[Physical activity and cancer survival]. / Activité physique et survie après cancer.

Physical activity has been shown in large cohort studies to positively impact survival in cancer survivors. Existing randomized controlled trials showed a beneficial effect of physical activity on physical fitness, quality of life, anxiety and self-esteem; however, the small sample size, the short follow-up and the lack of standardization of physical activity intervention across studies impaired definite conclusion in terms of survival. Physical activity reduces adiposity and circulating estrogen levels and increases insulin sensitivity among other effects. A workshop was conducted at the International Agency for Research on Cancer in April 2011 to discuss the role of physical activity on cancer survival and the methodology to develop multicentre randomized intervention trials, including the type of physical activity to implement and its association with nutritional recommendations. The authors discuss the beneficial effect of physical activity on cancer survival with a main focus on breast cancer and report the conclusions from this workshop.

Development of Eimeria ninakohlyakimovae in vitro in primary and permanent cell lines.

Infections with Eimeria ninakohlyakimovae represent important coccidian diseases of goats severely affecting animal health and profitability of goat industry. For the development of suitable vaccination strategies basic research is needed for which one important prerequisite is the establishment of in vitro cultures guaranteeing the availability of parasitic material. Therefore, primary cell cultures [caprine, bovine and human umbilical vein endothelial cells (CUVEC, BUVEC, HUVEC)] as well as permanent cell lines [bovine foetal gastrointestinal cells (BFGC), bovine colonic epithelial cells (BCEC), African green monkey kidney cells (VERO)] were exposed to vital sporozoites of E. ninakohlyakimovae. The parasites invaded all different cell types used, irrespective of their origin, but further development into macromeronts and subsequent release of viable merozoites I were restricted to ruminant cells. Mature macromeronts developed in both, endothelial (CUVEC, BUVEC) and epithelial cells (BCEC). VERO cells were non-permissive for parasite development, nevertheless sporozoites survived for 21 days p.i. within an enlarged parasitophorous vacuole. Best in vitro development of E. ninakohlyakimovae macromeronts with respect to the production of viable merozoites I was observed in BCEC, followed by BUVEC. However, the largest macromeronts developed in CUVEC. Mature macromeronts were also detected in BFGC, but these cells were less effective concerning infection rates and productivity. The complete life-cycle of E. ninakohlyakimovae leading to oocyst production was not accomplished in any cell type used. In conclusion, we established suitable in vitro systems for the culture of E. ninakohlyakimovae macromeronts, e.g., for the mass production of merozoites I, for basic studies on parasite/host endothelial cell interactions or for pharmaceutical screenings.

Neutrophil extracellular trap formation as innate immune reactions against the apicomplexan parasite Eimeria bovis.

Eimeria bovis infections are under immunological control and recent studies have emphasized the role of early PMN-mediated innate immune responses in infected calves. Neutrophil extracellular traps (NETs) have recently been demonstrated to act as a killing mechanism of PMN against several pathogens. In the present study, the interactions of bovine PMN with sporozoites of E. bovis were investigated in this respect in vitro. For demonstration and quantification of NET formation, extracellular DNA was stained by Sytox Orange. Fluorescence images after Sytox Orange staining as well as scanning electron microscopy (SEM) showed NET formation to occur upon contact with E. bovis sporozoites. Exposure of PMN to viable sporozoites induced stronger NET formation than to dead or homogenized parasites. NET formation was abolished by treatment with DNase and could be reduced by diphenylene iodonium, which is described as a potent inhibitor of NADPH oxidase. After sporozoite and PMN co-culture, extracellular fibres were found attached to sporozoites and seemed to trap them, strongly suggesting that NETs immobilize E. bovis sporozoites and thereby prevent them from infecting host cells. Thus, transfer of sporozoites, previously being confronted with PMN, to adequate host cells resulted in clearly reduced infection rates when compared to PMN-free controls. NET formation by PMN may therefore represent an effector mechanism in early innate immune reactions against E. bovis. This is the first report indicating Eimeria-induced NET formation.

Monocyte- and macrophage-mediated immune reactions against Eimeria bovis.

Innate immune reactions conducted by macrophages may affect the outcome of primary infections and are crucial for the transition to adaptive immune responses. In bovine coccidiosis little is known on early monocyte/macrophage-mediated responses. We therefore investigated in vivo, in vitro and ex vivo reactions of monocytes and macrophages against Eimeria bovis, one of the most pathogenic Eimeria species in cattle. Macrophages significantly infiltrate the gut mucosa of E. bovis-infected calves, particularly after challenge infection. Furthermore, peripheral monocytes of infected animals, as precursor cells of macrophages, exhibited enhanced ex vivo phagocytic and oxidative burst activities. Enhanced levels of both activities were found early after infection and towards the end of first merogony. In vitro exposure of macrophages to sporozoites led to phagocytosis of the pathogen, whilst monocytes failed to do so. Phagocytosis occurred independently of the viability of the sporozoites, indicating that active invasion by the parasites was negligible. Phagocytosis occurred in the absence of immune serum, but could clearly be enhanced by addition of immune serum, suggesting macrophage-derived antibody-dependent cytotoxicity. Furthermore, co-culture of macrophages with sporozoites and stimulation with merozoite I antigen induced distinct levels of cytokine and chemokine gene transcription. Thus, the transcription of genes encoding for IFN-gamma, IL-12, TNF-alpha, IL-6, CXCL1, CXCL8, CXCL10 and COX-2 was upregulated after sporozoite encounter. In contrast, soluble merozoite I antigen only induced the gene transcription of IL-6 and IL-12 and failed to upregulate IFN-gamma and TNF-alpha gene transcripts. In monocytes, IFN-gamma and CXCL10 were found upregulated, all other immunoregulatory molecules tested were not affected. In summary, our results strongly suggest that macrophage-mediated, innate immune reactions play an important role in the early immune response to E. bovis infections in calves.

Cytoskeletal changes in Eimeria bovis-infected host endothelial cells during first merogony.

The first merogony of Eimeria bovis takes place in lymphatic endothelial cells of the ileum, resulting in the formation of macromeronts up to 250 microm. In this study, we investigated the host cell cytoskeleton (actin filaments, microtubules, spectrin, vimentin intermediate filaments) associated with parasitic development in vitro by confocal laser scanning microscopy (CLSM) using primary bovine umbilical vein endothelial cells (BUVEC) and bovine spleen lymphatic endothelial cells (BSLEC) as host cells. No prominent changes in the host cell cytoskeleton were detected 1-3 days after E. bovis sporozoite invasion. With ongoing meront maturation a significant increase in microtubules and actin filaments close to the parasitophorous vacuole (PV) was found. Mature macromeronts within the PV were completely enclosed by these cytoskeletal elements. Our findings suggest, that in order to guarantee the survival of the host cell on the enlargement of macromeronts, E. bovis needs not only to augment but also to rearrange its cytoskeletal system.

Studies on synchronous egress of coccidian parasites (Neospora caninum, Toxoplasma gondii, Eimeria bovis) from bovine endothelial host cells mediated by calcium ionophore A23187.

Neospora caninum, Toxoplasma gondii and Eimeria bovis are coccidian parasites of veterinary importance. Tachyzoites of N. caninum and T. gondii and sporozoites of E. bovis are able to invade and replicate in endothelial cells in vivo and in vitro. As it holds true for all eukaryotic cells, the survival of parasitized host cells and the parasites themselves should be dependent on ion balances, especially on extra- and intracellular calcium concentrations. Addition of the calcium ionophore A23187 reliably did release merozoites from mature N. caninum and T. gondii meronts grown in cultured primary bovine umbilical vein endothelial cells (BUVEC). Extent and time course of merozoite release depended on both, maturity of the meronts and concentration of the calcium ionophore. Attempts, however, to achieve synchronous release of merozoites from E. bovis first generation meronts by ionophore treatment failed, suggesting a different biological behaviour of this parasite. According to microscopical observations, the quite variable time of E. bovis macromeront maturation and a hampered merozoite exit owing to dense parasite-induced cytoskeleton elements surrounding the meront may be a reason for the lack of inducible synchronous release.

PMN-mediated immune reactions against Eimeria bovis.

For successful in vivo infection, Eimeria bovis sporozoites have to traverse the mucosal layer of the ileum to infect lymphatic endothelial cells and may, thereby, be exposed to the interstitial fluid and to the lymph representing potential targets for leukocytes. To mimic this situation in vitro, we exposed E. bovis sporozoites to bovine PMN and found enhanced elimination of the parasites. Addition of immune serum clearly increased these reactions, whereas neonatal calf serum had no effect, thus proposing a PMN-derived antibody-dependent cytotoxicity. Scanning and transmission electron microscopy showed PMN engulfing sporozoites or extending filopodia towards them and occasionally incorporating the parasites. PMN reacted with enhanced transcription of IL-6, MCP-1, GROalpha, TNF-alpha, and iNOS genes after exposure to sporozoites while stimulation with merozoite-antigen, in addition, upregulated IL-8, IP-10 and IL-12 gene transcription. Furthermore, enhanced in vitro oxidative burst and phagocytic activities were observed after contact of PMN with viable sporozoites. To verify the potential role of PMN in the in vivo situation, we analysed the general phagocytic and oxidative burst activities of PMN obtained ex vivo from E. bovis experimentally infected calves. Enhanced levels of both activities were found early p.i. (1-5 days) and towards the end of the first schizogony (days 13-22 p.i.) underlining the in vitro data. Our results suggest that PMN-mediated, innate immune reactions play an important role in the early immune response to E. bovis infections in calves.

[Reaction of bovine endothelial cells in vitro to coccidia (Eimeria bovis, Toxoplasma gondii, Neospora caninum) infections as the expression of a non-adaptive immune response]. / Reaktion boviner Endothelzellen in vitro auf Kokzidieninfektionen (Eimeria bovis, Toxoplasma gondii, Neospora caninum) als Ausdruck einer nicht-adaptativen Immunantwort.

Eimeria (E.) bovis sporozoites as well as Toxoplasma (T.) gondii and Neospora (N.) caninum tachyzoites can invade bovine endothelial cells (BUVEC) in vitro and develop to next stage meronts within 15-20 and 3-4 days, respectively. The latter differences suggest different immune evasion strategies, particularly concerning innate reactions. Realtime RT-PCR techniques were used to determine transcript levels of genes relevant in this sense, i.e. adhesion molecule, chemokine, growth factor GM-CSF, cyclooxygenase 2 (COX-2) and iNOS genes in infected cells. In addition, adhesion of neutrophils (PMN) to infected BUVEC monolayers was quantified. Effects differed between E. bovis and T. gondii/N. caninum as the latter two species strongly enhance the transcription of all genes in question and induce PMN adhesion to infected BUVEC whereas E. bovis either caused only weak responses or failed to enhance gene transcription as in case of CXC chemokines and COX-2. It even down regulates adhesion molecule expression in response to cytokines. The differences between the species may reflect differing immune evasion strategies, in case of E. bovis favouring its long lasting development to macromeronts.