Convergent evolution of diverse Bacillus anthracis outbreak strains toward altered surface oligosaccharides that modulate anthrax pathogenesis.

Bacillus anthracis, a spore-forming gram-positive bacterium, causes anthrax. The external surface of the exosporium is coated with glycosylated proteins. The sugar additions are capped with the unique monosaccharide anthrose. The West African Group (WAG) B. anthracis have mutations rendering them anthrose deficient. Through genome sequencing, we identified 2 different large chromosomal deletions within the anthrose biosynthetic operon of B. anthracis strains from Chile and Poland. In silico analysis identified an anthrose-deficient strain in the anthrax outbreak among European heroin users. Anthrose-deficient strains are no longer restricted to West Africa so the role of anthrose in physiology and pathogenesis was investigated in B. anthracis Sterne. Loss of anthrose delayed spore germination and enhanced sporulation. Spores without anthrose were phagocytized at higher rates than spores with anthrose, indicating that anthrose may serve an antiphagocytic function on the spore surface. The anthrose mutant had half the LD50 and decreased time to death (TTD) of wild type and complement B. anthracis Sterne in the A/J mouse model. Following infection, anthrose mutant bacteria were more abundant in the spleen, indicating enhanced dissemination of Sterne anthrose mutant. At low sample sizes in the A/J mouse model, the mortality of ΔantC-infected mice challenged by intranasal or subcutaneous routes was 20% greater than wild type. Competitive index (CI) studies indicated that spores without anthrose disseminated to organs more extensively than a complemented mutant. Death process modeling using mouse mortality dynamics suggested that larger sample sizes would lead to significantly higher deaths in anthrose-negative infected animals. The model was tested by infecting Galleria mellonella with spores and confirmed the anthrose mutant was significantly more lethal. Vaccination studies in the A/J mouse model showed that the human vaccine protected against high-dose challenges of the nonencapsulated Sterne-based anthrose mutant. This work begins to identify the physiologic and pathogenic consequences of convergent anthrose mutations in B. anthracis.

In vitro immunocompetence of two compounds isolated from Polygala tenuifolia and development of resistance against grass carp reovirus (GCRV) and Dactylogyrus intermedius in respective host.

The present study was undertaken to isolate some compounds from methanol extract of Polygala tenuifolia and evaluate their immunostimulatory properties and antiviral activity using grass carp Ctenopharyngodon idella kidney (CIK) cells and GCRV. By applying insecticidal bioassay-guided, chromatography techniques and successive recrystallization, two purified compounds were obtained. The changes of expression of selected immune genes (Mx1, IL-1ß, TNFα, MyD88 and IgM) in C. idella kidney cell lines were evaluated after exposure to these isolated compounds. The results showed that compound 1 and 2 up-regulated to varying degrees of Mx1, IL-1ß, TNFα, and MyD88 in C. idella kidney cells. WST-8 kit assay verified the two compounds has no toxic effects on CIK cell, and furthermore, have in vitro antivirus activity. Especially, that there is keeping 79% cell viability when exposure to compound 2 (100 mg L(-1)). According to in vivo insecticidal assays against Dactylogyrus intermedius, compound 2 exhibited higher efficacy than compound 1, which was found to be 87.2% effective at the concentrations of 5 mg L(-1) and safe to goldfish (Carassius auratus). Besides, the purified compounds were identified by spectral data as: (1) 1,5-Anhydro-D-glucitol and (2) 3,4,5-trimethoxy cinnamic acid. Overall, the results indicate that bath administration of these compounds modulates the immune related genes in C. idella kidney cells and to some extent, eliminate the virus and parasitic infections.

Anthrax spore detection by a luminex assay based on monoclonal antibodies that recognize anthrose-containing oligosaccharides.

The similarity of endospore surface antigens between bacteria of the Bacillus cereus group complicates the development of selective antibody-based anthrax detection systems. The surface of B. anthracis endospores exposes a tetrasaccharide containing the monosaccharide anthrose. Anti-tetrasaccharide monoclonal antibodies (MAbs) and anti-anthrose-rhamnose disaccharide MAbs were produced and tested for their fine specificities in a direct spore enzyme-linked immunosorbent assay (ELISA) with inactivated spores of a broad spectrum of B. anthracis strains and related species of the Bacillus genus. Although the two sets of MAbs had different fine specificities, all of them recognized the tested B. anthracis strains and showed only a limited cross-reactivity with two B. cereus strains. The MAbs were further tested for their ability to be implemented in a highly sensitive and specific bead-based Luminex assay. This assay detected spores from different B. anthracis strains and two cross-reactive B. cereus strains, correlating with the results obtained in direct spore ELISA. The Luminex assay (detection limit 10(3) to 10(4) spores per ml) was much more sensitive than the corresponding sandwich ELISA. Although not strictly specific for B. anthracis spores, the developed Luminex assay represents a useful first-line screening tool for the detection of B. anthracis spores.

Development of antibodies against anthrose tetrasaccharide for specific detection of Bacillus anthracis spores.

Methods for the immunological detection of Bacillus anthracis in various environmental samples and the discrimination of B. anthracis from other members of the B. cereus group are not yet well established. To generate specific discriminating antibodies, we immunized rabbits, mice, and chickens with inactivated B. anthracis spores and, additionally, immunized rabbits and mice with the tetrasaccharide beta-Ant-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-L-Rhap. It is a constituent of the exosporium glycoprotein BclA and contains the newly discovered sugar anthrose 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-beta-D-glucose. The BclA protein is a major component of the exosporium of B. anthracis spores and is decorated by the tetrasaccharide indicated above. The anthrose-containing tetrasaccharide chain seems to be highly specific for B. anthracis, which makes it a key biomarker for the detection of these spores. The different immunizations led to anthrose-reactive polyclonal and monoclonal antibodies which were analyzed by various methods to characterize their ability to discriminate between B. anthracis and other Bacillus spp. Multiple applications, such as enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and electron microscopy, revealed the specificities of the polyclonal and monoclonal antibodies generated for B. anthracis spore detection. All polyclonal antibodies were able to correctly identify the B. anthracis strains tested and showed only minimal cross-reactivities with other Bacillus strains. Moreover, the antibodies generated proved functional in a new capture assay for B. anthracis spores and could therefore be useful for the detection of spores in complex samples.

Immuno-detection of anthrose containing tetrasaccharide in the exosporium of Bacillus anthracis and Bacillus cereus strains.

AIMS: Bacillus anthracis strains of various origins were analysed with the view to describe intrinsic and persistent structural components of the Bacillus collagen-like protein of anthracis glycoprotein associated anthrose containing tetrasaccharide in the exosporium. METHODS AND RESULTS: The tetrasaccharide consists of three rhamnose residues and an unique monosaccharide--anthrose. As anthrose was not found in spores of related strains of bacteria, we envisioned the detection of B. anthracis spores based on antibodies against anthrose-containing polysaccharides. Carbohydrate-protein conjugates containing the synthetic tetrasaccharide, an anthrose-rhamnose disaccharide or anthrose alone were employed to immunize mice. All three formulations were immunogenic and elicited IgG responses with different fine specificities. All sera and monoclonal antibodies derived from tetrasaccharide immunized mice cross-reacted not only with spore lysates of a panel of virulent B. anthracis strains, but also with some of the B. cereus strains tested. CONCLUSIONS: Our results demonstrate that antibodies to synthetic carbohydrates are useful tools for epitope analyses of complex carbohydrate antigens and for the detection of particular target structures in biological specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: Although not strictly specific for B. anthracis spores, antibodies against the tetrasaccharide may have potential as immuno-capturing components for a highly sensitive spore detection system.

Pro-atherogenic alterations in T-lymphocyte subpopulations related to acute hyperglycaemia in type 2 diabetic patients.

BACKGROUND: T cells are among the earliest cells to infiltrate the arterial intima during the initial stages of atherosclerosis. Alterations in the peripheral blood lymphocyte distribution might be associated with intensive lymphocytes extravasation and stimulation of atherosclerotic plaque development. Epidemiological data reveal that short-term postprandial hyperglycemia is a significant risk factor for coronary heart disease. Using a parameter that indicates recently-past acute hyperglycemia, 1,5-anhydro-D-glucitol (1,5-AG), the aim of the present study was to elucidate which alterations in peripheral blood T-lymphocytes, if any, are associated with acute hyperglycemia in patients with type 2 diabetes mellitus (DM) and, thus, might be involved in the progression of atherosclerosis. METHODS AND RESULTS: Measurement of fasting glucose level, glycated hemoglobin A(1c), 1,5-AG, lipid profile and lymphocyte receptors expression (CD3+, CD4+, CD8+, CD8+28+, CD+28 -) was performed in 97 patients with type 2 DM, 23 patients with coronary heart disease, and 15 healthy controls. The mean CD3+, CD4+, CD8+28 - and CD8+28+ lymphocyte counts were significantly higher in the DM patients than in both control groups. Multiple regression analysis revealed that CD4+ and CD8+28- lymphocyte counts primarily were dependent on 1,5-anhydro-D-glucitol plasma levels. CONCLUSIONS: These results suggest that acute hyperglycemia results in the progression of atherosclerosis in type 2 DM, at least in part through changes in CD4+ and CD8+28- lymphocyte subsets.

Immune alterations in three mouse strains following 2-deoxy-D-glucose administration.

Using 2-deoxy-D-glucose (2-DG)-induced stress, our laboratory has developed studies to define stress effects on immune responses. Here, we report effects of increasing doses of 2-DG on the immune response of BALB/c, C57BL/6 and BDF(1) mice 2 h after three injections of 0 to 2000 mg/kg of 2-DG. Female 4- to 5-week-old mice were euthanized and blood and spleens were collected. A suspension of partially purified mature T splenocytes was obtained by negative selection using J11.d2 antibodies. Glucose and corticosterone levels were measured in the plasma of each mouse. Splenocyte and mature T splenocyte suspensions were tested in in vitro proliferation assays with or without concanavalin A. Splenocytes were analyzed for the following cell-surface markers: CD3, TCR alpha/beta, CD4, CD8 and major histocompatibility complex (MHC) Class II. Significant increases in blood glucose levels were observed in C57BL/6 and BALB/c strains with the highest 2-DG dose (p<0.05). Corticosterone levels were higher in BDF(1) mice and C57BL/6 mice following the administration of 1000 and 2000 mg/kg of 2-DG, respectively (p<0.01). In vitro proliferation of mature T splenocytes in the presence of concanavalin A was decreased in BDF(1) (p<0.05) but not in BALB/c and C57BL/6 mice. In addition, in BDF(1) mice the decrease was highly correlated with an increase of CD3+ and TCR alpha/beta+ cells in the spleen. These results demonstrated high variability in the response of different mouse strains to 2-DG-induced stress.

Neuropeptide Y antibody attenuates 2-deoxy-D-glucose induced feeding in rats.

2-deoxy-D-glucose (2-DG) has been shown to induce increased feeding responses in animals. Recent studies suggest the possible involvement of neuropeptide Y (NPY) in 2-DG-induced feeding. The present study examined the effect of immunoneutralization of endogenous NPY on 2-DG-induced feeding. NPY antibody injected into the paraventricular nucleus of the rats significantly attenuated 2-DG-induced feeding, suggesting that hypothalamic NPY may mediate, at least partly, the effect of 2-DG on food intake.

[Migration and adhesiveness of human and rabbit blood leukocytes under the influence of metabolic blocking agents and hirudin]. / Untersuchungen über Migration und Haftung von Blutleukozyten des Menschen und Kaninchens bei Einwirkung von Stoffwechsel-blockern und von Hirudin.

The adherence inhibition by lymphokines and by hirudin do not correspond to another, in the first case it is connected with a migration inhibition, the second with a slight increase of migration. There are significant differences between granulocytes and lymphocytes in relation to adherence and migration caused by the influence of inhibitors on the metabolism. The possible reason is the different basic metabolism of these two kinds of cells. But the two inhibitors of glycolysis NaF and monoiodoacetic acid acts in a similar way as inhibitors on the migration of granulocytes and lymphocytes. The negative influence of hirudin on the adherence of lymphocytes and the promoting effect in the migration experiments account for participation of components of the coagulation system in both cell functions. Possibly mechanisms of the attachment are directly concerned. From the wide conformity of the experiments with metabolism inhibitors and reactions of cell-mediated immunity under influence of lymphokines we like to conclude that lymphokine-caused migration inhibition and adherence inhibition were produced by an impairement of metabolism.

Antigen-enhanced glucosamine incorporation by peritoneal macrophages in cell-mediated hypersensitivity. I. Studies on biology and mechanism.

The interaction between sensitized lymphocytes and specific antigen occurring in classic delayed hypersensitivity causes guinea pig peritoneal macrophages to incorporate increased amounts of glucosamine into TCA precipitable, membrane-associated, cell surface material. Antigen-induced stimulation of glucosamine also occurred in peritoneal exudate cells (PEC) isolated from animals primed for cutaneous basophil hypersensitivity with certain strong antigens (KLH, vaccinia virus) in incomplete Freund's adjuvant (IFA), and lymphocytes from such animals elaborated MIF when cultured with specific antigen. Thus, the use of complete Freund's adjuvant is not obligatory for the induction of sensitized lymphocytes capable of secreting MIF or stimulating macrophage glucosamine incorporation; however, the potency of the immunogen employed is a critical variable since lymphocytes from animals primed with weaker antigens (HSA, BGG) in IFA did not have these capabilities. Significantly enhanced incorporation of radioactive glucosamine by macrophages occurred when normal PEC were cultured in lymphokine-containing supernatants, but the magnitude of incorporation was smaller than that of sensitized PEC stimulated by antigen. The final 24 hr of macrophage culture was critically important because lymphokines were equally effective in promoting glucosamine incorporation when present for only this interval. The kinetics of this response are thus very similar to those reported for macrophage "activation". The mechanism by which sensitized lymphocytes and their products stimulate glucosamine incorporation is not established, but at least part of the increment may be attributed to enhanced transport of glucosamine across the macrophage plasma membrane. The plant lectins Con A and PHA stimulated unsensitized plastic-adherent cells to increased glucosamine in corporation and exerted a further additive stimulation on sensitized PEC when nonadherent sensitized lymphocytes were present. It is likely that these mitogens stimulate glucosamine incorporation by two distinct mechanisms, one involving sensitized nonadherent lymphocytes and a second involving only adherent cells (macrophages and/or plastic adherent lymphocytes.