Toll-like receptor 2 and Toll-like receptor 4 polymorphisms in invasive pneumococcal disease

BACKGROUND: Toll-like receptors (TLRs) recognize distinct pathogen-associated molecular patterns and trigger anti-microbial host defense responses. Several in vitro and in vivo studies in mice indicate that TLR2 and TLR4 are involved in the defense against Streptococcus pneumoniae. Studies have revealed associations between polymorphisms in TLRs and human diseases. The effect of polymorphisms in TLR2 and TLR4 in the human defense to S. pneumoniae has not been studied. METHODS: We genotyped 99 Caucasian patients with invasive pneumococcal disease and 178 Caucasian controls for the known R579H, P631H and R753Q polymorphisms in TLR2 and the D299G polymorphism in TLR4 with PCR-RFLP methods. RESULTS: The distribution of the TLR2 R579H, P631H and R753Q and TLR4 D299G variants was not significantly different between the patients and the controls. After stratification of the patient population by age, sex, diagnosis, and mortality no significant differences for the TLR2 R753Q genotype and TLR4 D299G genotype were found between various patient subgroups and between patient subgroups and the control population. It should be mentioned that for the TLR2 polymorphisms neither the control group nor the patient group contains homozygous mutant individuals. CONCLUSION: We found no association between TLR2 and TLR4 polymorphisms and invasive pneumococcal infection.     

Dolichol pathway in lymphocytes from rat spleen. Influence of the glucosylation on the cleavage of dolichyl diphosphate oligosaccharides into phosphooligosaccharides

Incubation of rat-spleen lymphocytes with UDP-glucose together with GDP-mannose and UDP-N-acetylglucosamine leads to the formation of glucosylated lipid intermediates characterized as dolichyl phosphate glucose and dolichyl diphosphate oligosaccharides. This latter can be either transferred onto endogenous protein acceptors or cleaved into phosphooligosaccharides. The striking fact is that phosphooligosaccharide populations contain far less glucosylated products than the dolichyl diphosphate oligosaccharide ones from which they are derived. Two hypotheses have been investigated: either a rapid action of glucosidases on the liberated phosphooligosaccharides or a preferential splitting of the non-glucosylated population of dolichyl diphosphate oligosaccharides. Addition of p-nitrophenyl-alpha-D-glucoside inhibits glucosidase activities and allows the production of a major population of dolichyl diphosphate oligosaccharides containing three glucose residues. Using these conditions, it is shown that the amount of phosphooligosaccharides generated from the splitting of dolichyl diphosphate oligosaccharides is greatly decreased and that the major part of these remaining phosphooligosaccharides do not contain glucose. These results show that the presence of glucosyl units prevent dolichyl diphosphate oligosaccharides from further degradation into phosphooligosaccharides.     

Receptor and antibody interactions of human interleukin-3 characterized by mutational analysis.

Human interleukin-3 (hIL-3) is a regulator of proliferation and differentiation of multipotent hemopoietic progenitor cells. Mutants of hIL-3 have been constructed by oligonucleotide-directed mutagenesis and expressed in Escherichia coli and Bacillus licheniformis. Purified muteins were assayed for induction of DNA synthesis in IL-3-dependent human cells and for binding to the IL-3 receptor. Residues at the NH2 and COOH termini together comprising one-quarter of the molecule could be removed without loss of biological function. Deletions of 6-15 residues within the central part of the molecule caused a large reduction (up to 5 logs) but no complete loss of activity. Substitution of evolutionary conserved residues resulted in a strong decrease of biological activity and demonstrated that the S-S bridge is an essential structural element in hIL-3. Interestingly, four muteins displayed a significantly higher potency of binding to the IL-3 receptor than in stimulating DNA synthesis. These results demonstrate that receptor binding may be (partly) disconnected from activation of DNA synthesis. Analysis of hIL-3 muteins demonstrated that the majority of monoclonal antibodies are directed against a small portion of the IL-3 molecule. The neutralizing potential of individual monoclonal antibodies could be increased by a combination of antibodies directed against nonoverlapping epitopes.     

Different fates of the oligosaccharide moieties of lipid intermediates.

We have previously described that the N-glycosylation process was accompanied by the release of oligosaccharide-phosphates and neutral oligosaccharides. The relationship between oligosaccharide-P-P-dolichol and its metabolic products (glycoproteins, oligosaccharide-phosphates and neutral oligosaccharides) was investigated by analysing the structure of the oligosaccharide moieties and the kinetic behaviour of the various species in pulse and pulse/chase experiments. For these studies, a glycosylation mutant of Chinese hamster ovary cells (B3F7) which does not synthesize mannosylphosphoryldolichol was utilized. Evidence was obtained for the presence of two pools of oligosaccharide-P-P-dolichol which have different fates. One pool is not glucosylated, is rapidly labelled and immediately chased by mannose, and generates the oligosaccharide-phosphate species. The second pool is glucosylated, exhibits a lag time (5-10 min) prior to being labelled, and is utilized in the glycosylation of proteins and in the production of neutral oligosaccharides. We postulate that the cleavage of non-glycosylated lipid intermediates generating oligosaccharide-phosphates represents a 'bypass' in the dolichol cycle which allows direct regeneration of dolichyl phosphate. The other metabolic fate of non-glucosylated oligosaccharide-lipids, glucosylation, results in their use as effective substrates for the glycosylation of proteins or in the generation of neutral oligosaccharides.     

Biogenic Silver for Disinfection of Water Contaminated with Viruses

The presence of enteric viruses in drinking water is a potential health risk. Growing interest has arisen in nanometals for water disinfection, in particular the use of silver-based nanotechnology. In this study, Lactobacillus fermentum served as a reducing agent and bacterial carrier matrix for zerovalent silver nanoparticles, referred to as biogenic Ag⁰. The antiviral action of biogenic Ag⁰ was examined in water spiked with an Enterobacter aerogenes-infecting bacteriophage (UZ1). Addition of 5.4 mg liter⁻¹ biogenic Ag⁰ caused a 4.0-log decrease of the phage after 1 h, whereas the use of chemically produced silver nanoparticles (nAg⁰) showed no inactivation within the same time frame. A control experiment with 5.4 mg liter⁻¹ ionic Ag⁺ resulted in a similar inactivation after 5 h only. The antiviral properties of biogenic Ag⁰ were also demonstrated on the murine norovirus 1 (MNV-1), a model organism for human noroviruses. Biogenic Ag⁰ was applied to an electropositive cartridge filter (NanoCeram) to evaluate its capacity for continuous disinfection. Addition of 31.25 mg biogenic Ag⁰ m⁻² on the filter (135 mg biogenic Ag⁰ kg⁻¹ filter medium) caused a 3.8-log decline of the virus. In contrast, only a 1.5-log decrease could be obtained with the original filter. This is the first report to demonstrate the antiviral efficacy of extracellular biogenic Ag⁰ and its promising opportunities for continuous water disinfection.At least 1 billion people do not have access to safe drinking water, according to the WHO (41). Contamination of drinking water and the subsequent outbreak of waterborne diseases are the leading cause of death in many developing nations. Moreover, the spectrum and incidence of some infectious diseases are increasing worldwide (40). Among them, the transmission of waterborne human noroviruses is considered to be the major cause of acute nonbacterial gastroenteritis (22). Numerous outbreaks of norovirus-associated gastroenteritis have been linked with ingestion of contaminated drinking water, in developed countries also (6; M. Kukkula, L. Maunula, E. Silvennoinen, and C. H. von Bonsdorff, presented at the International Workshop on Human Caliciviruses, Atlanta, GA, 29 to 31 March 1999). Therefore, the development of innovative drinking water quality control strategies is of the utmost importance in this decade.Recent interest has arisen in the use of nanotechnology for water disinfection (20). In particular the formation of by-products by conventional disinfection techniques (e.g., chlorination), has encouraged researchers to explore the antimicrobial activity of several nanomaterials, such as silver (18, 31). Silver-containing nanoparticles have previously been demonstrated to be effective against bacteria and viral particles (10, 28, 34). Several mechanisms of the antiviral activity have been ascribed to (chemically produced) zerovalent silver nanoparticles (nAg⁰) but still remain not fully understood. On the one hand, nAg⁰ can release Ag⁺ ions, which interact with thiol groups in proteins and interfere with DNA replication (11, 21, 24). On the other hand, the adhesion of nAg⁰ as such is responsible for the inactivation of HIV-1 virions (10).Previous studies showed that chemically produced nAg⁰ were unstable in solution and would easily aggregate with average particle sizes of <40 nm or at high concentrations (23). As a consequence, the specific surface of the nanomaterial decreases. Moreover, there is a need for environmentally friendly approaches to production of nanoparticles. To cope with these demands, biological processes have been developed using microorganisms. Microbial approaches to obtain nanoscale Ag⁰ have been demonstrated for the bacterium Pseudomonas stutzeri AG259 (17) and for fungi, e.g., Verticillium sp. (26), Phoma sp. (5), Fusarium sp. (2, 16), and Aspergillus sp. (12, 29, 39). However, these enzymatic reduction processes are slow and yield low concentrations of silver. Moreover, if the nanoparticles are produced intracellularly, specific treatments (e.g., heat treatment at 600°C for 6 h) are necessary to make the nanoparticles accessible for antibacterial or antiviral applications (39).Recently, lactic acid bacteria have been used as reducing agents for the fast, nonenzymatic, and extracellular production of nanoscale-sized Ag⁰ particles (33). The bacterial cell wall hereby serves as a microscale carrier matrix for the nanoparticles. The unique association of the nanoparticles with the (dead) bacterial carrier matrix, called biogenic Ag⁰, prevents them from aggregating and makes the association promising for disinfection technologies. In the case of virus inactivation, smaller nanoparticles are known to be more efficient due to a more effective binding to the glycoproteins of the viral envelope (10, 28). For biogenic Ag⁰ production using lactic acid bacteria, it was demonstrated that different particle sizes could be obtained, depending on the species used (33). Production by Lactobacillus fermentum resulted in the smallest average diameter and a narrow size distribution, potentially favorable for antimicrobial applications (33).The objective of the present study was to examine the inactivation of a bacteriophage (UZ1), isolated from hospital sewage, by biogenic Ag⁰. This DNA phage, a T7-like coliphage of the genus Podovirida (order Caudovirales) (38), is infective for Enterobacter aerogenes BE1, a species belonging to the normal digestive microbiota (30). The virucidal action of biogenic Ag⁰ was evaluated in drinking water and compared with the use of ionic Ag⁺ and chemically produced nAg⁰. To test the antiviral activity of biogenic Ag⁰ against noroviruses as well, the murine norovirus 1 (MNV-1) was used as a surrogate organism for human noroviruses (43). Finally, continuous disinfection by the biogenic nanoparticles was evaluated in a flowthrough system with a coated cartridge filter. To our knowledge, this is the first report to demonstrate the antiviral effect of extracellular biogenic Ag⁰.     

Tracking the Evolution of Multiple In Vitro Hepatitis C Virus Replicon Variants under Protease Inhibitor Selection Pressure by 454 Deep Sequencing

Resistance to hepatitis C virus (HCV) inhibitors targeting viral enzymes has been observed in in vitro replicon studies and during clinical trials. The factors determining the emergence of resistance and the changes in the viral quasispecies population under selective pressure are not fully understood. To assess the dynamics of variants emerging in vitro under various selective pressures with TMC380765, a potent macrocyclic HCV NS3/4A protease inhibitor, HCV genotype 1b replicon-containing cells were cultured in the presence of a low, high, or stepwise-increasing TMC380765 concentration(s). HCV replicon RNA from representative samples thus obtained was analyzed using (i) population, (ii) clonal, and (iii) 454 deep sequencing technologies. Depending on the concentration of TMC380765, distinct mutational patterns emerged. In particular, culturing with low concentrations resulted in the selection of low-level resistance mutations (F43S and A156G), whereas high concentrations resulted in the selection of high-level resistance mutations (A156V, D168V, and D168A). Clonal and 454 deep sequencing analysis of the replicon RNA allowed the identification of low-frequency preexisting mutations possibly contributing to the mutational pattern that emerged. Stepwise-increasing TMC380765 concentrations resulted in the emergence and disappearance of multiple replicon variants in response to the changing selection pressure. Moreover, two different codons for the wild-type amino acids were observed at certain NS3 positions within one population of replicons, which may contribute to the emerging mutational patterns. Deep sequencing technologies enabled the study of minority variants present in the HCV quasispecies population present at baseline and during antiviral drug pressure, giving new insights into the dynamics of resistance acquisition by HCV.Chronic hepatitis C virus (HCV) infection can lead to liver fibrosis, cirrhosis, hepatocellular carcinoma, and ultimately liver failure. Approximately 170 million people worldwide are infected with HCV (54a). The current standard of care consists of pegylated alpha interferon (Peg-IFN) plus ribavirin (RBV), providing limited efficacy for genotype 1-infected patients, i.e., a sustained virological response (SVR) in 40 to 50% of the patients. Moreover, Peg-IFN/RBV therapy is associated with significant adverse events (9). Therefore, direct antiviral agents (DAA) (previously also known as “specifically targeted antiviral therapies for hepatitis C” or STAT-C) have been a major focus of drug discovery efforts over the last 2 decades. Several NS3/4A (protease), NS5A, and NS5B (polymerase) inhibitors either alone or in combination with Peg-IFN/RBV have recently shown potent antiviral effects in HCV-infected patients (22, 36). However, viral resistance to these novel agents can occur rapidly when they are dosed as monotherapy (43, 49).Because of the high mutation rate of the HCV polymerase (10⁻³ to 10⁻⁵ misincorporations per nucleotide copied [11]) and the high viral production rates in vivo (approximately 10¹² viruses per patient per day [37]), it can be assumed that HCV exists as a diverse population of nonidentical but closely related viral genomes, referred to as a quasispecies (10). A viral quasispecies is characterized by a dominant nucleotide sequence, called a master sequence, and a surrounding mutant spectrum, which can harbor minority subpopulations (42). Although in theory all single and double mutants are produced daily in an infected person (6, 40), it is important to note that mutation rates are not equally distributed over the entire genome and that additional factors, such as viral fitness and the replication environment, determine whether a mutation becomes fixed in a viral quasispecies population (12). The diversity of the viral variants present in an infected individual facilitates the adaptation of the quasispecies to external pressure, such as antiviral treatment, improving the survival chances of the population (53). The speed of such adaptation depends mainly on the turnover of the viral nucleic acid acting as a source of new viral genomes. Whereas in HIV the rapid turnover of infected CD4⁺ T lymphocytes is responsible for the rapid turnover of nucleic acids, in HCV rapid turnover is explained by the short half-life (∼10 h) of HCV RNA strands in the hepatocyte (47). However, if mutation rates exceed a certain limit, called the error threshold, deleterious mutations will accumulate and the viral population will become extinct (4).Recent reports have demonstrated that mutations known to affect the activities of DAA compounds in vitro are present in some treatment-naive patients as either dominant or minority species (6, 13, 19, 21, 27). With the eradication of variants susceptible to the antiviral drugs, resistant viruses initially present as minority species may expand to occupy the freed replicative space, thus becoming the dominant master sequence (1); this may lead to failure of the antiviral regimen. In HIV it has been shown that minority species can play an important role in the accelerated evolution toward resistance to antiretroviral drugs (5). The extent to which preexisting HCV variants may compromise treatment with DAAs, however, is not yet fully understood (3). Depending on the concentration of the antiviral agent, different resistance profiles seem to emerge. In clinical studies, a correlation was noted between the plasma trough levels of the NS3/4A inhibitor telaprevir, the virological response, and the mutations responsible for the drug-resistant phenotype (43). In patients with a low exposure to telaprevir, variants carrying mutations with low resistance to telaprevir in vitro were observed, while higher drug levels were associated with variants conferring a greater degree of resistance in vitro. Correlations between the inhibitor concentration and the mutation profile were also described in in vitro studies (44, 50, 51).HCV replicon cell culture systems have been widely used to characterize resistance against antiviral inhibitors and to assess the impact of resistance mutations on drug susceptibility and replication fitness in vitro (8, 15). Although the information on resistance mutations observed with DAA during clinical trials is still limited, mutations identified in vitro appear to be predictive for those mutations that may emerge in patients (17, 20). In addition, analysis of the genetic variability and diversity of a long-term HCV replicon-containing cell culture has shown that mutations accumulate over time at rates comparable to those observed in vivo: (3.5 to 4.8) × 10⁻³ in vitro versus (1.4 to 1.9) × 10⁻³ in vivo base substitutions/site/year (16). Hence, HCV replicon systems are considered a useful and relevant surrogate system for analyzing the evolutionary dynamics and variations of HCV in response to selection pressure.The detailed study of the dynamics of viral variants present in a quasispecies population has long been hampered by the lack of sensitive sequencing methods. The recent development of deep sequencing technologies may facilitate a better understanding of the genetic composition and natural evolution of viral quasispecies in the presence of antiviral drugs (30, 34, 54). Indeed, studies of HIV suggest that these more-sensitive sequencing technologies detect additional minority variants for both treatment-naive and treatment-experienced patients which could impact the clinical outcome of antiretroviral therapy and may provide important information for treatment planning (2, 23, 41, 46).TMC380765 (Fig. ​(Fig.1)1) is a macrocyclic inhibitor of the HCV NS3/4A protease and a potent inhibitor of HCV RNA replication in vitro, with median 50% effective concentration (EC₅₀) and 90% effective concentration (EC₉₀) values of 35 nM and 106 nM, respectively, in the Huh7-Luc replicon using a luciferase readout (25, 39). Other examples of macrocyclic NS3/4A inhibitors include BILN-2061, ITMN-191, MK7009, and TMC435. To assess the effect of its selective pressure on the composition of the replicon population, selection experiments were performed with different concentrations of TMC380765 and sequence changes were determined with population, clonal, and 454 deep sequencing technologies.Open in a separate windowFIG. 1.Structural formulae of TMC380765.     

Context-Dependency of Agricultural Legacies in Temperate Forest Soils

Ecosystems - Anthropogenic activities have affected forests for centuries, leading to persistent legacies. Observations of agricultural legacies on forest soil properties have been site specific...