR) of SFFVp and the envelope gene region (gp55) of SFFVp (designated neo2 SFFVp) was found to be fully active as a selectable retroviral vector with identical biological properties to the wild type SFFVp neo2 SFFVp induced erythroid differentiation in vivo, with infected cells no longer requiring erythropoietin for differentiation. Furthermore neo2 SFFVp infected spleen cells could be used to generate immortal Friend leukaemia cells which were selectable with geneticin (neo2). A further construct, neo2 SFFV-M, which had a U3 region originating from NPSV was able to cause erythropoietin independent erythroid differentiation. However as compared to neo2 SFFVp and indeed the wild type SFFVp, neo2 SFFV-M was able to induce the erythroproliferative disease with a different kinetics. All attempts to isolate transformed Friend cells with this construct failed