Recurrent Sites for New Centromere Seeding

Using comparative FISH and genomics, we have studied and compared the evolution of chromosome 3 in primates and two human neocentromere cases on the long arm of this chromosome. Our results show that one of the human neocentromere cases maps to the same 3q26 chromosomal region where a new centromere emerged in a common ancestor of the Old World monkeys ∼25-40 million years ago. Similarly, the locus in which a new centromere was seeded in the great apes' ancestor was orthologous to the site in which a new centromere emerged in the New World monkeys' ancestor. These data suggest the recurrent use of longstanding latent centromeres and that there is an inherent potential of these regions to form centromeres. The second human neocentromere case (3q24) revealed unprecedented features. The neocentromere emergence was not accompanied by any chromosomal rearrangement that usually triggers these events. Instead, it involved the functional inactivation of the normal centromere, and was present in an otherwise phenotypically normal individual who transmitted this unusual chromosome to the next generation. We propose that the formation of neocentromeres in humans and the emergence of new centromeres during the course of evolution share a common mechanism

Recognition of Neisseria meningitidis by the long pentraxin PTX3 and its role as an endogenous adjuvant.

Long pentraxin 3 (PTX3) is a non-redundant component of the humoral arm of innate immunity. The present study was designed to investigate the interaction of PTX3 with Neisseria meningitidis. PTX3 bound acapsular meningococcus, Neisseria-derived outer membrane vesicles (OMV) and 3 selected meningococcal antigens (GNA0667, GNA1030 and GNA2091). PTX3-recognized microbial moieties are conserved structures which fulfil essential microbial functions. Ptx3-deficient mice had a lower antibody response in vaccination protocols with OMV and co-administration of PTX3 increased the antibody response, particularly in Ptx3-deficient mice. Administration of PTX3 reduced the bacterial load in infant rats challenged with Neisseria meningitidis. These results suggest that PTX3 recognizes a set of conserved structures from Neisseria meningitidis and acts as an amplifier/endogenous adjuvant of responses to this bacterium

FACS analysis of PTX3 binding to <i>Neisseria meningitidis</i>.

<p>Live bacteria were incubated with different doses of human bPTX3 for 1h at room temperature. Results were assessed by flow cytometry. a) Histograms from one out of three experiments performed, showing the negative control (Streptavidin-PE) and two doses of bPTX3 (22 nM and 222 nM); b) Dose response of human bPTX3 (2.2–2200 nM) binding to Nm. Results are MFI ± SD from three independent experiments. Preincubation with cold human PTX3 (1.1 μM—10 min at room temperature) before addition of bPTX3 (222 nM) results in a 86.5% reduction of MFI.</p

Interaction of PTX3 with recombinant proteins from <i>Neisseria meningitidis</i>.

<p>Binding was evaluated on plastic-immobilized proteins and expressed as mean A₄₅₀ ± SD from triplicate wells. a) Plastic wells were coated with the different recombinant proteins (1 μg/well) and incubated with human or murine PTX3, human CRP and SAP (22 pmoles of all proteins) for 1 h at 37°C before addition of the different antibodies. Results are from one out of three independent experiments. b) plastic wells were coated with the indicated Nm antigens (1 μg/well) and incubated with specific polyclonal antibodies (all diluted 1:1000) against the different Nm proteins. c) Plates were coated with recombinant PTX3 (1 μg/well) and binding was evaluated incubating with the indicated Nm antigens (1 μg/well) followed by incubation with specific antibodies. As background control, incubation with Neisserial antigens was omitted while wells were incubated with the specific antibodies.</p

Interaction of PTX3 with OMV.

<p>Microtiter plates were coated with OMV (1 μg/well) and binding of PTX3 (a, b) or CRP (c) is presented as mean absorbance at 450 nm (A₄₅₀) ± SD of triplicate wells. For each experimental setting data are from one out of three independent experiments. a) dose response of bPTX3; b) binding of bPTX3 to coated OMV in the presence or absence of calcium; c) Interaction of CRP with OMV. Microtiter plates were coated with 1 μg/well of OMV or Histone H1 (used as positive control for CRP binding), and incubated with different doses of CRP.</p

Characterization of PTX3 interaction with recombinant proteins from <i>Neisseria meningitidis</i>.

<p>a) Microtiter plate assay of the binding of different doses of bPTX3 to recombinant GNA0667, GNA1030 and GNA2091 (each used at 1 μg/well). b) Binding was performed in the presence or not of calcium. Results are from four independent experiments (mean ± SD of triplicate wells). c) Microtiter plate assay of the binding of recombinant N-terminal or C-terminal PTX3 domains (22 pmoles) to GNA0667, GNA1030 and GNA2091. Data are expressed as percentage of binding compared to recombinant full length PTX3 (mean ± SD from three independent experiments). d) Affinity of the interaction between GNA0667, GNA1030 and GNA2091, and various amount of bPTX3: specific binding was measured in accordance with a standard curve of bPTX3 with non-linear fitting analysis. Data are representative of three experiments.</p

Antibody titer in mice immunized with OVA.

<p>Immune response to OVA was analyzed in WT and <i>ptx3-/-</i> mice. a) OVA was immobilized on plastic wells and presence of coated protein was confirmed by incubation with anti-OVA antibody. b) Binding to immobilized OVA or OMV as control (both at 1 μg/well) was analysed using human bPTX3. Results are reported as A₄₅₀ (mean ± SD of triplicate wells). c) WT and <i>ptx3-/-</i> mice were immunized im with OVA (100 μg/mice ± 2 μg PTX3; each group n = 5) following the same setting used for OMV. Antibody titer was evaluated by ELISA.</p

Protective effect of PTX3 in the infant rat model.

<p>Infant rats were infected ip with 4x10⁴ CFU MenB strain 2996 in association with 0.2 or 2 mg/Kg of human recombinant PTX3 (n = 16 and n = 9 respectively) or vehicle (n = 13). Blood CFU were evaluated 18 hrs after challenge. * p<0.05 (unpaired Student’s <i>t</i> test).</p

Bactericidal activity of serum from mice immunized with OMV.

<p>WT and <i>ptx3-/-</i> mice were immunized by ip or im treatment with OMV and sera were collected two weeks after the last immunization. Data are presented as mean SBA titres ± SD a) comparison of log SBA titres in WT and <i>ptx3-/-</i> mice (** p<0.01, paired Student’s t test; pooled data from ip immunized animals). b) mean SBA titres in representative experiments performed in WT and <i>ptx3-/-</i> mice immunized ip (WT n = 8 <i>ptx3-/-</i> n = 6) or im (WT n = 15, <i>ptx3-/-</i> n = 16) with 0.05 (ip) or 0.5 (im) μg OMV. c) mean SBA titres in WT and <i>ptx3-/-</i> mice immunized with 0.05 μg OMV ± 2 μg PTX3 [one out of three experiments; WT(OMV), n = 8; WT(OMV+PTX3), n = 7; <i>ptx3-/-</i>(OMV), n = 6; <i>ptx3-/-</i>(OMV+PTX3), n = 8]. *p<0.5; ** p<0.01 (Student’s <i>t</i> test).</p