In vivo fate and distribution of poly-gamma-D-glutamic acid, the capsular antigen from Bacillus anthracis.

Bacillus anthracis is surrounded by an antiphagocytic capsule composed of poly-gamma-d-glutamic acid (gammaDPGA). Bacterial and fungal capsular polysaccharides are shed into body fluids in large amounts during infection. The goal of our study was to examine the in vivo fate and distribution of the gammaDPGA capsular polypeptide. Mice were injected via the intravenous route with various amounts of purified gammaDPGA. Blood, urine, and various organs were harvested at different times after treatment. Sites of gammaDPGA accumulation were determined by immunoassay using monoclonal antibodies specific for gammaDPGA. The results showed that the liver and spleen were the primary sites for the accumulation of gammaDPGA. As found in previous studies of capsular polysaccharides, the Kupffer cells of the liver and splenic macrophages were sites for the cellular accumulation of gammaDPGA. Unlike capsular polysaccharides, the hepatic sinusoidal endothelial cells were also sites for gammaDPGA accumulation. gammaDPGA was rapidly cleared from serum and was excreted into the urine. gammaDPGA in the urine showed a reduced molecular size relative to native gammaDPGA. The results indicate that in vivo clearance of the polypeptide capsular antigen of B. anthracis shares several features with the clearance of capsular polysaccharides. Key differences between the in vivo behaviors of gammaDPGA and capsular polysaccharides include the accumulation of gammaDPGA in hepatic sinusoidal endothelial cells and a gammaDPGA clearance rate that was more rapid than the clearance reported for capsular polysaccharides.

Upregulation of profilin, cofilin-2 and LIMK2 in cultured pulmonary artery smooth muscle cells and in pulmonary arteries of monocrotaline-treated rats.

Pulmonary hypertension is associated with remodeling of the smooth muscle layer of pulmonary arteries, manifested by reduced smooth muscle cell (SMC) contractility and enhanced motility and growth. These responses are underlied by increased dynamics of the peripheral actin network. Thus, we hypothesized that pulmonary hypertension is associated with upregulation of two proteins that regulate the dynamics of peripheral actin filaments, i.e., profilin and cofilin. We also analyzed the expression of LIMK2, which regulates the actin remodeling capacity of cofilin by phosphorylation. Experimental inflammation was induced by incubation of cultured pulmonary artery SMCs (PASMCs) with inflammatory mediators in vitro, and by subcutaneous administration of monocrotaline to Sprague-Dawley rats in vivo. Expression of messenger RNA (mRNA) was assessed by quantitative RT-PCR, protein levels and phosphorylation were analyzed by immunoblotting. Immune and Masson trichrome stained lung cryosections were analyzed by microscopy. PDGF, IL-1beta, ET-1 and TNFalpha upregulated the profilin, cofilin-2 and LIMK2 mRNA in cultured pulmonary artery SMCs (PASMCs). Along with the development of rat pulmonary artery and right ventricular hypertrophy, monocrotaline treatment also induced the mRNA and protein contents of profilin, cofilin-2 and LIMK2 in PASMCs. The cofilin upregulation was paralleled by a relative decrease of the phospho-cofilin content. The upregulation of profilin, cofilin and LIMK2 in experimental inflammation suggests that by intensifying the remodeling of subcortical actin filaments these proteins may contribute to the enhanced invasiveness and growth of SMCs, and to the development of increased vascular resistance and pulmonary hypertension.