Barrier Disrupting Effects of Alternaria Alternata Extract on Bronchial Epithelium from Asthmatic Donors

Sensitization and exposure to the allergenic fungus Alternaria alternata has been associated with increased risk of asthma and asthma exacerbations. The first cells to encounter inhaled allergens are epithelial cells at the airway mucosal surface. Epithelial barrier function has previously been reported to be defective in asthma. This study investigated the contribution of proteases from Alternaria alternata on epithelial barrier function and inflammatory responses and compared responses of in vitro cultures of differentiated bronchial epithelial cells derived from severely asthmatic donors with those from non-asthmatic controls. Polarised 16HBE cells or air-liquid interface (ALI) bronchial epithelial cultures from non-asthmatic or severe asthmatic donors were challenged apically with extracts of Alternaria and changes in inflammatory cytokine release and transepithelial electrical resistance (TER) were measured. Protease activity in Alternaria extracts was characterised and the effect of selectively inhibiting protease activity on epithelial responses was examined using protease inhibitors and heat-treatment. In 16HBE cells, Alternaria extracts stimulated release of IL-8 and TNFα, with concomitant reduction in TER; these effects were prevented by heat-treatment of the extracts. Examination of the effects of protease inhibitors suggested that serine proteases were the predominant class of proteases mediating these effects. ALI cultures from asthmatic donors exhibited a reduced IL-8 response to Alternaria relative to those from healthy controls, while neither responded with increased thymic stromal lymphopoietin (TSLP) release. Only cultures from asthmatic donors were susceptible to the barrier-weakening effects of Alternaria. Therefore, the bronchial epithelium of severely asthmatic individuals may be more susceptible to the deleterious effects of Alternaria.     

Generation of Inducible Immortalized Dendritic Cells with Proper Immune Function In Vitro and In Vivo

Dendritic cells are the professional antigen presenting cells of innate immunity and key players in maintaining the balance of immune responses. Studies with dendritic cells are mainly limited by their low numbers in vivo and their difficult maintenance in vitro. We differentiated bone marrow cells from transgenic mice expressing an inducible SV40 large T-antigen into dendritic cells. When immortalized by dexamethasone and doxycycline, these cells were stable in long-term culture. In the absence of dexamethasone and doxycycline (de-induction), dendritic cells displayed properties of primary cells, characterized by expression of classical dendritic cell surface markers CD11c, CD11b, MHCII, CD40 and CD86. Furthermore, de-induced lipopolysaccharide activated dendritic cells secreted IL-1β, IL-6, TNFα and IL-12. De-induced, Ovalbumin-loaded dendritic cells polarize CD4⁺ T cells into Th1, Th17 and Th2 cells, indicating their correct antigen presenting property. Consistent with intratracheal application of Ovalbumin-loaded primary dendritic cells into mice, the application of de-induced dendritic cells resulted in recruitment of lymphocytes to the lungs. In summary, we successfully expanded dendritic cells using conditional immortalization. The generated dendritic cells demonstrate the characteristic immunophenotype of primary dendritic cells and will facilitate further studies on immunomodulatory properties of dendritic cells.     

Oral Perceptions of Fat and Taste Stimuli Are Modulated by Affect and Mood Induction

This study examined the impact of three clinical psychological variables (non-pathological levels of depression and anxiety, as well as experimentally manipulated mood) on fat and taste perception in healthy subjects. After a baseline orosensory evaluation, ‘sad’, ‘happy’ and ‘neutral’ video clips were presented to induce corresponding moods in eighty participants. Following mood manipulation, subjects rated five different oral stimuli, appearing sweet, umami, sour, bitter, fatty, which were delivered at five different concentrations each. Depression levels were assessed with Beck’s Depression Inventory (BDI) and anxiety levels were assessed via the Spielberger’s STAI-trait and state questionnaire. Overall, subjects were able to track the concentrations of the stimuli correctly, yet depression level affected taste ratings. First, depression scores were positively correlated with sucrose ratings. Second, subjects with depression scores above the sample median rated sucrose and quinine as more intense after mood induction (positive, negative and neutral). Third and most important, the group with enhanced depression scores did not rate low and high fat stimuli differently after positive or negative mood induction, whereas, during baseline or during the non-emotional neutral condition they rated the fat intensity as increasing with concentration. Consistent with others’ prior observations we also found that sweet and bitter stimuli at baseline were rated as more intense by participants with higher anxiety scores and that after positive and negative mood induction, citric acid was rated as stronger tasting compared to baseline. The observation that subjects with mild subclinical depression rated low and high fat stimuli similarly when in positive or negative mood is novel and likely has potential implications for unhealthy eating patterns. This deficit may foster unconscious eating of fatty foods in sub-clinical mildly depressed populations.     

Import and export of biological samples from tropical countries–considerations and guidelines for research teams

‘Biodiversity’ is increasingly perceived as an important resource for research and conservation, but also for economy. Conservation, access and sustainable use of biodiversity (genetic resources, species, samples) are negotiated on different political levels, resulting in an internationally binding legal framework. Resulting legislation is binding for all parties involved in biological sampling, i.e. researches and (and in italics) countries, and especially applies for tissue or DNA samples and dervied products thereof. Understanding and awareness of export and import permits for biological samples is increasingly important for biologists to perform research projects legally and timely. Nevertheless, some biologists are still exporting and importing biological samples ignoring or non-compliant with national and international legislation, conventions, and regulations. Resulting difficulties may not only cause serious problems during field work, but may also delay the export, import or exchange of samples. Comprehensive a priori information regarding legal requirements helps to avoid or at least diminish potential problems. We identified four major factors facilitating export/import permits: (1) good personal (mutually trusted) contacts in the country of origin, (2) understanding and compliance with all relevant laws and regulations; (3) access to information regarding knowledge on permits, regulations and laws including their circulation within the researcher communities; and (4) access to consistent and up to date regulations     

Testosterone Increases Susceptibility to Amebic Liver Abscess in Mice and Mediates Inhibition of IFNγ Secretion in Natural Killer T Cells

Amebic liver abscess (ALA), a parasitic disease due to infection with the protozoan Entamoeba histolytica, occurs age and gender dependent with strong preferences for adult males. Using a mouse model for ALA with a similar male bias for the disease, we have investigated the role of female and male sexual hormones and provide evidence for a strong contribution of testosterone. Removal of testosterone by orchiectomy significantly reduced sizes of abscesses in male mice, while substitution of testosterone increased development of ALA in female mice. Activation of natural killer T (NKT) cells, which are known to be important for the control of ALA, is influenced by testosterone. Specifically activated NKT cells isolated from female mice produce more IFNγ compared to NKT cells derived from male mice. This high level production of IFNγ in female derived NKT cells was inhibited by testosterone substitution, while the IFNγ production in male derived NKT cells was increased by orchiectomy. Gender dependent differences were not a result of differences in the total number of NKT cells, but a result of a higher activation potential for the CD4⁻ NKT cell subpopulation in female mice. Taken together, we conclude that the hormone status of the host, in particular the testosterone level, determines susceptibility to ALA at least in a mouse model of the disease.     

Does early learning drive ecological divergence during speciation processes in parasitoid wasps?

Central to the concept of ecological speciation is the evolution of ecotypes, i.e. groups of individuals occupying different ecological niches. However, the mechanisms behind the first step of separation, the switch of individuals into new niches, are unclear. One long-standing hypothesis, which was proposed for insects but never tested, is that early learning causes new ecological preferences, leading to a switch into a new niche within one generation. Here, we show that a host switch occurred within a parasitoid wasp, which is associated with the ability for early learning and the splitting into separate lineages during speciation. Lariophagus distinguendus consists of two genetically distinct lineages, most likely representing different species. One attacks drugstore beetle larvae (Stegobium paniceum (L.)), which were probably the ancestral host of both lineages. The drugstore beetle lineage has an innate host preference that cannot be altered by experience. In contrast, the second lineage is found on Sitophilus weevils as hosts and changes its preference by early learning. We conclude that a host switch has occurred in the ancestor of the second lineage, which must have been enabled by early learning. Because early learning is widespread in insects, it might have facilitated ecological divergence and associated speciation in this hyperdiverse group.     

Characterization of Thi9, a novel thiamine (Vitamin B1) transporter from Schizosaccharomyces pombe

Thiamine is an essential component of the human diet and thiamine diphosphate-dependent enzymes play an important role in carbohydrate metabolism in all living cells. Although the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe can derive thiamine from biosynthesis, both are also able to take up thiamine from external sources, leading to the down-regulation of the enzymes involved in its formation. We have isolated the S. pombe thiamine transporter Thi9 by genetic complementation of mutants defective in thiamine biosynthesis and transport. Thi9 localizes to the S. pombe cell surface and works as a high-affinity proton/thiamine symporter. The uptake of thiamine was reduced in the presence of pyrithiamine, oxythiamine, amprolium, and the thiazole part of thiamine, indicating that these compounds are substrates of Thi9. In pyrithiamine-resistant mutants, a conserved glutamate residue close to the first of the 12 transmembrane domains is exchanged by a lysine and this causes aberrant localization of the protein. Thiamine uptake is significantly increased in thiamine-deficient medium and this is associated with an increase in thi9(+) mRNA and protein levels. Upon addition of thiamine, the thi9(+) mRNA becomes undetectable within minutes, whereas the Thi9 protein appears to be stable. The protein is distantly related to transporters for amino acids, gamma-aminobutyric acid and polyamines, and not to any of the known thiamine transporters. We also found that the pyridoxine transporter Bsu1 has a marked contribution to the thiamine uptake activity of S. pombe cells.     

Adaptation of aerobically growing Pseudomonas aeruginosa to copper starvation

Restricted bioavailability of copper in certain environments can interfere with cellular respiration because copper is an essential cofactor of most terminal oxidases. The global response of the metabolically versatile bacterium and opportunistic pathogen Pseudomonas aeruginosa to copper limitation was assessed under aerobic conditions. Expression of cioAB (encoding an alternative, copper-independent, cyanide-resistant ubiquinol oxidase) was upregulated, whereas numerous iron uptake functions (including the siderophores pyoverdine and pyochelin) were expressed at reduced levels, presumably reflecting a lower demand for iron by respiratory enzymes. Wild-type P. aeruginosa was able to grow aerobically in a defined glucose medium depleted of copper, whereas a cioAB mutant did not grow. Thus, P. aeruginosa relies on the CioAB enzyme to cope with severe copper deprivation. A quadruple cyo cco1 cco2 cox mutant, which was deleted for all known heme-copper terminal oxidases of P. aeruginosa, grew aerobically, albeit more slowly than did the wild type, indicating that the CioAB enzyme is capable of energy conservation. However, the expression of a cioA'-'lacZ fusion was less dependent on the copper status in the quadruple mutant than in the wild type, suggesting that copper availability might affect cioAB expression indirectly, via the function of the heme-copper oxidases.