Detection of airborne genetically modified maize pollen by real-time PCR

The cultivation of genetically modified (GM) crops has raised numerous concerns in the European Union and other parts of the world about their environmental and economic impact. Especially outcrossing of genetically modified organisms (GMO) was from the beginning a critical issue as airborne pollen has been considered an important way of GMO dispersal. Here, we investigate the use of airborne pollen sampling combined with microscopic analysis and molecular PCR analysis as an approach to monitor GM maize cultivations in a specific area. Field trial experiments in the European Union and South America demonstrated the applicability of the approach under different climate conditions, in rural and semi-urban environment, even at very low levels of airborne pollen. The study documents in detail the sampling of GM pollen, sample DNA extraction and real-time PCR analysis. Our results suggest that this 'GM pollen monitoring by bioaerosol sampling and PCR screening' approach might represent an useful aid in the surveillance of GM-free areas, centres of origin and natural reserves.     

Ly49h-deficient C57BL/6 mice: a new mouse cytomegalovirus-susceptible model remains resistant to unrelated pathogens controlled by the NK gene complex

Cmv1 was the first mouse cytomegalovirus (MCMV) resistance locus identified in C57BL/6 mice. It encodes Ly49H, a NK cell-activating receptor that specifically recognizes the m157 viral protein at the surface of MCMV-infected cells. To dissect the effect of the Ly49h gene in host-pathogen interactions, we generated C57BL/6 mice lacking the Ly49h region. We found that 36 h after MCMV infection, the lack of Ly49h resulted in high viral replication in the spleen and dramatically enhanced proinflammatory cytokine production in the serum and spleen. At later points in time, we observed that MCMV induced a drastic loss in CD8(+) T cells in B6.Ly49h(-/-) mice, probably reflecting severe histological changes in the spleen. Overall, our results indicate that Ly49H(+) NK cells contain a systemic production of cytokines that may contribute to the MCMV-induced pathology and play a central role in maintaining normal spleen cell microarchitecture. Finally, we tested the ability of B6.Ly49h(-/-) mice to control replication of Leishmania major and ectromelia virus. Resistance to these pathogens has been previously mapped within the NK gene complex. We found that the lack of Ly49H(+) NK cells is not associated with an altered resistance to L. major. In contrast, absence of Ly49H(+) NK cells seems to afford additional protection against ectromelia infection in C57BL/6 mice, suggesting that Ly49H may recognize ectromelia-infected cells with detrimental effects. Taken together, these results confirm the pivotal role of the Ly49H receptor during MCMV infection and open the way for further investigations in host-pathogen interactions.     

Membrane fusogenic activity of the Alzheimer's peptide A beta(1-42) demonstrated by small-angle neutron scattering

Amyloid-β peptide (Aβ) is considered a triggering agent of Alzheimer's disease. In relation to a therapeutic treatment of the disease, the interaction of Aβ with the cell membrane has to be elucidated at the molecular level to understand its mechanism of action. In previous works, we had ascertained by neutron diffraction on stacked lipid multilayers that a toxic fragment of Aβ is able to penetrate and perturb the lipid bilayer. Here, the influence of Aβ(1-42), the most abundant Aβ form in senile plaques, on unilamellar lipid vesicles of phospholipids is investigated by small-angle neutron scattering. We have used the recently proposed separated form factor method to fit the data and to obtain information about the vesicle diameter and structure of the lipid bilayer and its change upon peptide administration. The lipid membrane parameters were obtained with different models of the bilayer profile. As a result, we obtained an increase in the vesicle radii, indicating vesicle fusion. This effect was particularly enhanced at pH 7.0 and at a high peptide/lipid ratio. At the same time, a thinning of the lipid bilayer occurred. A fusogenic activity of the peptide may have very important consequences and may contribute to cytotoxicity by destabilizing the cell membrane. The perturbation of the bilayer structure suggests a strong interaction and/or insertion of the peptide into the membrane, although its localization remains beyond the limit of the experimental resolution.     

Conformational plasticity of the Gerstmann-Sträussler-Scheinker disease peptide as indicated by its multiple aggregation pathways

The existence of several prion strains and their capacity of overcoming species barriers seem to point to a high conformational adaptability of the prion protein. To investigate this structural plasticity, we studied here the aggregation pathways of the human prion peptide PrP82-146, a major component of the Gerstmann-Sträussler-Scheinker amyloid disease.By Fourier transform infrared (FT-IR) spectroscopy, electron microscopy, and atomic force microscopy (AFM), we monitored the time course of PrP82-146 fibril formation. After incubation at 37 °C, the unfolded peptide was found to aggregate into oligomers characterized by intermolecular β-sheet infrared bands. At a critical oligomer concentration, the emergence of a new FT-IR band allowed to detect fibril formation. A different intermolecular β-sheet interaction of the peptides in oligomers and in fibrils is, therefore, detected by FT-IR spectroscopy, which, in addition, suggests a parallel orientation of the cross β-sheet structures of PrP82-146 fibrils. By AFM, a wide distribution of PrP82-146 oligomer volumes—the smallest ones containing from 5 to 30 peptides—was observed. Interestingly, the statistical analysis of AFM data enabled us to detect a quantization in the oligomer height values differing by steps of ∼ 0.5 nm that could reflect an orientation of oligomer β-strands parallel with the sample surface. Different morphologies were also detected for fibrils that displayed high heterogeneity in their twisting periodicity and a complex hierarchical assembly.Thermal aggregation of PrP82-146 was also investigated by FT-IR spectroscopy, which indicated for these aggregates an intermolecular β-sheet interaction different from that observed for oligomers and fibrils. Unexpectedly, random aggregates, induced by solvent evaporation, were found to display a significant α-helical structure as well as several β-sheet components.All these results clearly point to a high plasticity of the PrP82-146 peptide, which was found to be capable of undergoing several aggregation pathways, with end products displaying different secondary structures and intermolecular interactions.     

Structural basis of the honey bee PBP pheromone and pH-induced conformational change

The behavior of insects and their perception of their surroundings are driven, in a large part, by odorants and pheromones. This is especially true for social insects, such as the honey bee, where the queen controls the development and the caste status of the other individuals. Pheromone perception is a complex phenomenon relying on a cascade of recognition events, initiated in antennae by pheromone recognition by a pheromone-binding protein and finishing with signal transduction at the axon membrane level. With to the objective of deciphering this initial step, we have determined the structures of the bee antennal pheromone-binding protein (ASP1) in the apo form and in complex with the main component of the queen mandibular pheromonal mixture, 9-keto-2(E)-decenoic acid (9-ODA) and with nonpheromonal components. In the apo protein, the C terminus obstructs the binding site. In contrast, ASP1 complexes have different open conformations, depending on the ligand shape, leading to different volumes of the binding cavity. The binding site integrity depends on the C terminus (111-119) conformation, which involves the interplay of two factors; i.e. the presence of a ligand and a low pH. Ligand binding to ASP1 is favored by low pH, opposite to what is observed with other pheromone-binding proteins, such as those of Bombyx mori and Anopheles gambiae.     

Seroepidemiology of Toxoplasma gondii in zoo animals in selected zoos in the midwestern United States

Toxoplasma gondii infections in zoo animals are of interest because many captive animals die of clinical toxoplasmosis and because of the potential risk of exposure of children and elderly to T. gondii oocysts excreted by cats in the zoos. Seroprevalence of T. gondii antibodies in wild zoo felids, highly susceptible zoo species, and feral cats from 8 zoos of the midwestern United States was determined by using the modified agglutination test (MAT). A titer of 1:25 was considered indicative of T. gondii exposure. Among wild felids, antibodies to T. gondii were found in 6 (27.3%) of 22 cheetahs (Acynonyx jubatus jubatus), 2 of 4 African lynx (Caracal caracal), 1 of 7 clouded leopards (Neofelis nebulosa), 1 of 5 Pallas cats (Otocolobus manul), 12 (54.5%) of 22 African lions (Panthera leo), 1 of 1 jaguar (Panthera onca), 1 of 1 Amur leopard (Panthera pardus orientalis), 1 of 1 Persian leopard (Panthera pardus saxicolor), 5 (27.8%) of 18 Amur tigers (Panthera tigris altaica), 1 of 4 fishing cats (Prionailurus viverrinus), 3 of 6 pumas (Puma concolor), 2 of 2 Texas pumas (Puma concolor stanleyana), and 5 (35.7%) of 14 snow leopards (Uncia uncia). Antibodies were found in 10 of 34 feral domestic cats (Felis domesticus) trapped in 3 zoos. Toxoplasma gondii oocysts were not found in any of the 78 fecal samples from wild and domestic cats. Among the macropods, antibodies were detected in 1 of 3 Dama wallabies (Macropus eugenii), 1 of 1 western grey kangaroo (Macropus fuliginosus), 1 of 2 wallaroos (Macropus robustus), 6 of 8 Bennett's wallabies (Macropus rufogriseus), 21 (61.8%) of 34 red kangaroos (Macropus rufus), and 1 of 1 dusky pademelon (Thylogale brunii). Among prosimians, antibodies were detected in 1 of 3 blue-eyed black lemurs (Eulemur macaco flavifrons), 1 of 21 ring-tailed lemurs (Lemur catta), 2 of 9 red-ruffed lemurs (Varecia variegata rubra), and 2 of 4 black- and white-ruffed lemurs (Varecia variegata variegata). Among the avian species tested, 2 of 3 bald eagles (Haliaeetus leucocephalus) were seropositive. Among 7 possible risk factors, sex, freezing meat temperature (above -13 C vs. below -13 C), washing vegetables thoroughly, frequency of feral cat sightings on zoo grounds (occasionally vs. frequently), frequency of feral cat control programs, capability of feral cats to enter hay/grain barn, and type of animal exhibit, exhibiting animals in open enclosures was the only factor identified as a significant risk (OR 3.22, P = 0.00).     

Comparison of proteins expressed on secretory vesicle membranes and plasma membranes of human neutrophils

Secretory vesicles are neutrophil intracellular storage granules formed by endocytosis. Understanding the functional consequences of secretory vesicle exocytosis requires knowledge of their membrane proteins. The current study was designed to use proteomic technologies to develop a more complete catalog of secretory vesicle membrane proteins and to compare the proteomes of secretory vesicle and plasma membranes. A total of 1118 proteins were identified, 573 (51%) were present only in plasma membrane-enriched fractions, 418 (37%) only in secretory vesicle-enriched membrane fractions, and 127 (11%) in both fractions. Gene Ontology categorized 373 of these proteins as integral membrane proteins. Proteins typically associated with other intracellular organelles, including nuclei, mitochondria, and ribosomes, were identified in both membrane fractions. Ingenuity Pathway Knowledge Base analysis determined that the majority of canonical and functional pathways were significantly associated with proteins from both plasma membrane-enriched and secretory vesicle-enriched fractions. There were, however, some canonical signaling pathways that involved proteins only from plasma membranes or secretory vesicles. In conclusion, a number of proteins were identified that may elucidate mechanisms and functional consequences of secretory vesicle exocytosis. The small number of common proteins suggests that the hypothesis that secretory vesicles are formed from plasma membranes by endocytosis requires more critical evaluation.     

IFN-gamma-producing human invariant NKT cells promote tumor-associated antigen-specific cytotoxic T cell responses

CD1d-restricted invariant NKT (iNKT) cells can enhance immunity to cancer or prevent autoimmunity, depending on the cytokine profile secreted. Antitumor effects of the iNKT cell ligand alpha-galactosylceramide (alphaGC) and iNKT cell adoptive transfer have been demonstrated in various tumor models. Together with reduced numbers of iNKT cells in cancer patients, which have been linked to poor clinical outcome, these data suggest that cancer patients may benefit from therapy aiming at iNKT cell proliferation and activation. Herein we present results of investigations on the effects of human iNKT cells on Ag-specific CTL responses. iNKT cells were expanded using alphaGC-pulsed allogeneic DC derived from the acute myeloid leukemia cell line MUTZ-3, transduced with CD1d to enhance iNKT cell stimulation, and with IL-12 to stimulate type 1 cytokine production. Enhanced activation and increased IFN-gamma production was observed in iNKT cells, irrespective of CD4 expression, upon stimulation with IL-12-overexpressing dendritic cells. IL-12-stimulated iNKT cells strongly enhanced the MART-1 (melanoma Ag recognized by T cell 1)-specific CD8(+) CTL response, which was dependent on iNKT cell-derived IFN-gamma. Furthermore, autologous IL-12-overexpressing dendritic cells, loaded with Ag as well as alphaGC, was superior in stimulating both iNKT cells and Ag-specific CTL. This study shows that IL-12-overexpressing allogeneic dendritic cells expand IFN-gamma-producing iNKT cells, which may be more effective against tumors in vivo. Furthermore, the efficacy of autologous Ag-loaded DC vaccines may well be enhanced by IL-12 overexpression and loading with alphaGC.     

Leukemia inhibitory factor deficiency modulates the immune response and limits autoimmune demyelination: a new role for neurotrophic cytokines in neuroinflammation

The neurotrophic cytokines ciliary neurotrophic factor and leukemia inhibitory factor (LIF) play a key role in neuronal and oligodendrocyte survival and as protective factors in neuroinflammation. To further elucidate the potential of endogenous LIF in modulating neuroinflammation, we studied myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in LIF knockout mice (LIF(-/-) mice). In the late phase of active myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, LIF(-/-) mice exhibited a markedly milder disease course. The inflammatory infiltrate in LIF(-/-) mice was characterized by an increase in neutrophilic granulocytes early and fewer infiltrating macrophages associated with less demyelination later in the disease. In good correlation with an effect of endogenous LIF on the immune response, we found an Ag-specific T cell-priming defect with impaired IFN-gamma production in LIF(-/-) mice. On the molecular level, the altered recruitment of inflammatory cells is associated with distinct patterns of chemokine production in LIF(-/-) mice with an increase of CXCL1 early and a decrease of CCL2, CCL3, and CXCL10 later in the disease. These data reveal that endogenous LIF is an immunologically active molecule in neuroinflammation. This establishes a link between LIF and the immune system which was not observed in the ciliary neurotrophic factor knockout mouse.