Ewing sarcoma-primitive neuroectodermal tumor (EWS) is associated with the most unfavorable prognosis of all primary musculoskeletal tumors. The objective of the present study was to investigate whether tumor-associated macrophages (TAMs) affect the development of EWS. TAMs were isolated from mouse xenografts using CD11b magnetic beads and examined for their cytokine expression and osteoclastic differentiation. To evaluate the role of TAMs in xenograft formation, liposome-encapsulated clodronate was used to deplete TAMs in mice. Macrophage infiltration and tumor microvascular density were histologically evaluated in 41 patients with EWS, and association with prognosis was examined using Kaplan-Meier survival analysis. In mouse EWS xenografts, TAMs expressed higher concentrations of cytokines including interleukin-6, keratinocyte-derived chemokine, and monocyte chemotactic protein-1. TAMs were more capable than normal monocytes of differentiating into tartrate-resistant acid phosphatase-positive giant cells. Depleting macrophages using liposome-encapsulated clodronate significantly inhibited development of EWS xenografts. In human EWS samples, higher levels of CD68-positive macrophages were associated with poorer overall survival. In addition, enhanced vascularity, increase in the amount of C-reactive protein, and higher white blood cell counts were also associated with poor prognosis and macrophage infiltration. TAMs seem to enhance the progression of EWS by stimulating both angiogenesis and osteoclastogenesis. Further investigation of the behavior of TAMs may lead to development of biologically targeted therapies for EWS.

Streptococcus pyogenes strains can be divided into two classes, one capable and the other incapable of producing H 2 O 2 (M. Saito, S. Ohga, M. Endoh, H. Nakayama, Y. Mizunoe, T. Hara, and S. Yoshida, Microbiology 147: [2469][2470][2471][2472][2473][2474][2475][2476][2477] 2001). In the present study, this dichotomy was shown to parallel the presence or absence of H 2 O 2 -producing lactate oxidase activity in permeabilized cells. Both lactate oxidase activity and H 2 O 2 production under aerobic conditions were detectable only after glucose in the medium was exhausted. Thus, the glucose-repressible lactate oxidase is likely responsible for H 2 O 2 production in S. pyogenes. Of the other two potential H 2 O 2 -producing enzymes of this bacterium, NADH and ␣-glycerophosphate oxidase, only the former exhibited low but significant activity in either class of strains. This activity was independent of the growth phase, suggesting that the protein may serve in vivo as a subunit of the H 2 O 2 -scavenging enzyme NAD(P)H-linked alkylhydroperoxide reductase. The activity of lactate oxidase was associated with the membrane while that of NADH oxidase was in the soluble fraction, findings consistent with their respective physiological roles, i.e., the production and scavenging of H 2 O 2 . Analyses of fermentation end products revealed that the concentration of lactate initially increased with time and decreased on glucose exhaustion, while that of acetate increased during the culture. These results suggest that the lactate oxidase activity of H 2 O 2 -producing cells oxidizes lactate to pyruvate, which is in turn converted to acetate. This latter process proceeds presumably via acetyl coenzyme A and acetyl phosphate with formation of extra ATP.The gram-positive microorganism Streptococcus pyogenes is the causative agent of a variety of important human diseases. These include not only the direct consequences of infections such as pharyngitis, impetigo, cellulitis, necrotizing fasciitis, and toxic-shock-like syndrome but also secondary pathologies called poststreptococcal sequelae. The physiological features of S. pyogenes place it as a member of lactic acid bacteria, which are generally believed to be almost totally dependent for growth on the lactic mode of fermentation under both aerobic and anaerobic conditions.In a number of species of lactic acid bacteria, it has been known that cells growing aerobically produce and excrete copious amounts of H 2 O 2. In a previous study, it was found that S. pyogenes strains could be divided into two classes with respect to the production of H 2 O 2 , i.e., producers and nonproducers (18). However, the metabolic basis and biological significance of bacterial H 2 O 2 production are largely unexplored. The presence of H 2 O 2 -producing oxidases in lactic acid bacteria has been demonstrated, and the possibility of their involvement in this phenomenon has been suggested (6,14,15,21). Notably, a homology search against the S. pyogenes genome database revealed that putative H 2 O 2 -produc...

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Ethidium monoazide (EMA) is a DNA intercalating agent and a eukaryotic topoisomerase II poison. We found that EMA treatment and subsequent visible light irradiation (photoactivation or photolysis) shows a bactericidal effect, hence the mechanism was analyzed. When bacterial cells were treated with more than 10 µ µg/ml of EMA for 1 hr plus photoactivation for 20 min, cleavage of bacterial DNA was confirmed by agarose gel electrophoresis and electron microscopic studies. The cleavage of chromosomal DNA was seen when it was treated in vitro with EMA and photolysis, which showed that the cleavage directly took place without the assistance of DNA gyrase/topoisomerase IV and the DNA repair enzymes of bacteria. It was also verified, by using negatively supercoiled pBR322 DNA, that medium/high concentrations of EMA (1 to 100 µ µg/ml) led to breaks of double-stranded DNA and that low concentrations of EMA (10 to 100 ng/ml) generated a single-stranded break. EMA is known to easily penetrate dead but not live bacteria. After treatment of 10 µ µg/ml of EMA for 30 min and photoactivation for 5 min, EMA cleaved the DNA of dead but not live Klebsiella oxytoca. When the cleaved DNA was used for templates in PCR targeting 16S rDNA, PCR product from the dead bacteria was completely suppressed. We demonstrated that EMA and photolysis directly cleaved bacterial DNA and are effective tools for discriminating live from dead bacteria by PCR.

Streptococcus pneumoniae was shown to possess lactate oxidase in addition to well-documented pyruvate oxidase. The activities of both H 2 O 2 -forming oxidases in wild-type cultures were detectable even in the early exponential phase of growth and attained the highest levels in the early stationary phase. For each of these oxidases, a defective mutant was constructed and compared to the parent regarding the dynamics of pyruvate and lactate in aerobic cultures. The results obtained indicated that the energy-yielding metabolism in the wild type could be best described by the following scheme. (i) As long as glucose is available, approximately one-fourth of the pyruvate formed is converted to acetate by the sequential action of pyruvate oxidase and acetate kinase with acquisition of additional ATP; (ii) the rest of the pyruvate is reduced by lactate dehydrogenase to form lactate, with partial achievement of redox balance; (iii) the lactate is oxidized by lactate oxidase back to pyruvate, which is converted to acetate as described above; and (iv) the sequential reactions mentioned above continue to occur as long as lactate is present. As predicted by this model, exogenously added lactate was shown to increase the final growth yield in the presence of both oxidases.The gram-positive bacterium Streptococcus pneumoniae, also known as pneumococcus, is often a commensal resident of the human upper respiratory tract but also represents an important human pathogen, causing invasive diseases such as pneumonia, otitis media, and meningitis (18,20,22). Recently, a sharp rise in the incidence of drug resistance among clinical isolates of S. pneumoniae has been posing serious problems (13).A member of lactic acid bacteria, S. pneumoniae is aerotolerant but lacks the cytochromes necessary for aerobic respiration. Under anaerobic conditions, it is believed to be totally dependent on homolactic fermentation for the acquisition of energy required for growth, in which glucose is metabolized to pyruvate and then to the final product lactate. Under aerobiosis, however, pyruvate is also known to be converted to acetate, with acetyl phosphate being the intermediate capable of phosphorylating ADP to yield ATP by the action of acetate kinase (30). The H 2 O 2 -forming flavoprotein pyruvate oxidase (EC 1.2.3.3, the product of the spxB gene), which catalyzes the formation of acetyl phosphate, CO 2 and H 2 O 2 from pyruvate, orthophosphate, and O 2 , has been shown to be involved in this pathway and also to account for most of the H 2 O 2 produced by aerobically growing S. pneumoniae cells (23, 30). Massive production of H 2 O 2 has been a well-known hallmark of this bacterium since the time of Oswald Avery (2, 19), usually necessitating the addition of catalase to the culture medium to obtain full growth under aerobic conditions. S. pneumoniae possesses another H 2 O 2 -forming flavoprotein, L-lactate oxidase (the product of the lox gene; formerly EC 1.1.3.2 but now sharing the EC number 1.13.12.4 with lactate monooxigenase) catalyzing the ...