Effects of endogenous D-alanine synthesis and autoinhibition of Bacillus anthracis germination on in vitro and in vivo infections

Bacillus anthracis transitions from a dormant spore to a vegetative bacillus through a series of structural and biochemical changes collectively referred to as germination. The timing of germination is important during early steps in infection and may determine if B. anthracis survives or succumbs to responsive macrophages. In the current study experiments determined the contribution of endogenous D-alanine production to the efficiency and timing of B. anthracis spore germination under in vitro and in vivo conditions. Racemase-mediated production of endogenous D-alanine by B. anthracis altered the kinetics for initiation of germination over a range of spore densities and exhibited a threshold effect wherein small changes in spore number resulted in major changes in germination efficiency. This threshold effect correlated with D-alanine production, was prevented by an alanine racemase inhibitor, and required L-alanine. Interestingly, endogenous production of inhibitory levels of D-alanine was detected under experimental conditions that did not support germination and in a germination-deficient mutant of B. anthracis. Racemase-dependent production of D-alanine enhanced survival of B. anthracis during interaction with murine macrophages, suggesting a role for inhibition of germination during interaction with these cells. Finally, in vivo experiments revealed an approximately twofold decrease in the 50% lethal dose of B. anthracis spores administered in the presence of D-alanine, indicating that rates of germination may be directly influenced by the levels of this amino acid during early stages of disease.     

Protective and immunochemical activities of monoclonal antibodies reactive with the Bacillus anthracis polypeptide capsule

Bacillus anthracis is surrounded by a polypeptide capsule composed of poly-gamma-d-glutamic acid (gammaDPGA). In a previous study, we reported that a monoclonal antibody (MAb F26G3) reactive with the capsular polypeptide is protective in a murine model of pulmonary anthrax. The present study examined a library of six MAbs generated from mice immunized with gammaDPGA. Evaluation of MAb binding to the capsule by a capsular "quellung" type reaction showed a striking diversity in capsular effects. Most MAbs produced a rim type reaction that was characterized by a sharp increase followed directly by a decrease in refractive index at the capsular edge. Some MAbs produced a second capsular reaction well beneath the capsular edge, suggesting complexity in capsular architecture. Binding of MAbs to soluble gammaDPGA was assessed by a fluorescence perturbation assay in which a change in the MAb intrinsic fluorescence produced by ligand binding was used as a reporter for antigen-antibody interaction. The MAbs differed considerably in the complexity of the binding curves. MAbs producing rim type capsule reactions typically produced the more complex binding isotherms. Finally, the protective activity of the MAbs was compared in a murine model of pulmonary anthrax. One MAb was markedly less protective than the remaining five MAbs. Characteristics of the more protective MAbs included a relatively high affinity, an immunoglobulin G3 isotype, and a complex binding isotherm in the fluorescence perturbation assay. Given the relatively monotonous structure of gammaDPGA, the results demonstrate a striking diversity in the antigen binding behavior of gammaDPGA antibodies.     

Influence of Nicotine on Myocardial Stiffness and Fibrosis During Chronic Ethanol Use

Cardiomyopathy related to ethanol abuse is often accompanied by cigarette use. To examine if the major cardioactive component may intensify the abnormal function and composition induced by chronic ethanol, nicotine was administered orally, 2.5 mg bid, to a canine model receiving 36% of calories as ethanol for 6 months (group III). These animals were compared with group II receiving ethanol alone, group IV on nicotine alone, and controls (group I). In the intact, ventilated, anesthetized dog, left ventricular pressures and volumes were measured before and after dextran infusion and related to left ventricular collagen alterations. Basal heart rate, aortic pressure, and ejection fraction were comparable with controls. End-diastolic pressure and diastolic chamber stiffness (KPV) were significantly higher in the basal state and during dextran infusion in the three experimental groups, compared with group I. The increment was largest in the ethanol-nicotine group. Analysis of left ventricular myocardium revealed a rise of collagen concentrations in all three experimental groups, with an interstitial distribution on histochemical examination. Moreover, determination of advanced glycosylation endproducts, as a measure of alterations in collagen cross-links, revealed higher concentrations versus controls. The greater increase of diastolic stiffness in the nicotine-ethanol group occurred despite a similar concentration of fluorescent products as group II. Because the former had a larger increase of collagen concentration, total cross-linked collagen content was presumably greater after the combined use of nicotine-ethanol. Thus, nicotine in relatively high dose when combined with ethanol, elicited a modest further increase in the left ventricular chamber stiffness and collagen concentration.     

Signalling lymphocyte activation molecule family member 9 is found on select subsets of antigen-presenting cells and promotes resistance to Salmonella infection

Signalling lymphocyte activation molecule family member 9 (SLAMF9) is an orphan receptor of the CD2/SLAM family of leucocyte surface proteins. Examination of SLAMF9 expression and function indicates that SLAMF9 promotes inflammation by specialized subsets of antigen-presenting cells. Within healthy liver and circulating mouse peripheral blood mononuclear cells, SLAMF9 is expressed on CD11b⁺, Ly6C⁻, CD11c^low, F4/80^low, MHC-II⁺, CX₃CR1⁺ mononuclear phagocytes as well as plasmacytoid dendritic cells. In addition, SLAMF9 can be found on peritoneal B1 cells and small (F4/80^low), but not large (F4/80^high), peritoneal macrophages. Upon systemic challenge with Salmonella enterica Typhimurium, Slamf9^−/− mice were impaired in their ability to clear the infection from the liver. In humans, SLAMF9 is up-regulated upon differentiation of monocytes into macrophages, and lipopolysaccharide stimulation of PMA-differentiated, SLAMF9 knockdown THP-1 cells showed an essential role of SLAMF9 in production of granulocyte–macrophage colony-stimulating factor, tumour necrosis factor-α, and interleukin-1β. Taken together, these data implicate SLAMF9 in the initiation of inflammation and clearance of bacterial infection.     

Receptor-mediated calcium signalling in glial cells from mouse corpus callosum slices

We addressed the question of whether glial cells in intact white matter tracts express neurotransmitter receptors and we used Ca⁺⁺ signalling as a probe to detect the receptor activation. Corpus callosum slices from postnatal mice were bulk-loaded with the Ca⁺⁺- sensitive fluorescent dye fluo-3, and confocal microscopy was used to measure Ca⁺⁺ transients in response to neuroligands. Glial cell bodies were intensely dye-loaded and could be discriminated from the diffuse fluorescence of axons. Subpopulations of glial cells from slices obtained at postnatal days 3 to 7 responded with Ca⁺⁺ signals to ATP, glutamate, histamine, GABA, norepinephrine, serotonin, angiotensin II, bradykinin, and substance P. These subpopulations showed a distinct overlap; cells which were responsive to substance P always showed Ca⁺⁺ signalling in response to histamine, ATP, GABA, and high K⁺ (membrane depolarization). GABA-responsive cells almost always showed a [Ca⁺⁺], increase after membrane depolarization. In brain slices from postnatal day 11 to 18 animals, the Ca⁺⁺ responses were evident for glutamate, ATP, and norepinephrine, while GABA, substance P, serotonin, histamine, or angiotensin II rarely elicited a response. This study demonstrates that white matter glial cells in slices exhibit a large repertoire of neurotransmitter responses linked to Ca⁺⁺ signalling and that these receptor systems are differentially distributed on sub-populations of glial cells. © 1996 Wiley-Liss, Inc.