The impact of nitrite and antioxidants on ultraviolet-A-induced cell death of human skin fibroblasts

Nitrite (NO(2)(-)) occurs ubiquitously in biological fluids such as blood and sweat. Ultraviolet A-induced nitric oxide formation via decomposition of cutaneous nitrite, accompanied by the production of reactive oxygen (ROS) or nitrogen species (RNS), represents an important source for NO in human skin physiology. Examining the impact of nitrite and the antioxidants glutathione (GSH), Trolox (TRL), and ascorbic acid (ASC) on UVA-induced toxicity of human skin fibroblasts (FB) we found that NO(2)(-) concentration-dependently enhances the susceptibility of FB to the toxic effects of UVA by a mechanism comprising enhanced induction of lipid peroxidation. While ASC completely protects FB cultures from UVA/NO(2)(-)-induced cell damage, GSH or TRL excessively enhances UVA/NO(2)(-)-induced cell death by a mechanism comprising nitrite concentration-dependent TRL radical formation or GSH-derived oxidative stress. Simultaneously, in the presence of GSH or TRL the mode of UVA/NO(2)(-)-induced cell death changes from apoptosis to necrosis. In summary, during photodecomposition of nitrite, ROS or RNS formation may act as strong toxic insults. Although inhibition of oxidative stress by NO and other antioxidants represents a successful strategy for protection from UVA/NO(2)(-)-induced injuries, GSH and TRL may nitrite-dependently aggravate the injurious impact by TRL or GSH radical formation, respectively.     

Seasonal release from competition explains partial migration in European moose

Partial migration, whereby a proportion of a population migrates between distinct seasonal ranges, is common throughout the animal kingdom. However, studies linking existing theoretical models of migration probability, with empirical data are lacking. The competitive release hypothesis for partial migration predicts that due to density-dependent habitat selection, the proportion of migrants increases as the relative quality and size of the seasonal range increases, but decreases with increasing migration cost and population density. To test this prediction, we developed a quantitative framework to predict the proportion of migrants, using empirical data from 545 individually GPS-marked moose Alces alces from across Fennoscandia, spanning latitudes of 56° to 68°N. Moose contracted their ranges to common and spatially limited winter areas (typically at lower elevation), but expanded them during summer due to an increase in suitable habitat (at highland ranges). As predicted from our model, a better and larger highland range relative to the lowland range corresponded to a higher proportion of migrants in an area. Quantitative predictions coupling the balance of habitat availability of seasonal ranges with the probability of migrating in a large herbivore is a necessary step towards an enhanced understanding of the mechanisms underlying migration at the population level.     

Seed bank offers potential for active restoration of mountain meadows

The nitrogen‐fixing legume Lupinus polyphyllus invaded semi‐natural mountainous grasslands across Europe during the last decades. This invasion resulted in degraded habitats through changes in the structure and function of the mountain meadow vegetation. In our study, we analyzed (1) the effects of increasing cover of L. polyphyllus on the seed bank of mountain meadows, and (2) the potential of the seed bank of these stands for active restoration of mountain meadows in terms of species composition and number. We conducted a seed bank analysis on 84 plots with increasing cover of L. polyphyllus in three mountain‐meadow types of the Rhön Biosphere Reserve, Germany. Seedlings from 119 species germinated from the seed bank samples, including 17 Red List species but only a few seedlings of L. polyphyllus. The species composition of the seed bank matched distinct patterns of the three meadow types, but differed from the species composition of the current aboveground vegetation in a nonmetric multidimensional scaling ordination. While the influence of L. polyphyllus on the current vegetation was visible, no effects on the seed bank were apparent. L. polyphyllus had no influence on total seed density, seed density of typical mountain‐meadow species, or species numbers in the seed bank. Only the seeds of the Red List species were significantly related to the cover of L. polyphyllus. We conclude that the seed bank offers potential for active restoration of species‐rich mountain meadows, but species absent from the seed bank have to be added by other measures.     

pH effects on growth and lipid accumulation of the biofuel microalgae Nannochloropsis salina and invading organisms

Biofuels derived from non-crop sources, such as microalgae, offer their own advantages and limitations. Despite high growth rates and lipid accumulation, microalgae cultivation still requires more energy than it produces. Furthermore, invading organisms can lower efficiency of algae production. Simple environmental changes might be able to increase algae productivity while minimizing undesired organisms like competitive algae or predatory algae grazers. Microalgae are susceptible to pH changes. In many production systems, pH is kept below 8 by CO₂ addition. Here, we uncouple the effects of pH and CO₂ input, by using chemical pH buffers and investigate how pH influences Nannochloropsis salina growth and lipid accumulation as well as invading organisms. We used a wide range of pH levels (5, 6, 7, 8, 9, and 10). N. salina showed highest growth rates at pH 8 and 9 (0.19?±?0.008 and 0.19?±?0.011, respectively; mean ± SD). Maximum cell densities in these treatments were reached around 21 days into the experiment (95.6?×?10⁶?±?9?×?10⁶ cells mL^?1 for pH 8 and 92.8?×?10⁶?±?24?×?10⁶ cells mL^?1 for pH 9). Lipid accumulation of unbuffered controls were 21.8?±?5.8 % fatty acid methyl esters content by mass, and we were unable to trigger additional significant lipid accumulation by manipulating pH levels at the beginning of stationary phase. Ciliates (grazing predators) occurred in significant higher densities at pH 6 (56.9?±?39.6?×?10⁴ organisms mL^?1) than higher pH treatments (0.1–6.8?×?10⁴ organisms mL^?1). Furthermore, the addition of buffers themselves seemed to negatively impact diatoms (algal competitors). They were more abundant in an unbuffered control (12.7?±?5.1?×?10⁴ organisms mL^?1) than any of the pH treatments (3.6–4.7?×?10⁴ organisms mL^?1). In general, pH values of 8 to 9 might be most conducive to increasing algae production and minimizing invading organisms. CO₂ addition seems more valuable to algae as an inorganic carbon source and not as an essential mechanism to reduce pH.     

Foxp3+ Regulatory T Cells Delay Expulsion of Intestinal Nematodes by Suppression of IL-9-Driven Mast Cell Activation in BALB/c but Not in C57BL/6 Mice

Accumulating evidence suggests that IL-9-mediated immunity plays a fundamental role in control of intestinal nematode infection. Here we report a different impact of Foxp3⁺ regulatory T cells (Treg) in nematode-induced evasion of IL-9-mediated immunity in BALB/c and C57BL/6 mice. Infection with Strongyloides ratti induced Treg expansion with similar kinetics and phenotype in both strains. Strikingly, Treg depletion reduced parasite burden selectively in BALB/c but not in C57BL/6 mice. Treg function was apparent in both strains as Treg depletion increased nematode-specific humoral and cellular Th2 response in BALB/c and C57BL/6 mice to the same extent. Improved resistance in Treg-depleted BALB/c mice was accompanied by increased production of IL-9 and accelerated degranulation of mast cells. In contrast, IL-9 production was not significantly elevated and kinetics of mast cell degranulation were unaffected by Treg depletion in C57BL/6 mice. By in vivo neutralization, we demonstrate that increased IL-9 production during the first days of infection caused accelerated mast cell degranulation and rapid expulsion of S. ratti adults from the small intestine of Treg-depleted BALB/c mice. In genetically mast cell-deficient (Cpa3-Cre) BALB/c mice, Treg depletion still resulted in increased IL-9 production but resistance to S. ratti infection was lost, suggesting that IL-9-driven mast cell activation mediated accelerated expulsion of S. ratti in Treg-depleted BALB/c mice. This IL-9-driven mast cell degranulation is a central mechanism of S. ratti expulsion in both, BALB/c and C57BL/6 mice, because IL-9 injection reduced and IL-9 neutralization increased parasite burden in the presence of Treg in both strains. Therefore our results suggest that Foxp3⁺ Treg suppress sufficient IL-9 production for subsequent mast cell degranulation during S. ratti infection in a non-redundant manner in BALB/c mice, whereas additional regulatory pathways are functional in Treg-depleted C57BL/6 mice.     

Loss of FADS2 Function Severely Impairs the Use of HeLa Cells as an In Vitro Model for Host Response Studies Involving Fatty Acid Effects

Scope

Established epithelial cell lines equipped with pattern recognition receptors such as the Toll-like receptor (TLR)-2 are common tools for immune response studies on invading pathogens, e.g. the obligate intracellular species of Chlamydia. Moreover, such models are widely used to elucidate fatty acid-mediated immune effects. In several transformed cell lines, however, unusual loss of metabolic functions was described. The cell lines A549 and HeLa are poorly characterized in this respect. Therefore, we comparatively assessed the metabolic capacity of A549 and HeLa prior to proposed application as in vitro model for fatty acid effects on chlamydial infection.

Methodology/Principal Findings

We incubated both cell lines either with substrates (C18∶2n−6 or C18∶3n−3) or products (C18∶3n−6, C18∶4n−3) of fatty acid desaturase-2 (FADS2), and analysed the fatty acid profiles after 24 h and 72 h by gas chromatography. Based on these data, we suspected that the complete discontinuation of normal biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) in HeLa was due to loss of FADS2 function. Consequently, prostaglandin E2 (PGE2) formation was less inducible by TLR2 stimulation in HeLa, likely as a result of not only insufficient supply of precursors but also weak cyclooxygenase-2 (COX-2) response. In accordance, Chlamydia infection rates were consistently lower in HeLa than in A549. Sequence analysis revealed no alteration within the FADS2 gene in HeLa. The FADS2 expression level, however, was significantly lower and, in contrast to A549, not regulated by C18∶2n−6. A549 exhibited regular fatty acid metabolism and enzyme functionality.

Conclusions/Significance

Our data show that HeLa cells considerably differ from A549 at several stages of fatty acid metabolism. The poor metabolic potential of HeLa, mainly concerning FADS2 upstream of COX-2 function, calls into question whether these cells represent a good model to unveil fatty acid or downstream eicosanoid effects in the course of intracellular bacterial infection.     

Nematode-Induced Interference with Vaccination Efficacy Targets Follicular T Helper Cell Induction and Is Preserved after Termination of Infection

One-third of the human population is infected with parasitic worms. To avoid being eliminated, these parasites actively dampen the immune response of their hosts. This immune modulation also suppresses immune responses to third-party antigens such as vaccines. Here, we used Litomosoides sigmodontis-infected BALB/c mice to analyse nematode-induced interference with vaccination. Chronic nematode infection led to complete suppression of the humoral response to thymus-dependent vaccination. Thereby the numbers of antigen-specific B cells as well as the serum immunoglobulin (Ig) G titres were reduced. T_H2-associated IgG1 and T_H1-associated IgG2 responses were both suppressed. Thus, nematode infection did not bias responses towards a T_H2 response, but interfered with Ig responses in general. We provide evidence that this suppression indirectly targeted B cells via accessory T cells as number and frequency of vaccine-induced follicular B helper T cells were reduced. Moreover, vaccination using model antigens that stimulate Ig response independently of T helper cells was functional in nematode-infected mice. Using depletion experiments, we show that CD4⁺Foxp3⁺ regulatory T cells did not mediate the suppression of Ig response during chronic nematode infection. Suppression was induced by fourth stage larvae, immature adults and mature adults, and increased with the duration of the infection. By contrast, isolated microfilariae increased IgG2a responses to vaccination. This pro-inflammatory effect of microfilariae was overruled by the simultaneous presence of adults. Strikingly, a reduced humoral response was still observed if vaccination was performed more than 16 weeks after termination of L. sigmodontis infection. In summary, our results suggest that vaccination may not only fail in helminth-infected individuals, but also in individuals with a history of previous helminth infections.     

Map2k4δ — Identification and functional characterization of a novel Map2k4 splice variant

Mitogen-activated protein kinase kinase 4 (Map2k4) is a dual specificity serin/threonine protein kinase that is unique among all MAP2Ks in activating two different subfamilies of mitogen-activated protein kinases, the c-Jun N-terminal kinases (JNKs) and p38 kinases. Map2k4 is essential during embryogenesis and involved in a variety of physiological and pathological processes. However, studies on its role in cancer development revealed partially conflicting data. In the present study, we report the identification of a novel splice variant of Map2k4, Map2k4δ, with an additional exon in front of the substrate binding D-domain. Map2k4δ is expressed together with Map2k4 in various tissues from rat, mouse and human. In PC12 cells, both splice variants control cell cycle progression and basal apoptosis by using different signaling pathways. If expression and activation of Map2k4 and Map2k4δ are at a certain, cell type-specific equilibrium, an appropriate cell growth is ensured. Overexpression of one kinase disrupts the intricate balance and either results in a highly proliferative or pro-apoptotic phenotype, partially reflecting the discrepancies in the literature on Map2k4 and its role in tumor development. Our findings contribute to the understanding of previous studies and point out that Map2k4 has not always a definite function, but rather triggers a cellular reaction in concert with other modulators.     

Autocrine GM-CSF transcription in the leukemic progenitor cell line KG1a is mediated by the transcription factor ETS1 and is negatively regulated through SECTM1 mediated ligation of CD7

Background

CD7 expression is found on ~ 30% of acute myeloblastic leukemias (AML). The leukemic progenitor cell line KG1a (CD7 +) constitutively expresses GM-CSF while the parental KG1 (CD7-) cell line does not. This study focuses on the molecular basis of CD7 mediated GM-CSF regulation.

Methods

KG1a cells were treated with recombinant SECTM1-Fc protein, the PI3K kinase inhibitors wortmannin, LY292004, or PI4K activator spermine. Stable KG1-CD7 +, KG1a-shCD7, KG1a-shETS1 as well as KG1a-GFP, KG1a-PKCβII-GFP cell lines were generated and the levels of CD7, GM-CSF and ETS-1 mRNA and protein were compared by real-time-PCR, western blotting, flow cytometry and ELISA.

Results

SECTM1 is expressed in Human Bone Marrow Endothelial Cells (HBMEC) and its expression can be upregulated by both IFN-γ. KG1a cells demonstrated high expression levels of CD7 and ETS-1 allowing a constitutative signaling through the PI3K/Atk pathway to promote GM-CSF expression, while KG1 cells with low expression of CD7 and ETS-1 showed low GM-CSF expression. On KG1a cells GM-CSF expression could be negatively regulated by PI3K inhibitors or by recombinant SECTM1-Fc. Overexpression of CD7 in KG1 cells was insufficient to promote GM-CSF expression, while silencing of CD7 or ETS-1 resulted in reduced GM-CSF expression levels. Differentiation capable KG1a cells overexpressing PKCβII illustrated complete loss of CD7, but maintained normal levels of both ETS-1 and GM-CSF expression.

Conclusion

These findings add an additional layer to the previously described autocrine/paracrine signaling between leukemic progenitor cells and the bone marrow microenvironment and highlight a role for SECTM1 in both normal and malignant hematopoiesis.

General Significance

This work shows that SECTM1 secreted from bone marrow stromal cells may interact with CD7 to influence GM-CSF expression in leukemic cells.     

Estimating sampling efficiency of diurnal Lepidoptera in farmland

Setting up effective survey strategies for biodiversity monitoring in agro-ecosystems is a major task in order to detect adverse effects on biodiversity before negative changes will manifest. Here, we studied the relative costs required for the monitoring of butterflies and selected diurnal moths (Papilionoidea et Hesperioidea; Zygaenoidea: Zygaenidae) in farmland. Analysing data from a well-established Lepidoptera monitoring system in Switzerland, we assessed the influence of inspection periods, inspection frequency and transect length on counts of diurnal Lepidoptera. Furthermore, we estimated the number of transects in relation to sampling effort necessary to detect changes of a given effect size for recorded species number (and abundance). Reducing the counting frequency from seven to four inspections per season still yielded 80–90 % of the species, as long as peak abundances in summer months were included. The variation in observed species number was mostly independent of inspection frequency, but strongly increased when transect length was reduced to less than 1 km. Sedentary Lepidoptera species are especially valuable indicators as their occurrences are directly linked to local effects on biodiversity, and the proportion of recorded sedentary species was not substantially affected by reduced inspection frequency. Transects of 1–1.5 km length were generally the most cost-efficient to detect an effect on total species number of diurnal Lepidoptera in arable landscapes, given that travelling distances between transects were short. Studying effects on rare species or selected species groups would involve higher sampling intensity and costs. Surveying schemes with reduced inspection frequency and transect lengths can detect changes in species richness and total abundance of diurnal Lepidoptera cost-effectively. Facing expected changes in agricultural policy and management, the results and recommendations presented here will help to implement and improve cost-efficient Lepidoptera schemes to monitor changes in arable landscapes.