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Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (Tb), has a complex cell envelope which forms an efficient barrier to antibiotics, thus contributing to the challenges of anti-tuberculosis therapy. However, the unique Mtb cell wall can be considered an advantage and be utilized to selectively label Mtb bacteria. Here we introduce three azido pentoses as new compounds for metabolic labeling of Mtb: 3-azido arabinose (3AraAz), 3-azido ribose (3RiboAz), and 5-azido arabinofuranose (5AraAz). 5AraAz demonstrated the highest level of Mtb labeling and was efficiently incorporated into the Mtb cell wall. All three azido pentoses can be easily used to label a variety of Mtb clinical isolates without influencing Mtb-dependent phagosomal maturation arrest in infection studies with human macrophages. Thus, this metabolic labeling method offers the opportunity to attach desired molecules to the surface of Mtb bacteria in order to facilitate investigation of the varying virulence characteristics of different Mtb clinical isolates, which influence the outcome of a Tb infection.

Plant pollen are an important source of antigens that evoke allergic responses. Protein antigens have been the focus of studies aiming to elucidate the mechanisms responsible for allergic reactions to pollen. However, proteins are not the sole active agent present in pollen. It is known that pollen grains contain lipids essential for its reproduction and bioactive lipid mediators. These small molecular compounds are co-delivered with the allergens and hence have the potential to modulate the immune response of subjects by activating their innate immune cells. Previous reports showed that pollen associated lipid mediators exhibited neutrophil- and eosinophil-chemotactic activity and induced polarization of dendritic cells (DCs) toward a Th2-inducing phenotype. In our study we performed chemical analyses of the pollen associated lipids, that are rapidly released upon hydration. As main components we have identified different types of phytoprostanes (PhytoPs), and for the first time phytofurans (PhytoFs), with predominating 16-F 1t -PhytoPs (PPF 1 -I), 9-F 1t -PhytoPs (PPF 1 -II), 16-E 1t -PhytoPs (PPE 1 -I) and 9-D 1t -PhytoPs (PPE 1 -II), and 16( RS )-9- epi -ST-Δ 14 -10-PhytoFs. Interestingly 16-E 1t -PhytoP and 9-D 1t -PhytoPs were found to be bound to glycerol. Lipid-containing samples (aqueous pollen extract, APE) induced murine mast cell chemotaxis and IL-6 release, and enhanced their IgE-dependent degranulation, demonstrating a role for these lipids in the immediate effector phase of allergic inflammation. Noteworthy, mast cell degranulation seems to be dependent on glycerol-bound, but not free phytoprostanes. On murine dendritic cells, APE selectively induced the upregulation of CD1d, likely preparing lipid-antigen presentation to iNKT cells. Our report contributes to the understanding of the activity of lipid mediators in the immediate effector phase of allergic reactions but identifies a yet undescribed pathway for the recognition of pollen-derived glycolipids by iNKT cells.

Changes in microbial biomass and activity were determined in a sandy-loam soil treated with successive dosages of oxytetracycline (a bactericide) or captan (a fungicide) throughout 98 days of incubation under laboratory conditions. The numbers of culturable bacteria and fungi, total bacterial and fungal biomass (as amounts of phospholipid fatty acids, PLFA), the fungal/bacterial ratio, activities of acid and alkaline phosphatases and urease as well as concentrations of N-NH 4 + and N-NO 3 -were assessed. Both oxytetracycline and captan significantly decreased numbers of culturable bacteria whereas total bacterial biomass (bactPLFA) was not affected. Oxytetracycline did not effect on the fungal biomass, however their numbers were reduced after the first and second time of soil amendment with the bactericide. Conversely, fungal numbers and biomass (PLFA 18:2x6,9) significantly decreased in response to soil treatment with the fungicide. Compared to oxytetracycline, captan significantly decreased activities of acid and alkaline phosphatases. For urease activity, the decreased activity was only observed in the soil after the third dosage of captan. Both biocides significantly increased concentrations of N-NH 4 + and decreased concentrations of N-NO 3 -after the soil treatments. The results of this study indicate that successive soil treatment with oxytetracycline or captan dosages may negatively affect non-target soil microorganisms and their activities.

M embers of the mycobacterial genus are renowned for their waxy, lipid-rich outer envelope. Under physiological conditions, this outer layer is likely to be the first point of contact between the bacterial cell and the host's immune system, and the outcome of this interaction is pivotal in the establishment of infection. One of the most important groups of membrane bound lipids consists of the phosphatidylinositol mannosides (PIMs). Interest in PIMs was stimulated since it was shown that phosphatidylinositol dimannoside (PIM 2 ) forms the phosphoglycolipid anchor which tethers a large array of glycolipids and lipoglycans, including lipomannan (LM) and lipoarabinomannan (LAM), to the cellular membrane (1). PIMs have been shown to interact with a variety of immune components and mediate significant effects on the host. Ever since the realization that NKT cells could respond to lipid antigens (2-4) and the subsequent discovery of the CD1d-restricted lipid antigen ␣-galactosylceramide (␣-GalCer) (5, 6), much research effort has been concentrated on understanding lipid antigens. Although work was initially focused on invariant NKT cells, it has since been shown that great diversity exists in the lipid-responsive T cell receptor (TCR) repertoire (7-9) and that these diverse NKT cells contribute to the Th1/Th2 balance (7, 10). It has been shown that CD1d-restricted NKT cells are capable of recognizing a variety of lipid antigens, including phospholipids (11). More recently, a CD1b-restricted subset of T cells has been found (12). Similarly to CD1d-restricted invariant NKT (iNKT) cells, the CD1b-restricted variant cells require CD1B for their development and produce proinflammatory cytokines in response to CD1b-expressing dendritic cells (DCs) (12). It is clear that NKT-like cells have a significant role to play in lipid-mediated responses, and the hunt for their antigens has continued (13-15).Given the ability of lipid molecules to generate responses in peripheral blood mononuclear cells (PBMC), we decided to assess the ability of individual, highly purified natural PIMs to activate bovine lymphocytes. In this study, we developed a novel extraction method which allowed us to extract and highly purify a variety of PIM molecules from virulent Mycobacterium tuberculosis H37Rv. The ability of these molecules to induce lymphocyte responses in Mycobacterium bovis-infected cattle was investigated by measuring lymphocyte proliferation and gamma interferon (IFN-␥) production. Furthermore, flow cytometry techniques were utilized to characterize responding cell populations. MATERIALS AND METHODS Extraction of PIMs.Using the novel methodology outlined below, highly pure phosphatidylinositol dimannoside (PIM 2 ), acylphosphatidylinositol dimannoside (AcPIM 2 ), diacyl-phosphatidylinositol dimannoside (Ac 2 PIM 2 ), acylphosphatidylinositol hexamannoside (AcPIM 6 ), and diacylphosphatidylinositol hexamannoside (Ac 2 PIM 6 ) were successfully isolated. Individual PIM molecules were analyzed by electrospray ionization mass spectrometr...

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