Neutrophils, dendritic cells and Toxoplasma

Toxoplasma gondii rapidly elicits strong Type 1 cytokine-based immunity. The necessity for this response is well illustrated by the example of IFN-gamma and IL-12 gene knockout mice that rapidly succumb to the effects of acute infection. The parasite itself is skilled at sparking complex interactions in the innate immune system that lead to protective immunity. Neutrophils are one of the first cell types to arrive at the site of infection, and the cells release several proinflammatory cytokines and chemokines in response to Toxoplasma. Dendritic cells are an important source of IL-12 during infection with T. gondii and other microbial pathogens, and they are also specialized for high-level antigen presentation to T lymphocytes. Tachyzoites express at least two types of molecules that trigger innate immune cell cytokine production. One of these involves Toll-like receptor/MyD88 pathways common to many microbial pathogens. The second pathway is less conventional and involves molecular mimicry between a parasite cyclophilin and host CC chemokine receptor 5-binding ligands. Neutrophils, dendritic cells and Toxoplasma work together to elicit the immune response required for host survival. Cytokine and chemokine cross-talk between parasite-triggered neutrophils and dendritic cells results in recruitment, maturation and activation of the latter. Neutrophil-empowered dendritic cells possess properties expected of highly potent antigen presenting cells that drive T helper 1 generation.     

Toxoplasma gondii triggers myeloid differentiation factor 88-dependent IL-12 and chemokine ligand 2 (monocyte chemoattractant protein 1) responses using distinct parasite molecules and host receptors

Toll-like receptors (TLR) that signal through the common adaptor molecule myeloid differentiation factor 88 (MyD88) are essential in proinflammatory cytokine responses to many microbial pathogens. In this study we report that Toxoplasma gondii triggers neutrophil IL-12 and chemokine ligand 2 (CCL2; monocyte chemoattractant protein 1) production in strict dependence upon functional MyD88. Nevertheless, the responses are distinct. Although we identify TLR2 as the receptor triggering CCL2 production, parasite-induced IL-12 release did not involve this TLR. The production of both IL-12 and CCL2 was increased after neutrophil activation with IFN-gamma. However, the synergistic effect of IFN-gamma on IL-12, but not CCL2, was dependent upon Stat1 signal transduction. Although IL-10 was a potent down-regulator of Toxoplasma-triggered neutrophil IL-12 release, the cytokine had no effect on parasite-induced CCL2 production. Soluble tachyzoite Ag fractionation demonstrated that CCL2- and IL-12 inducing activities are biochemically distinct. Importantly, Toxoplasma cyclophilin-18, a molecule previously shown to induce dendritic cell IL-12, was not involved in neutrophil IL-12 production. Our results show for the first time that T. gondii possesses multiple molecules triggering distinct MyD88-dependent signaling cascades, that these pathways are independently regulated, and that they lead to distinct profiles of cytokine production.     

Large duplication 4q25-q34 with mild clinical effect

We report on a 5-year-old Tunisian boy with particular dysmorphic features and mild mental retardation limited in delayed and poor language acquisition. Cytogenetic analysis using RHG banding and FISH using whole chromosome four painting probe showed a partial duplication in the long arm of chromosome four. Locus specific probes and CGH confirmed the presence of a 'pure' partial trisomy 4q due to de novo direct tandem dup(4)(q25q34). Comparative analysis of our case with those published previously, suggests that region 4q31-q33 may be involved in the development of the 4q characteristic dysmorphic features and the distal band 4q35 may be involved in the development of microcephaly and severe mental and growth retardation.     

Activation of the RegB endoribonuclease by the S1 ribosomal protein is due to cooperation between the S1 four C-terminal modules in a substrate-dependant manner

The RegB protein, encoded by the T4 bacteriophage genome, is a ribonuclease involved in the inactivation of the phage early messenger RNAs. Its in vitro activity is very low but can be enhanced up to 100-fold in the presence of the ribosomal protein S1. The latter is made of six repeats of a conserved module found in many other proteins of RNA metabolism. Considering the difference between its size (556 amino acids) and that of several RegB substrates (10 nucleotides), we wondered whether all six modules are necessary for RegB activation. We studied the influence of twelve S1 fragments on the cleavage efficiency of three short substrates. RegB activation requires the cooperation of different sets of modules depending on the substrates. Two RNAs are quite well cleaved in the presence of the fragment formed by the fourth and fifth modules, whereas the third requires the presence of the four C-terminal domains. However, NMR interaction experiments showed that, despite these differences, the interactions of the substrates with either the bi- or tetra-modules are similar, suggesting a common interaction surface. In the case of the tetra-module the interactions involve all four domains, raising the question of the spatial organization of this region.     

Cross-talk in the innate immune system: neutrophils instruct recruitment and activation of dendritic cells during microbial infection

Type I inflammatory cytokines are essential for immunity to many microbial pathogens, including Toxoplasma gondii. Dendritic cells (DC) are key to initiating type 1 immunity, but neutrophils are also a source of chemokines and cytokines involved in Th1 response ignition. We found that T. gondii triggered neutrophil synthesis of CC chemokine ligand (CCL)3, CCL4, CCL5, and CCL20, chemokines that were strongly chemotactic for immature DC. Moreover, supernatants obtained from parasite-stimulated polymorphonuclear leukocytes induced DC IL-12(p40) and TNF-alpha production. Parasite-triggered neutrophils also released factors that induced DC CD40 and CD86 up-regulation, and this response was dependent upon parasite-triggered neutrophil TNF-alpha production. In vivo evidence that polymorphonuclear leukocytes exert an important influence on DC activation was obtained by examining splenic DC cytokine production following infection of neutrophil-depleted mice. These animals displayed severely curtailed splenic DC IL-12 and TNF-alpha production, as revealed by ex vivo flow cytometric analysis and in vitro culture assay. Our results reveal a previously unrecognized regulatory role for neutrophils in DC function during microbial infection, and suggest that cross-talk between these cell populations is an important component of the innate immune response to infection.     

Microbial antigen triggers rapid mobilization of TNF-alpha to the surface of mouse neutrophils transforming them into inducers of high-level dendritic cell TNF-alpha production

Neutrophils play a critical role in early immunity to many microbial pathogens, and this may in part be due to their ability to release immunoregulatory cytokines and chemokines during infection. Here, we demonstrate by flow cytometric analysis that mouse polymorphonuclear leukocytes (PMN) up-regulate surface expression of TNF-alpha within 10 min of stimulation with LPS, and that this is followed by gradual loss over a period of 18 h. Early increases in surface TNF-alpha expression correlated with loss of intracellular pools of preformed TNF-alpha. Nevertheless, extended incubation with LPS resulted in increased levels of TNF-alpha mRNA synthesis and replenishment of intracellular cytokine. After triggering with LPS, PMN acquired the ability to induce dendritic cell (DC) TNF-alpha and IL-12 production. Transwell assays demonstrated that high-level DC TNF-alpha production induced by LPS-triggered neutrophils was dependent upon cell-to-cell contact and neutrophil TNF-alpha, but neither was required for neutrophil instruction of DC IL-12 synthesis. The data suggest that microbial Ag-triggered mouse PMN acquire the capacity to deliver potent DC-activating signals through elaboration of cytokines and direct interactions at the cell surface.     

The Amaryllidaceae Alkaloid Haemanthamine Binds the Eukaryotic Ribosome to Repress Cancer Cell Growth

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In vivo study of engineered G-domain mutants of Escherichia coli translation initiation factor IF2

During the IF2-catalysed formation of the 30S initiation complex, the GTP requirement and Its subsequent hydrolysis during 70S complex formation are considered to be essential for translation initiation in Escherichia coli. In order to clarify the role of certain amino acid residues believed to be crucial for the GTP hydrolytic activity of E. coli IF2, we have introduced seven single amino acid substitutions into its GTP-binding site (Gly for Val-400; Thr for Pro-446; Gly, Glu, Gin for His-448; and Asn, Glu for Asp-501). These mutated IF2 proteins were expressed in vivo in physiological quantities and tested for their ability to maintain the growth of an E. coli strain from which the functional chromosomal copy of the infB gene has been deleted. Only one of the mutated proteins (Asp-501 to Giu) was able to sustain cell viability and several displayed a dominant negative effect. These results emphasize that the amino acid residues we substituted are essential for the iF2 functions and demonstrate the importance of GTP hydrolysis in translation initiation. These findings are discussed in relation to a previously proposed theoretical model for the IF2 G-domain.     

Antibacterial and cytotoxic activities of extracts from (Tunisian)Rhamnus alaternus (Rhamnaceae)

The petroleum ether, chloroformic, ethyl acetate, methanolic, Total Oligomers Flavonoids (TOF) enriched extracts, water extract as well as its fractions A₁, A₂, A₃ obtained from aerial parts ofRhamnus alaternus, a Tunisian-Mediterranean medicinal species, were investigated for the contents of phenolic compounds, cytotoxic activity against the K562 human chronic myelogenous leukaemia cell line and L1210 leukaemia murine cells and for antibacterial activity against Gram positive and Gram negative bacterial reference strains. A pronounced cytotoxic effect on both the cell lines was shown in the TOF, ethyl acetate, methanolic, aqueous extracts and A₂ fraction, with respectively IC₅₀ values 75, 232, 298, 606 and 571 μg/ml on K562 cells and 198, 176, 767, 560 and 614 μg/ml on L1210 cell line. Significant activity against bacterial reference strains:Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Salmonella enteritidis andSalmonella typhimurium was shown with ethyl acetate, TOF extracts and A2 fraction. The antimicrobial and cytotoxic activities showed byR. alatemus depended on the chemical composition of the tested extracts.