Dual role of TLR2 and myeloid differentiation factor 88 in a mouse model of invasive group B streptococcal disease

Toll-like receptors (TLRs) are involved in pathogen recognition by the innate immune system. Different TLRs and the adaptor molecule myeloid differentiation factor 88 (MyD88) were previously shown to mediate in vitro cell activation induced by group B streptococcus (GBS). The present study examined the potential in vivo roles of TLR2 and MyD88 during infection with GBS. When pups were infected locally with a low bacterial dose, none of the TLR2- or MyD88-deficient mice, but all of the wild-type ones, were able to prevent systemic spread of GBS from the initial focus. Bacterial burden was higher in MyD88- than in TLR2-deficient mice, indicating a more profound defect of host defense in the former animals. In contrast, a high bacterial dose induced high level bacteremia in both mutant and wild-type mice. Under these conditions, however, TLR2 or MyD88 deficiency significantly protected mice from lethality, concomitantly with decreased circulating levels of TNF-alpha and IL-6. Administration of anti-TNF-alpha Abs to wild-type mice could mimic the effects of TLR2 or MyD88 deficiency and was detrimental in the low dose model, but protective in the high dose model. In conclusion, these data highlight a dual role of TLR2 and MyD88 in the host defense against GBS sepsis and strongly suggest TNF-alpha as the molecular mediator of bacterial clearance and septic shock.     

Design of mineral medium for growth of <Emphasis Type="Italic">Actinomadura</Emphasis> sp. ATCC 39727, producer of the glycopeptide A40926: effects of calcium ions and nitrogen sources

Actinomadura sp. ATCC 39727 produces the glycopeptide antibiotic A40926, structurally similar to teicoplanin, with significant activity against Neisseria gonorrhoeae and precursor of the semi-synthetic antibiotic dalbavancin. In this study the production of A40926 by Actinomadura under a variety of growth conditions was investigated. The use of chemically defined mineral media allowed us to analyze the influence of carbon and nitrogen sources, phosphate, ammonium and calcium on the growth and the antibiotic productivity of Actinomadura. We confirm recent data [Gunnarsson et al. (2003) J Ind Microbiol Biotechnol 30:150–156] that low initial concentrations of phosphate and ammonium are beneficial for growth and A40926 production, and we provide new evidence that the production of A40926 is depressed by calcium, but promoted when l-glutamine or l-asparagine are used as nitrogen sources instead of ammonium salts.     

Flow cytometric screening of aldolase catalytic antibodies

High-throughput screening of cells expressing active catalytic antibody clones by flow cytometry is described. A fluorogenic retro-aldol retro-Michael substrate was designed and synthesized with incorporation of a chloromethyl moiety for intracellular retention. Hybridoma or transfected mammalian cells expressing catalytic antibody molecules could be rapidly isolated.     

Role of GM3-enriched microdomains in signal transduction regulation in T lymphocytes

Gangliosides, sialic acid containing glycosphigolipids, are ubiquitous constituents of cell plasma membranes. Each cell type shows a peculiar ganglioside expression pattern. In human T lymphocytes monosialoganglioside GM3 represents the main ganglioside constituent of cell plasma membrane where it is concentrated in glycosphingolipid-enriched microdomains (GEM). The presence of tyrosine kinase receptors, mono- (Ras, Rap) and heterotrimeric G proteins, Src-like tyrosine kinases (lck, lyn, fyn), PKC isozymes, glycosylphosphatidylinositol (GPI)-anchored proteins and, after T cell activation, the Syk-family kinase Zap-70, prompts these portions of the plasma membrane to be considered as "glycosignaling domains." In particular, during T cell activation and/or other dynamic functions of the cell, such as apoptosis, key signaling molecules are recruited to these microdomains, where they strictly interact with GM3. The association of transducer proteins with GM3 in microdomains suggests that this ganglioside is the main marker of GEM in human lymphocytes and is a component of a cell plasma membrane multimolecular signaling complex involved in cell-cell interaction, signal transduction, and cell activation.     

Caspase inhibition protects against reovirus-induced myocardial injury in vitro and in vivo

Viral myocarditis is a disease with a high morbidity and mortality. The pathogenesis of this disease remains poorly characterized, with components of both direct virus-mediated and secondary inflammatory and immune responses contributing to disease. Apoptosis has increasingly been viewed as an important mechanism of myocardial injury in noninfectious models of cardiac disease, including ischemia and failure. Using a reovirus murine model of viral myocarditis, we characterized and targeted apoptosis as a key mechanism of virus-associated myocardial injury in vitro and in vivo. We demonstrated caspase-3 activation, in conjunction with terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and annexin binding, in cardiac myocytes after myocarditic viral infection in vitro. We also demonstrated a tight temporal and geographical correlation between caspase-3 activation, histologic injury, and viral load in cardiac tissue after myocarditic viral infection in vivo. Two pharmacologic agents that broadly inhibit caspase activity, Q-VD-OPH and Z-VAD(OMe)-FMK, effectively inhibited virus-induced cellular death in vitro. The inhibition of caspase activity in vivo by the use of pharmacologic agents as well as genetic manipulation reduced virus-induced myocardial injury by 40 to 60% and dramatically improved survival in infected caspase-3-deficient animals. This study indicates that apoptosis plays a critical role in mediating cardiac injury in the setting of viral myocarditis and is the first demonstration that caspase inhibition may serve as a novel therapeutic strategy for this devastating disease.     

Using stable isotope analysis to obtain dietary profiles from old hair: a case study from Plains Indians

Stable isotope composition of human tissue reflects that of foods consumed, and can provide information about diet independent of artifactual remains. Here we refine and test this method by analyzing nitrogen (delta(15)N) and carbon (delta(13)C) isotope ratios in historic North American Plains Indians hair. Gas-source isotope-ratio mass spectrometry provides high-precision data for both delta(15)N and delta(13)C (+/-0.2 per thousand, 1 sigma) in single hair strands as short as 2 cm (100-150 mug). Because hair contains more carbon than nitrogen, if only delta(13)C data are needed, shorter strands (<1 cm) can be analyzed. This reduction in sample size opens new opportunities for analysis of small hair fragments found in archaeological excavations, as well as for analysis of seasonal variations in long hair strands. We find distinct isotope profiles (delta(15)N vs. delta(13)C) for two cultural groups, the Lower Brule reservation Sioux of 1892 and the reservation Blackfoot of 1892 and 1935. The resultant dietary profiles indicate a higher consumption of meat by the Blackfoot and a higher consumption of maize (or of animals that had fed on maize or other C(4) plants) by the Lower Brule. The two groups of Blackfoot yield similar isotopic profiles despite the passage of four decades, suggesting a strong role for cultural preference even as food sources change. Such stable isotope profiles can be used to link samples from the same cultural tradition based on their similar diets.     

Enhanced biotransformations and product recovery in a membrane bioreactor through application of a direct electric current

The simultaneous enhancement of biotransformation coupled to product recovery, purification and concentration is presented. The nitrilase of Rhodococcus rhodochrous LL100-21 catalyses the single-step hydrolytic biotransformation of benzonitrile to benzoic acid and ammonia. When a direct electric current is applied across a bioreactor containing the bacterium and benzonitrile, the charged product (benzoic acid) can be removed in situ across an anion exchange membrane and recovered in a separate compartment. Over the course of a 24-hour biotransformation, benzonitrile was converted to benzoic acid which was completely removed from the bioreactor chamber and concentrated 3-fold in a separate chamber. The rate of production of benzoic acid increased by 42% when the current was applied (0.044 mmol/min/g dry cell weight in the presence of current as compared to 0.03 mmol/min/g dry cell weight in its absence). The enhanced reaction rate was achieved irrespective of product separation and therefore appears to be a direct effect upon the bacterial cells. This process has potential for enhanced productivity from biotransformations through a simultaneous increase in metabolic activity and in situ product recovery.