CpG drives human transitional B cells to terminal differentiation and production of natural antibodies

The receptor TLR9, recognizing unmethylated bacterial DNA (CpG), is expressed by B cells and plays a role in the maintenance of serological memory. Little is known about the response of B cells stimulated with CpG alone, without additional cytokines. In this study, we show for the first time the phenotypic modification, changes in gene expression, and functional events downstream to TLR9 stimulation in human B cell subsets. In addition, we demonstrate that upon CpG stimulation, IgM memory B cells differentiate into plasma cells producing IgM Abs directed against the capsular polysaccharides of Streptococcus pneumoniae. This novel finding proves that IgM memory is the B cell compartment responsible for the defense against encapsulated bacteria. We also show that cord blood transitional B cells, corresponding to new bone marrow emigrants, respond to CpG. Upon TLR9 engagement, they de novo express AID and Blimp-1, genes necessary for hypersomatic mutation, class-switch recombination, and plasma cell differentiation and produce Abs with anti-pneumococcal specificity. Transitional B cells, isolated from cord blood, have not been exposed to pneumococcus in vivo. In addition, it is known that Ag binding through the BCR causes apoptotic cell death at this stage of development. Therefore, the ability of transitional B cells to sense bacterial DNA through TLR9 represents a tool to rapidly build up the repertoire of natural Abs necessary for our first-line defense at birth.     

The antibiotic polymyxin B modulates P2X7 receptor function

The natural peptide polymyxin B (PMB) is a well-known and potent antibiotic that binds and neutralizes bacterial endotoxin (LPS), thus preventing its noxious effects among LPS-mediated endotoxin shock in animal models. We have investigated the effect of PMB on responses mediated by the P2X(7)R in HEK293 and K562 cells transfected with P2X(7) cDNA and in mouse and human macrophages. In addition, in view of the potential exploitation of P2X(7)-directed agonists in antitumor therapy, we also investigated the effect of PMB in B lymphocytes from patients affected by chronic lymphocytic leukemia. PMB, at an optimal concentration dependent on the given cell type, greatly potentiated the effect of nucleotide-mediated P2X(7) stimulation. In particular, ATP-mediated Ca(2+) influx, plasma membrane permeabilization, and cytotoxicity were enhanced to an extent that, in the presence of PMB, cells were killed by otherwise ineffective nucleotide concentrations. The synergistic effect due to the combined application of ATP and PMB was prevented by incubation with the irreversible P2X blocker oxidized ATP (oATP), but not with the reversible antagonist 1-(N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl)-4-phenilpiperazine (KN-62). Cells lacking P2X(7) were fully insensitive to the combined stimulation with PMB and ATP. Furthermore, PMB at the concentrations used had no untoward effects on cell viability. These results point to PMB as a useful tool for the modulation of P2X(7)R function and suggest that care should be used in the evaluation of ATP-stimulated immune cell responses in the presence of PMB as they may not solely be affected by removal of contaminating LPS.     

Estimated Glomerular Filtration Rate,All-Cause Mortality and Cardiovascular Diseases Incidence in a Low Risk Population: The MATISS Study

Background

Chronic kidney disease (CKD) independently increases the risk of death and cardiovascular disease (CVD) in the general population. However, the relationship between estimated glomerular filtration rate (eGFR) and CVD/death risk in a general population at low risk of CVD has not been explored so far.

Design

Baseline and longitudinal data of 1465 men and 1459 women aged 35-74 years participating to the MATISS study, an Italian general population cohort, were used to evaluate the role of eGFR in the prediction of all-cause mortality and incident CVD.

Methods

Bio-bank stored sera were used to evaluate eGFR at baseline. Serum creatinine was measured on thawed samples by means of an IDMS-calibrated enzymatic method. eGFR was calculated by the CKD-EPI formula.

Results

At baseline, less than 2% of enrolled persons had eGFR < 60 mL/min/1.73m² and more than 70% had a 10-year cardiovascular risk score < 10%. In people 60 or more years old, the first and the last eGFR quintiles (<90 and ≥109 mL/min/1.73m², respectively) were associated to an increased risk for both all-cause mortality (hazard ratio 1.6, 95% confidence interval 1.2-2.1 and 4.3, 1.6-11.7, respectively) and incident CVD (1.6, 1.0-2.4 and 7.0, 2.2-22.9, respectively), even if adjusted for classical risk factors.

Conclusions

These findings strongly suggest that in an elderly, general population at low risk of CVD and low prevalence of reduced renal filtration, even a modest eGFR reduction is related to all-cause mortality and CVD incidence, underlying the potential benefit to this population of considering eGFR for their risk prediction.     

Molecular characterization of the rpoB gene in Brucella species: new potential molecular markers for genotyping

The rpoB gene encoding the beta subunit of the DNA-dependent RNA polymerase was molecularly characterized by PCR amplification and DNA sequencing in 26 Brucella reference strains by using primers selected according to the B. melitensis 16 M rpoB published sequence. Comparison of the rpoB nucleotide sequence of all Brucella strains analysed revealed specific nucleotide variations associated with different Brucella species and biovars. 17 rpoB alleles were recognized and new Brucella typing is proposed. Our results suggest that the rpoB gene polymorphism can be used to identify all Brucella species and most of the biovars, offering an improvement over conventional typing methods.     

In vitro and in silico cloning of Xenopus laevis SOD2 cDNA and its phylogenetic analysis

By using the methodology of both wet and dry biology (i.e., RT-PCR and cycle sequencing, and biocomputational technology, respectively) and the data obtained through the Genome Projects, we have cloned Xenopus laevis SOD2 (MnSOD) cDNA and determined its nucleotide sequence. These data and the deduced protein primary structure were compared with all the other SOD2 nucleotide and amino acid sequences from eukaryotes and prokaryotes, published in public databases. The analysis was performed by using both Clustal W, a well known and widely used program for sequence analysis, and AntiClustAl, a new algorithm recently created and implemented by our group. Our results demonstrate a very high conservation of the enzyme amino acid sequence during evolution, which proves a close structure-function relationship. This is to be expected for very ancient molecules endowed with critical biological functions, performed through a specific structural organization. The nucleotide sequence conservation is less pronounced: this too was foreseeable, due to neutral mutations and to the species-specific codon usage. The data obtained by using AntiClustAl are comparable with those produced with Clustal W, which validates this algorithm as an important new tool for biocomputational analysis. Finally, it is noteworthy that evolutionary trees, drawn by using all the available data on SOD2 nucleotide sequences and amino acid and either Clustal W or AntiClustAl, are comparable to those obtained through phylogenetic analysis based on fossil records.     

Isolation and functional characterization of a cDNA coding a hydroxycinnamoyltransferase involved in phenylpropanoid biosynthesis in <Emphasis Type="Italic">Cynara cardunculus</Emphasis> L

Background  

Cynara cardunculus L. is an edible plant of pharmaceutical interest, in particular with respect to the polyphenolic content of its leaves. It includes three taxa: globe artichoke, cultivated cardoon, and wild cardoon. The dominating phenolics are the di-caffeoylquinic acids (such as cynarin), which are largely restricted to Cynara species, along with their precursor, chlorogenic acid (CGA). The scope of this study is to better understand CGA synthesis in this plant.