Photobehaviour of Paramecium bursaria infected with different symbiotic and aposymbiotic species of Chlorella

The endosymbiotic unit of Paramecium bursaria and Chlorella spec. shows two types of photobehaviour: 1) A step-up photophobic response which possibly depends on photosensitive agents in the ciliate cell itself — as is also shown by alga-free Paramecium bursaria - and can be drastically enhanced by photosynthetic activity of symbiotic algae; and 2) a step-down photophobic response. The step-down response leads to photoaccumulation of green paramecia. Both types of photobehaviour in Paramecium bursaria do not depend on any special kind of algal partners: The infection of alga-free Paramecium bursaria with different Chlorella species results in new ciliatealgae-associations. They are formed not only by combination of the original symbiotic algae with their host, but also by infection with other symbiotic or free-living (aposymbiotic) chlorellae, respecitively. Systems with other than the original algae are not permanently stable — algae are lost under stress conditions — but show the same types of photobehaviour. Photoaccumulation in general requires algal photosynthesis and occurs only with ciliates containing more than fifty algae/cell. It is not mediated by a chemotactic response to oxygen in the medium, since it occurs at light fluence rates not sufficient for a release of oxygen by the symbiotic system, e.g., below its photosynthetic compensation point. Photoresponses can be inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Sensory transduction does not depend on any special symbiotic features of the algae, e.g., sugar excretion. The participation of oxygen in the Paramecium cell, of its cytoplasmic pH and of ions released or taken up by endosymbiotic algae in sensory transduction is discussed.     

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory,GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.     

Inverse response of leukocyte heat shock proteins and DNA damage to exercise and heat

Elevated ambient temperature may exert an additional impact on the exercise-induced expression of heat shock proteins (HSP) and DNA damage in leukocytes. The protective functions of HSP include antioxidative and antiapoptotic effects and may prevent damage to DNA. Twelve athletes completed a continuous run (75% VO2max) on the treadmill, six at 28 degrees C and six at 18 degrees C room temperature. Leukocyte expression of HSP27 and inducible HSP70 was analyzed on mRNA- (RT-PCR) and protein-level (flow cytometry), while DNA damage was quantified by the comet assay. High ambient temperature induced an additional accumulation of HSP-mRNA and -protein in leukocytes compared with the exercise-induced expression at 18 degrees C. HSP27 showed a special heat sensitivity. Surprisingly, the increase of DNA damage was less pronounced after exercise at 28 degrees C compared to 18 degrees C although heat shock in vitro clearly induced DNA damage. The inverse relation between HSP and DNA damage may indicate functions of HSP which protect against exercise-induced DNA-damage in terms of thermotolerance or apoptosis.     

Dendritic cells in the recognition of intestinal microbiota

Mucosal dendritic cells (DCs) constantly survey the luminal microenvironment which contains commensal microbiota and potentially harmful organisms regulating pathogen recognition and adaptive as well as innate defense activation. Distinct mechanisms are beginning to emerge by which intestinal antigen sampling and handling is achieved ensuring specificity and contributing to redundancy in pathogen detection. Distinct DC subsets are associated with these mechanisms and regulate specific innate or adaptive immune responses to help distinguish between commensal microbiota, pathogens and self antigens. Understanding DC biology in the mucosal immune system may contribute to the unraveling of infection routes of intestinal pathogens and may aid in developing novel vaccines and therapeutic strategies for the treatment of infectious and inflammatory diseases.     

CD69 regulates type I IFN-induced tolerogenic signals to mucosal CD4 T cells that attenuate their colitogenic potential

CD69 is highly expressed by lymphocytes at mucosal surfaces. We aimed to investigate the role of CD69 in mucosal immune responses. The expression of CD69 by CD4 T cells isolated from the spleen, mesenteric lymph nodes, small intestinal lamina propria, and colonic lamina propria was determined in specific pathogen-free B6 and TCR transgenic animals, as well as in germ-free B6 mice. Transfer colitis was induced by transplanting RAG(-/-) mice with B6 or CD69(-/-)CD45RB(high) CD4 T cells. CD69 expression by CD4 T cells is induced by the intestinal microflora, oral delivery of specific Ag, and type I IFN (IFN-I) signals. CD4 T cells from CD69(-/-) animals produce higher amounts of the proinflammatory cytokines IFN-γ, TNF-α, and IL-21, whereas the production of TGF-β1 is decreased. CD69-deficient CD4 T cells showed reduced potential to differentiate into Foxp3(+) regulatory T cells in vivo and in vitro. The transfer of CD69(-/-)CD45RB(high) CD4 T cells into RAG(-/-) hosts induced an accelerated colitis. Oral tolerance was impaired in CD69(-/-) and IFN-I receptor 1-deficient mice when compared with B6 and OT-II × RAG(-/-) animals. Polyinosinic-polycytidylic acid treatment of RAG(-/-) mice transplanted with B6 but not CD69(-/-) or IFN-I receptor 1-deficient CD45RB(high) CD4 T cells attenuated transfer colitis. CD69 deficiency led to the increased production of proinflammatory cytokines, reduced Foxp3(+) regulatory T cell induction, impaired oral tolerance, and more severe colitis. Hence, the activation Ag CD69 plays an important role in regulating mucosal immune responses.     

Is Bagging Effective in the Classification of Small-Sample Genomic and Proteomic Data?

There has been considerable interest recently in the application of bagging in the classification of both gene-expression data and protein-abundance mass spectrometry data. The approach is often justified by the improvement it produces on the performance of unstable, overfitting classification rules under small-sample situations. However, the question of real practical interest is whether the ensemble scheme will improve performance of those classifiers sufficiently to beat the performance of single stable, nonoverfitting classifiers, in the case of small-sample genomic and proteomic data sets. To investigate that question, we conducted a detailed empirical study, using publicly-available data sets from published genomic and proteomic studies. We observed that, under t-test and RELIEF filter-based feature selection, bagging generally does a good job of improving the performance of unstable, overfitting classifiers, such as CART decision trees and neural networks, but that improvement was not sufficient to beat the performance of single stable, nonoverfitting classifiers, such as diagonal and plain linear discriminant analysis, or 3-nearest neighbors. Furthermore, as expected, the ensemble method did not improve the performance of these classifiers significantly. Representative experimental results are presented and discussed in this work.     

Electron availability in CO2, CO and H2 mixtures constrains flux distribution,energy management and product formation in Clostridium ljungdahlii

Acetogens such as Clostridium ljungdahlii can play a crucial role reducing the human CO₂ footprint by converting industrial emissions containing CO₂, CO and H₂ into valuable products such as organic acids or alcohols. The quantitative understanding of cellular metabolism is a prerequisite to exploit the bacterial endowments and to fine-tune the cells by applying metabolic engineering tools. Studying the three gas mixtures CO₂ + H₂, CO and CO + CO₂ + H₂ (syngas) by continuously gassed batch cultivation experiments and applying flux balance analysis, we identified CO as the preferred carbon and electron source for growth and producing alcohols. However, the total yield of moles of carbon (mol-C) per electrons consumed was almost identical in all setups which underlines electron availability as the main factor influencing product formation. The Wood–Ljungdahl pathway (WLP) showed high flexibility by serving as the key NAD⁺ provider for CO₂ + H_2, whereas this function was strongly compensated by the transhydrogenase-like Nfn complex when CO was metabolized. Availability of reduced ferredoxin (Fd_red) can be considered as a key determinant of metabolic control. Oxidation of CO via carbon monoxide dehydrogenase (CODH) is the main route of Fd_red formation when CO is used as substrate, whereas Fd_red is mainly regenerated via the methyl branch of WLP and the Nfn complex utilizing CO₂ + H₂. Consequently, doubled growth rates, highest ATP formation rates and highest amounts of reduced products (ethanol, 2,3-butanediol) were observed when CO was the sole carbon and electron source.     

Antioxidant intervention does not affect the response of plasma erythropoietin to short-term normobaric hypoxia in humans.

Recent research has demonstrated that reactive oxygen species (ROS) participate in intracellular signaling processes initiated during hypoxia. We investigated the role of ROS in the response of plasma erythropoietin (Epo) to short-term normobaric hypoxia in humans. Twelve male subjects were exposed twice to 4 h of normobaric hypoxia (H; inspired oxygen fraction 12.5%) with a period of 6 wk between both experiments (H1 and H2). With the use of a randomized placebo-controlled crossover design, the subjects received orally a combination of the antioxidants all-rac-alpha-tocopherol (800 mg/day for 3 wk) and alpha-lipoic acid (600 mg/day for 2 wk) or placebo before H1 and H2, respectively. Three weeks before H1, the subjects underwent one control experiment in normoxia (N; inspired oxygen fraction 20.9%) without any treatment. Serum alpha-tocopherol was significantly higher after treatment with antioxidants compared with placebo. Capillary Po(2) declined during H without significant differences between antioxidants and placebo. Plasma peroxide levels were lower under antioxidant treatment but not affected by hypoxia. The response of Epo to H did not show significant differences between antioxidant [maximum increase (means, 95% confidence interval): +121%, +66 to +176%] and placebo conditions (+108%, +68 to +149%). Similarly, hypoxia-induced increase of Epo corrected for diurnal variations, as revealed during N, did not differ between antioxidants and placebo. Individual variability of Epo in response to H was not related to the individual degree of hypoxemia during H. Our results do not support the assumption that ROS play a major modulating role in the response of Epo to short-term normobaric hypoxia in humans.