Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major

ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1β) release; otherwise, transforming growth factor-beta (TGF-β) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an appropriate molecular template for the construction of an efficient anti-infective agent.     

Isolation of macrocyclic and non-macrocyclic trichothecenes (stachybotrys and fusarium toxins) from the Environment of 200 III Sport Horses

Satratoxins H and G, verrucarin J, and roridin E were isolated from the bedding straw of 200 sport horses exhibiting typical symptoms of stachybotryo-toxicosis. At the same time, the oat feed consumed by the horses contained non-macrocyclicFusarium trichothecenes: T-2 toxin and diacetoxyscirpenol.     

Heymann antibodies induce complement-dependent injury of rat glomerular visceral epithelial cells

The aim of this study was to investigate the in vitro role of the complement membrane attack complex (MAC) in the injury induced by nephritogenic anti-brush border vesicle (Fx1A) antibodies on rat glomerular visceral epithelial cells (GEC). Both sheep and rabbit anti-rat brush border vesicle IgG-induced complement-dependent lysis of cultured GEC. Fab fragments of sheep anti-rat brush border vesicles and polyclonal or monoclonal gp330 IgG were devoid of lytic activity. Shedding of cell-surface antigens induced by sheep or rabbit anti-rat brush border vesicle IgG protected GEC from subsequent exposure to lytic antibodies and complement, an effect that was not obtained with Fab fragments. When GEC were incubated with sheep or rabbit anti-rat brush border vesicle IgG in capping conditions, the C3 component was co-redistributed with Heymann immune complexes; in contrast, the MAC remained diffusely bound to the cell surface, indicating that it was not associated with the antigen-antibody complexes. The MAC was demonstrated on the surface of GEC by immunofluorescence staining with anti-MAC neoantigen and by electron microscopy of negatively stained membranes showing focal clusters of 110 A MAC lesions. When GEC were treated with sheep IgG or rabbit IgG plus C6-deficient sera, the cells were not lysed and MAC was not demonstrable on the surface; however, lytic activity was restored when C6-deficient sera were reconstituted with purified C6. The results are consistent with the interpretation that injury induced by Heymann antibodies on GEC is MAC-dependent.     

Isolation ofBacillus schlegelii,a thermophilic,hydrogen oxidizing,aerobic autotroph,from geothermal and nongeothermal environments

Thermophilic hydrogen-oxidizing strains forming round, terminal endospores were isolated from geothermal areas. They were neutrophilic and facultatively autotrophic. They resembledBacillus schlegelii, a thermophilic hydrogen bacterium found so far only in cold environments. Phenotypic similarities, as well as DNA G+C content and DNA:DNA homologies, clearly revealed that the isolated strains belonged to the taxospeciesB. schlegelii. Hence, the strains ofB. schlegelii found in cold environments are probably allochthonous, their origin being geothermal and volcanic areas.     

Cytology of induced systemic resistance of cucumber to Colletotrichum lagenarium

The infection of cucumber leaves by Colletotrichum lagenarium was studied using cytological methods. Its progress in untreated plants was compared with that in plants in which systemic resistance had been induced by pre-infecting the first true leaf with the same fungus. In induced plants, a reduction of fungal development was observed at the leaf surface, in the epidermis, and in the mesophyll. On the leaf surface, formation of appressoria was slightly reduced. In the epidermis, enhanced formation of papillae beneath appressoria, and possibly increased lignification of entire cells, correlated with reduced development of infection hyphae. Papillae contained callose, identified by staining with aniline-blue fluorochrome and digestion with -1,3-glucanase, as a main structural component. In the mesophyll, reduced fungal development provided evidence for the existence of an additional induced defence reaction. The results imply that preinfection elicited a systemic, multicomponent defence reaction of the host plant against the fungus.Dedicated to the memory of Professor H. Grisebach     

Covalent immobilization of avidin on glassy carbon electrodes as the basis for multivalent biosensors.

One of the most crucial steps for the successful construction of a biosensor is the appropriate and reproducible coupling of the biological part (e.g. enzyme, antibody) to the inorganic moiety of the device (e.g. electrode, microchip). In this paper three methods of immobilization of avidin to a glassy carbon electrode are described. Depending on the type of immobilization, avidin may lose its biological activity as determined by an enzyme immunoassay, using biotinylated reagents. If avidin is covalently bound to the glassy carbon electrode via the bridge molecule 4.4'-diaminodiphenylamine, the biological activity is retained. About 1.5 pmol of avidin can be bound to the electrode (3 mm in diameter), resulting in a nearly complete monolayer of protein.     

Evidence for the presence of muscarinic acetylcholine receptors in bovine brain coated vesicles

Evidence obtained from quinuclidinylbenzilate binding determinations suggested that muscarinic acetylcholine receptor molecules are present in purified bovine brain coated vesicles. Immunoprecipitates formed from coated vesicles with polyclonal antibodies to clathrin bound on the surface of fixed Staphylococcus aureus cells also showed quinuclidinylbenzilate binding activity. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Muscarinic receptor sites for quinuclidinylbenzilate binding to coated vesicles displayed a Kd of 25 pM and a Bmax of about 191 fmol/mg of protein. Binding competition experiments using atropine, N-methylscopolamine, oxotremorine, and carbamylcholine confirmed the typical muscarinic nature of the binding site. Ranking order of potency for the receptors was: atropine greater than N-methylscopolamine greater than oxotremorine greater than carbachol Analysis of data using a two-site model revealed 13% high-affinity sites for oxotremorine, 66% high-affinity sites for carbachol, and 62% for the antagonist N-methylscopolamine. Heterogeneity of binding affinities for muscarinic drugs detected in the coated vesicles may be related to the presence of coated vesicle subpopulations in brain tissue, (Kohtz, D. S., Kohtz, J. D., Schook, W. J., and Puszkin, S. (1985) J. Cell Biol. 101, 48a; Pfeffer, S. R., and Kelly, R. B. (1985) Cell 40, 949-957).