Immunomodulatory effects of probiotic bacteria DNA: IL-1 and IL-10 response in human peripheral blood mononuclear cells

A new therapeutic approach for inflammatory bowel diseases is based on the administration of probiotic bacteria. Prokaryotic DNA contains unmethylated CpG motifs which can activate immune responses, but it is unknown whether bacterial DNA is involved in the beneficial effects obtained by probiotic treatment. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with pure DNA of eight probiotic strains and with total bacterial DNA from human feces collected before and after probiotic ingestion. Cytokine production was analyzed in culture supernatants. Modification of human microflora after probiotic administration was proven by polymerase chain reaction analysis. Here we show that Bifidobacterium genomic DNA induced secretion of the antiinflammatory interleukin-10 by PBMC. Total bacterial DNA from feces collected after probiotic administration modulated the immune response by a decrease of interleukin-1 beta and an increase of interleukin-10.     

Differential Localization of VE- and N-Cadherins in Human Endothelial Cells: VE-Cadherin Competes with N-Cadherin for Junctional Localization

The two major cadherins of endothelial cells are neural (N)-cadherin and vascular endothelial (VE)- cadherin. Despite similar level of protein expression only VE-cadherin is located at cell–cell contacts, whereas N-cadherin is distributed over the whole cell membrane. Cotransfection of VE-cadherin and N-cadherin in CHO cells resulted in the same distribution as that observed in endothelial cells indicating that the behavior of the two cadherins was not cell specific but related to their structural characteristics. Similar amounts of α- and β-catenins and plakoglobin were associated to VE- and N-cadherins, whereas p120 was higher in the VE-cadherin complex. The presence of VE-cadherin did not affect N-cadherin homotypic adhesive properties or its capacity to localize at junctions when cotransfectants were cocultured with cells transfected with N-cadherin only. To define the molecular domain responsible for the VE-cadherin–dominant activity we prepared a chimeric construct formed by VE-cadherin extracellular region linked to N-cadherin intracellular domain. The chimera lost the capacity to exclude N-cadherin from junctions indicating that the extracellular domain of VE-cadherin alone is not sufficient for the preferential localization of the molecule at the junctions. A truncated mutant of VE-cadherin retaining the full extracellular domain and a short cytoplasmic tail (Arg⁶²¹–Pro⁷⁰²) lacking the catenin-binding region was able to exclude N-cadherin from junctions. This indicates that the Arg⁶²¹–Pro⁷⁰² sequence in the VE-cadherin cytoplasmic tail is required for N-cadherin exclusion from junctions. Competition between cadherins for their clustering at intercellular junctions in the same cell has never been described before. We speculate that, in the endothelium, VE- and N-cadherin play different roles; whereas VE-cadherin mostly promotes the homotypic interaction between endothelial cells, N-cadherin may be responsible for the anchorage of the endothelium to other surrounding cell types expressing N-cadherin such as vascular smooth muscle cells or pericytes.     

MFR, a Putative Receptor Mediating the Fusion of Macrophages

We had previously identified a macrophage surface protein whose expression is highly induced, transient, and specific, as it is restricted to actively fusing macrophages in vitro and in vivo. This protein is recognized by monoclonal antibodies that block macrophage fusion. We have now purified this protein and cloned its corresponding cDNA. This protein belongs to the superfamily of immunoglobulins and is similar to immune antigen receptors such as the T-cell receptor, B-cell receptor, and viral receptors such as CD4. We have therefore named this protein macrophage fusion receptor (MFR). We show that the extracellular domain of MFR prevents fusion of macrophages in vitro and therefore propose that MFR belongs to the fusion machinery of macrophages. MFR is identical to SHPS-1 and BIT and is a homologue of P84, SIRPα, and MyD-1, all of which have been recently cloned and implicated in cell signaling and cell-cell interaction events.     

Exonic Sequences in the 5′ Untranslated Region of α-Tubulin mRNA Modulate trans Splicing in Trypanosoma brucei

Previous studies have identified a conserved AG dinucleotide at the 3′ splice site (3′SS) and a polypyrimidine (pPy) tract that are required for trans splicing of polycistronic pre-mRNAs in trypanosomatids. Furthermore, the pPy tract of the Trypanosoma brucei α-tubulin 3′SS region is required to specify accurate 3′-end formation of the upstream β-tubulin gene and trans splicing of the downstream α-tubulin gene. Here, we employed an in vivo cis competition assay to determine whether sequences other than those of the AG dinucleotide and the pPy tract were required for 3′SS identification. Our results indicate that a minimal α-tubulin 3′SS, from the putative branch site region to the AG dinucleotide, is not sufficient for recognition by the trans-splicing machinery and that polyadenylation is strictly dependent on downstream trans splicing. We show that efficient use of the α-tubulin 3′SS is dependent upon the presence of exon sequences. Furthermore, β-tubulin, but not actin exon sequences or unrelated plasmid sequences, can replace α-tubulin exon sequences for accurate trans-splice-site selection. Taken together, these results support a model in which the informational content required for efficient trans splicing of the α-tubulin pre-mRNA includes exon sequences which are involved in modulation of trans-splicing efficiency. Sequences that positively regulate trans splicing might be similar to cis-splicing enhancers described in other systems.     

Fallback foraging as a way of life: Using dietary toughness to compare the fallback signal among capuchins and implications for interpreting morphological variation

The genus Cebus is one of the best extant models for examining the role of fallback foods in primate evolution. Cebus includes the tufted capuchins, which exhibit skeletal features for the exploitation of hard and tough foods. Paradoxically, these seemingly “specialized” taxa belong to the most ubiquitous group of closely related primates in South America, thriving in a range of different habitats. This appears to be a consequence of their ability to exploit obdurate fallback foods. Here we compare the toughness of foods exploited by two tufted capuchin species at two ecologically distinct sites; C. apella in a tropical rainforest, and C. libidinosus in a cerrado forest. We include dietary data for one untufted species (C. olivaceus) to assess the degree of difference between the tufted species. These data, along with information on skeletal morphology, are used to address whether or not a fallback foraging species exhibits a given suite of morphological and behavioral attributes, regardless of habitat. Both tufted species ingest and masticate a number of exceedingly tough plant tissues that appear to be used as fallback resources, however, C. libidinosus has the toughest diet both in terms of median and maximal values. Morphologically, C. libidinosus is intermediate in absolute symphyseal and mandibular measurements, and in measures of postcranial robusticity, but exhibits a higher intermembral index than C. apella. We propose that this incongruence between dietary toughness and skeletal morphology is the consequence of C. libidinosus' use of tools while on the ground for the exploitation of fallback foods. Am J Phys Anthropol 140:687–699, 2009. © 2009 Wiley-Liss, Inc.     

Identification of Protein Tyrosine Phosphatase Receptor Gamma Extracellular Domain (sPTPRG) as a Natural Soluble Protein in Plasma

Background

PTPRG is a widely expressed protein tyrosine phosphatase present in various isoforms. Peptides from its extracellular domain have been detected in plasma by proteomic techniques. We aim at characterizing the plasmatic PTPRG (sPTPRG) form and to identify its source.

Methodology/Principal Findings

The expression of sPTPRG was evaluated in human plasma and murine plasma and tissues by immunoprecipitation and Western blotting. The polypeptides identified have an apparent Mr of about 120 kDa (major band) and 90 kDa (minor band) respectively. Full length PTPRG was identified in the 100.000×g pelleted plasma fraction, suggesting that it was present associated to cell-derived vesicles (exosomes). The release of sPTPRG by HepG2 human hepatocellular carcinoma cell line was induced by ethanol and sensitive to metalloproteinase and not to Furin inhibitors. Finally, increased levels of the plasmatic ∼120 kDa isoform were associated with the occurrence of liver damage.

Conclusions

These results demonstrate that sPTPRG represent a novel candidate protein biomarker in plasma whose increased expression is associated to hepatocyte damage. This observation could open a new avenue of investigation in this challenging field.