Neurodegenerative phosphoprotein signaling landscape in models of SCA3.

Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disorder resulting from an aberrant expansion of a polyglutamine stretch in the ataxin-3 protein and subsequent neuronal death. The underlying intracellular signaling pathways are currently unknown. We applied the Reverse-phase Protein MicroArray (RPMA) technology to assess the levels of 50 signaling proteins (in phosphorylated and total forms) using three in vitro and in vivo models expressing expanded ataxin-3: (i) human embryonic kidney (HEK293T) cells stably transfected with human ataxin-3 constructs, (ii) mouse embryonic fibroblasts (MEF) from SCA3 transgenic mice, and (iii) whole brains from SCA3 transgenic mice.

Immune-modulating Activity of Hydrogel Microparticles Contributes to the Host Defense in a Murine Model of Cutaneous Anthrax.

We recently reported that the open-mesh (0.7 μ) polyacrylamide microparticles (MPs) with internally-coupled Cibacron affinity dye demonstrate protective effect in mice challenged into footpads with high doses (200 LD50) of anthrax (Sterne) spores. A single injection of MPs before spore challenge reduces inflammatory response, delays onset of mortality and promotes survival.

Chemokine-Releasing Microparticles Improve Bacterial Clearance and Survival of Anthrax Spore-Challenged Mice.

In this study the hydrogel microparticles (MPs) were used to enhance migration of neutrophils in order to improve outcome of anthrax infection in a mouse model. Two MP formulations were tested. In the first one the polyacrylamide gel MPs were chemically coupled with Cibacron Blue (CB) affinity bait.

Cytotoxic Potential of Bacillus cereus Strains ATCC 11778 and 14579 Against Human Lung Epithelial Cells Under Microaerobic Growth Conditions.

Bacillus cereus, a food poisoning bacterium closely related to Bacillus anthracis, secretes a multitude of virulence factors including enterotoxins, hemolysins, and phospholipases. However, the majority of the in vitro experiments evaluating the cytotoxic potential of B. cereus were carried out in the conditions of aeration, and the impact of the oxygen limitation in conditions encountered by the microbe in natural environment such as gastrointestinal tract remains poorly understood.

Nitric oxide as a regulator of B. anthracis pathogenicity.

Nitric oxide (NO) is a key physiological regulator in eukaryotic and prokaryotic organisms. It can cause a variety of biological effects by reacting with its targets or/and indirectly inducing oxidative stress. NO can also be produced by bacteria including the pathogenic Bacillus anthracis; however, its role in the infectious process only begins to emerge.

Reverse-Phase Microarray Analysis Reveals Novel Targets in Lymph Nodes of Bacillus anthracis Spore-Challenged Mice.

Anthrax is a frequently fatal infection of many animal species and men. The causative agent Bacillus anthracis propagates through the lymphatic system of the infected host; however, the specific interactions of the host and microbe within the lymphatics are incompletely understood. We report the first description of the phosphoprotein signaling in the lymph nodes of DBA/2 mice using a novel technique combining the reverse-phase microarray with the laser capture microdissection.

Chemokine-Releasing Nanoparticles for Manipulation of Lymph Node Microenvironment.

Chemokines (CKs) secreted by the host cells into surrounding tissue establish concentration gradients directing the migration of leukocytes. We propose an CK gradient remodeling approach based on sustained release of CKs by the crosslinked poly(N-isopropylacrylamide) hydrogel open meshwork nano-particles (NPs) containing internal crosslinked dye affinity baits for a reversible CK binding and release. The sustained release is based on a new principle of affinity off-rate tuning.

Whole proteome analysis of mouse lymph nodes in cutaneous anthrax.

This study aimed to characterize a soluble proteome of popliteal lymph nodes during lymphadenitis induced by intradermal injection of Bacillus anthracis Sterne spores in mice using tandem LC-MS/MS and reverse-phase protein microarray with antibodies specific to epitopes of phosphorylated proteins. More than 380 proteins were detected in the normal intra-nodal lymph, while the infectious process resulted in the profound changes in the protein abundances and appearance of 297 unique proteins. These proteins belong to an array of processes reflecting response to wounding, inflammation and perturbations of hemostasis, innate immune response, coagulation and fibrinolysis, regulation of body fluid levels and vascular disturbance among others.

Bacillus anthracis co-opts nitric oxide and host serum albumin for pathogenicity in hypoxic conditions.

Bacillus anthracis is a dangerous pathogen of humans and many animal species. Its virulence has been mainly attributed to the production of Lethal and Edema toxins as well as the antiphagocytic capsule. Recent data indicate that the nitric oxide (NO) synthase (baNOS) plays an important pathogenic role at the early stage of disease by protecting bacteria from the host reactive species and S-nytrosylating the mitochondrial proteins in macrophages.

Bacillus anthracis-derived nitric oxide induces protein S-nitrosylation contributing to macrophage death.

Bacillus anthracis, a causative agent of anthrax, is able to germinate and survive within macrophages. A recent study suggested that B. anthracis-derived nitric oxide (bNO) is a key aspect of bacterial defense that protects bacterial DNA from oxidative burst in the macrophages.

Platelets, inflammatory cells, von Willebrand factor, syndecan-1, fibrin, fibronectin, and bacteria co-localize in the liver thrombi of Bacillus anthracis-infected mice.

Unlabelled: Vascular dysfunction and thrombosis have been described in association with anthrax infection in humans and animals but the mechanisms of these dysfunctions, as well as the components involved in thrombi formation are poorly understood. Immunofluorescent microscopy was used to define the composition of thrombi in the liver of mice challenged with the Bacillus anthracis Sterne spores. Lethal infection with the toxigenic Sterne strain, in contrast to the non-lethal, non-toxigenic delta-Sterne strain, demonstrated time-dependent increase in the number of vegetative bacteria inside the liver sinusoids and central vein.

Targeting the inflammasome and adenosine type-3 receptors improves outcome of antibiotic therapy in murine anthrax.

Aim: To establish whether activation of adenosine type-3 receptors (A3Rs) and inhibition of interleukin-1β-induced inflammation is beneficial in combination with antibiotic therapy to increase survival of mice challenged with anthrax spores.

Methods: DBA/2 mice were challenged with Bacillus anthracis spores of the toxigenic Sterne strain 43F2. Survival of animals was monitored for 15 d.

Bacillus anthracis interacts with plasmin(ogen) to evade C3b-dependent innate immunity.

The causative agent of anthrax, Bacillus anthracis, is capable of circumventing the humoral and innate immune defense of the host and modulating the blood chemistry in circulation to initiate a productive infection. It has been shown that the pathogen employs a number of strategies against immune cells using secreted pathogenic factors such as toxins. However, interference of B.

Bacillus anthracis protease InhA increases blood-brain barrier permeability and contributes to cerebral hemorrhages.

Hemorrhagic meningitis is a fatal complication of anthrax, but its pathogenesis remains poorly understood. The present study examined the role of B. anthracis-secreted metalloprotease InhA on monolayer integrity and permeability of human brain microvasculature endothelial cells (HBMECs) which constitute the blood-brain barrier (BBB).

Reverse-phase phosphoproteome analysis of signaling pathways induced by Rift valley fever virus in human small airway epithelial cells.

Rift valley fever virus (RVFV) infection is an emerging zoonotic disease endemic in many countries of sub-Saharan Africa and in Egypt. In this study we show that human small airway epithelial cells are highly susceptible to RVFV virulent strain ZH-501 and the attenuated strain MP-12. We used the reverse-phase protein arrays technology to identify phosphoprotein signaling pathways modulated during infection of cultured airway epithelium.

Anthrolysin O and fermentation products mediate the toxicity of Bacillus anthracis to lung epithelial cells under microaerobic conditions.

Bacillus anthracis generates virulence factors such as lethal and edema toxins, capsule, and hemolytic proteins under conditions of reduced oxygenation. Here, we report on the acute cytotoxicity of culture supernatants (Sups) of six nonencapsulated B. anthracis strains grown till the stationary phase under static microaerobic conditions.

Activation of plasminogen activator inhibitor implicates protease InhA in the acute-phase response to Bacillus anthracis infection.

Anthrax is a zoonotic disease caused by Bacillus anthracis. The infection is associated with inflammation and sepsis, but little is known about the acute-phase response during disease and the nature of the bacterial factors causing it. In this study, we examined the levels of the acute-phase proteins (APPs) in comparative experiments using mice challenged with spores and a purified B.

Neutrophil elastase and syndecan shedding contribute to antithrombin depletion in murine anthrax.

Bacillus anthracis infection is associated with severe hemostatic disturbances but their roles and contribution to fatality remain incompletely characterized. We undertook analyses of circulating antithrombin levels during the course of infection using a comparison of lethal and nonlethal murine anthrax models. Plasma samples were obtained from DBA/2 mice challenged intraperitoneally with the spores of either toxigenic B.

Degradation of circulating von Willebrand factor and its regulator ADAMTS13 implicates secreted Bacillus anthracis metalloproteases in anthrax consumptive coagulopathy.

Pathology data from the anthrax animal models show evidence of significant increases in vascular permeability coincident with hemostatic imbalances manifested by thrombocytopenia, transient leucopenia, and aggressive disseminated intravascular coagulation. In this study we hypothesized that anthrax infection modulates the activity of von Willebrand factor (VWF) and its endogenous regulator ADAMTS13, which play important roles in hemostasis and thrombosis, including interaction of endothelial cells with platelets. We previously demonstrated that purified anthrax neutral metalloproteases Npr599 and InhA are capable of cleaving a variety of host structural and regulatory proteins.

Secreted neutral metalloproteases of Bacillus anthracis as candidate pathogenic factors.

To evaluate the pathogenic potential of Bacillus anthracis-secreted proteases distinct from lethal toxin, two neutral zinc metalloproteases were purified to apparent homogeneity from the culture supernatant of a non-virulent delta Ames strain (pXO1-, pXO2-). The first (designated Npr599) is a thermolysin-like enzyme highly homologous to bacillolysins from other Bacillus species. The second (designated InhA) is a homolog of the Bacillus thuringiensis immune inhibitor A.

Acceleration of epithelial cell syndecan-1 shedding by anthrax hemolytic virulence factors.

Background: It has been recently reported that major pathogens Staphylococcus aureus and Pseudomonas aeruginosa accelerate a normal process of cell surface syndecan-1 (Synd1) ectodomain shedding as a mechanism of host damage due to the production of shedding-inducing virulence factors. We tested if acceleration of Synd1 shedding takes place in vitro upon treatment of epithelial cells with B. anthracis hemolysins, as well as in vivo during anthrax infection in mice.

Effective antiprotease-antibiotic treatment of experimental anthrax.

Background: Inhalation anthrax is characterized by a systemic spread of the challenge agent, Bacillus anthracis. It causes severe damage, including multiple hemorrhagic lesions, to host tissues and organs. It is widely believed that anthrax lethal toxin secreted by proliferating bacteria is a major cause of death, however, the pathology of intoxication in experimental animals is drastically different from that found during the infectious process.

Systemic cytokine response in murine anthrax.

Systemic pro-inflammatory cytokine release has been previously implicated as a major death-causing factor in anthrax, however, direct data have been absent. We determined the levels of IL-1 beta, IL-6 and TNF-alpha in serum of mice challenged with virulent (Ames) or attenuated (Sterne) strains of Bacillus anthracis. More than 10-fold increase in the IL-1beta levels was detected in Ames-challenged Balb/c mice, in contrast to more susceptible C57BL/6 mice, which showed no IL-1beta response.

Regulation of expression of the paralogous Mlp family in Borrelia burgdorferi.

The Mlp (multicopy lipoproteins) family is one of many paralogous protein families in Borrelia burgdorferi. To examine the extent to which the 10 members of the Mlp family in B. burgdorferi strain 297 might be differentially regulated, antibodies specific for each of the Mlps were developed and used to analyze the protein expression profiles of individual Mlps when B.