Vascular injury derived apoptotic exosome-like vesicles trigger autoimmunity.

According to a central tenet of classical immune theory, a healthy immune system must avoid self-reactive lymphocyte clones but we now know that B cells repertoire exhibit some level of autoreactivity. These autoreactive B cells are thought to rely on self-ligands for their clonal selection and survival. Here, we confirm that healthy mice exhibit self-reactive B cell clones that can be stimulated in vitro by agonists of toll-like receptor (TLR) 1/2, TLR4, TLR7 and TLR9 to secrete anti-LG3/perlecan. LG3/perlecan is an antigen packaged in exosome-like structures released by apoptotic endothelial cells (ApoExos) upon vascular injury. We demonstrate that the injection of ApoExos in healthy animals activates the IL-23/IL-17 pro-inflammatory and autoimmune axis, and produces several autoantibodies, including anti-LG3 autoantibodies and hallmark autoantibodies found in systemic lupus erythematosus. We also identify γδT cells as key mediators of the maturation of ApoExos-induced autoantibodies in healthy mice. Altogether we show that ApoExos released by apoptotic endothelial cells display immune-mediating functions that can stimulate the B cells in the normal repertoire to produce autoantibodies. Our work also identifies TLR activation and γδT cells as important modulators of the humoral autoimmune response induced by ApoExos.

X-linked hypophosphatemia: The value of feedback focus groups to assess patient and caregiver needs.

X-linked hypophosphatemia (XLH) is a rare, multi-systemic, invalidating disease requiring a multi-disciplinary approach. No specific action in XLH, neither for the patients' specific needs nor for the methodology for the evaluation of these were found. Thus, to identify the needs of XLH patients and their caregivers, we organised focus groups in our reference centre with a view to build educational sessions. Focus groups including either XLH children, XLH adults, or caregivers ran in parallel. Each group was led by a person trained in therapeutic education (nurse, paediatric nephrologist) with another healthcare provider specialised in XLH (rheumatologist, nephrologist). One additional person with knowledge of XLH (clinical research associate, paediatric resident) took minutes. The duration of each session was 1.5h; XLH patients/caregivers were asked to answer age-adapted "open questions" on their daily life and quality of life. At the end, a global restitution was made. The needs identified were later grouped and analysed, which allowed us to build the educational sessions. The XLH children group included 5 children, the XLH adults group included 10 adults, and the caregivers group included 6 parents or partners. Major needs were identified: knowledge of XLH, treatment, dental care and adapted physical activity, with additional questions on socio-professional adaptations and financial support in adults. Partner patients were also identified to co-build the support programme. The study allowed us to identify the needs of XLH patients and their caregivers using the focus group method and then, using these needs, to build educational sessions and a therapeutic education programme for XLH patients.

Dynamics of heme in hemoproteins: proton NMR study of myoglobin reconstituted with iron 3-ethyl-2-methylporphyrin.

The asymmetric 3-ethyl-2-methylporphyrin iron complex was synthetized and inserted into apomyoglobin. UV-visible spectroscopic studies demonstrated the capacity of iron to coordinate different exogenous axial ligands in ferrous and ferric forms. The position of synthetic heme into the hydrophobic pocket of the reconstituted myoglobin was investigated by ((1))H NMR spectroscopy. In absence of exogenous ligand, signals of the synthetic prosthetic group were not detected, suggesting a rotational disorder of the synthetic porphyrin into the heme pocket. This direct interconversion behavior is favored since site-specific interactions between the poorly substituted heme and protein in the chiral hydrophobic cavity were weak. Complexion of cyanide to the iron allowed to quench partially the heme reorientation and two interconvertible forms, around the meso-Cα-Cγ axis, were detected in solution.

13C NMR studies of the electronic structure of low-spin iron(III) tetraphenylchlorin complexes.

A series of low-spin six-coordinate (tetraphenylchlorinato)iron(III) complexes [Fe(TPC)(L)2]+/- (L = 1-MeIm, CN-, 4-CNPy, and (t)BuNC) have been prepared, and their (13)C NMR spectra have been examined to reveal the electronic structure. These complexes exist as the mixture of the two isomers with the (d(xy))2(d(xz), d(yz))3 and (d(xz), d(yz))4(d(xy))1 ground states. Contribution of the (d(xz), d(yz))4(d(xy))1 isomer has increased as the axial ligand changes from 1-MeIm, to CN(-) (in CD2Cl2 solution), CN- (in CD(3)OD solution), and 4-CNPy, and then to tBuNC as revealed by the meso and pyrroline carbon chemical shifts; the meso carbon signals at 146 and -19 ppm in [Fe(TPC)(1-MeIm)2]+ shifted to 763 and 700 ppm in [Fe(TPC)(tBuNC)2]+. In the case of the CN- complex, the population of the (d(xz), d(yz))4(d(xy))1 isomer has increased to a great extent when the solvent is changed from CD2Cl2 to CD3OD. The result is ascribed to the stabilization of the d(xz) and d(yz) orbitals of iron(III) caused by the hydrogen bonding between methanol and the coordinated cyanide ligand. Comparison of the 13C NMR data of the TPC complexes with those of the TPP, OEP, and OEC complexes has revealed that the populations of the (d(xz), d(yz))4(d(xy))1 isomer in TPC complexes are much larger than those in the corresponding TPP, OEC, and OEP complexes carrying the same axial ligands.

Proton NMR study of myoglobin reconstituted with 3, 7-diethyl-2, 8-dimethyl iron porphyrin: remarkable influence of peripheral substitution on heme rotation.

The iron complex of 3,7-diethyl-2,8-dimethylporphyrin was incorporated into horse heart apomyoglobin to investigate the influence of peripheral substitution on artificial heme rotation. The hyperfine-shifted 1H NMR spectrum of the reconstituted deoxymyoglobin (rMb) revealed the proximal imidazole N-H resonance at 82.5 ppm to indicate the formation of the Fe--N (His93) bond. The pyrrole-protons of the hemin of myoglobin in the absence of external ligand appeared as four resonances between -10 and -18 ppm, indicating a mainly low-spin ferric hemin, with a ligated distal histidine (His64). This also indicates the lost of the symmetry of the hemin, according to an absence of free rotation of the prosthetic group. The 1H NMR spectrum of reconstituted rMbCO revealed a set of four pyrrole-protons and a set of four meso-protons. Accordingly, the prosthetic group without acid side chains interacts specifically with the surrounding globin showing a unique heme orientation in the 1H NMR time-scale, despite the presence of only four alkyl substituents on the porphine ring. This also suggests that two ethyl groups are large enough to avoid the free rotation movement of the heme.