FoxP3+ regulatory T cells essentially contribute to peripheral CD8+ T-cell tolerance induced by steady-state dendritic cells.

Peripheral T-cell tolerance is thought to significantly contribute to the prevention of autoimmunity, and it has been shown that antigen-presenting steady-state dendritic cells efficiently induce peripheral tolerance. We previously showed that dendritic-cell-induced tolerance is a T-cell-intrinsic process that depends on coinhibitory molecules such as programmed death-1. Here we specifically analyze the involvement of FoxP3(+) regulatory T cells, which are known to be important for maintenance of self-tolerance. We show that antigen presentation by steady-state dendritic cells failed to induce peripheral tolerance in the absence of FoxP3(+) regulatory T cells but induced protective CD8(+) T-cell-mediated immunity instead. Regulatory T-cell-depleted mice had massively increased numbers of dendritic cells in lymph nodes. Dendritic cells isolated from mice without regulatory T cells had up-regulated costimulatory molecules and showed stronger T-cell stimulatory capacity ex vivo, suggesting that regulatory T cells contribute to peripheral tolerance by keeping the dendritic cells in an immature state. Using blocking antibodies, we demonstrate that CTLA-4 but not IL-10 is necessary for control of dendritic cells by regulatory T cells.

Estimation of joint incongruence in dysplastic canine elbows before and after dynamic proximal ulnar osteotomy.

OBJECTIVE: To characterize joint incongruence in dysplastic canine elbows before and after dynamic proximal ulnar osteotomy (DPUO). STUDY DESIGN: Clinical, prospective study. ANIMALS: Dogs (n = 10; 12 elbows) with ≥ 2 mm radioulnar incongruence (RUI), FCP, and/or OCD. METHODS: Computed tomography in a nonload bearing position was used to generate in silico 3D models of the elbow joint before DPUO, and this was repeated after DPUO union (median 3 months postoperatively). On these models, RUI, humeroradial and humeroulnar subchondral joint space width (SJSW) as well as alignment of the proximal ulnar segment were investigated. RESULTS: RUI at the medial coronoid process (MCP) decreased (P = .001), while it increased at the lateral coronoid process (P = .0005), and remained unchanged at the level of the trochlear ridge (P = .25). SJSW at the MCP increased (P = .001), changing from focal joint space collapse preoperatively to a more homogeneous pattern of moderate SJSW at follow-up. The proximal ulnar segment rotated in all 3 planes with caudal tipping at the level of the osteotomy and varus deformity, while no axial translation could be noted. CONCLUSIONS: DPUO results in reduction of RUI and amelioration of focal contact area at the MCP. This effect is the result of a complex three-dimensional rotation of the proximal ulnar segment, rather than axial shift.

CMV promoter is inadequate for expression of mutant human RyR2 in transgenic rabbits.

INTRODUCTION: Fundamental differences in Ca²+ homeostasis between mice and larger mammals require the validation of the mechanisms of arrhythmogenesis before translation into human pathophysiology. The purpose of this study was to create transgenic rabbits that express defective human cardiac ryanodine receptor (hRyR2) with a mutation (R4497C) causing a clinically relevant arrhythmogenic syndrome. METHODS: The construct pcDNA3-EGFP-hRyR2-R4497C with the CMV promoter was used to generate transgenic rabbits. The founder animals were created by microinjection and identified by PCR with specific primers for the EGFP sequence. The copy number of the transgene was quantified by real-time PCR using genomic DNA from blood cells. mRNA expression of EGFP-hRyR2-R4497C was quantified using RT-PCR with specific primers for the RyR2 and EGFP sequence. Protein expression of the transgene in heart and non-cardiac tissues was determined using immunoblots with antibodies directed against EGFP and RyR2. RESULTS: Real-time PCR in peripheral blood cells identified several rabbit lines with the construct integrated into their genome. Transcription levels of the transgene were low (Ct>30). On the protein level, neither EGFP nor hRyR2 R4497C was detected in either cardiac or non-cardiac tissue. A truncated gene product (3' end and central part of hRyR2 R4497C, but not EGFP) could be detected at the mRNA level in the heart. DISCUSSION: Lack of significant protein expression of the EGFP-RyR2 R4497C gene construct despite successful incorporation into the genomic DNA is due to combination of at least two factors: low mRNA expression, and truncation of the transgene on the mRNA level. Our results suggest that the CMV promoter may not be well suited for creating transgenic rabbits.

Oxytocin receptors: ligand binding, signalling and cholesterol dependence.

The G protein coupled oxytocin receptor (OTR) reveals some specific molecular and physiological characteristics. Ligand-receptor interaction has been analysed by photoaffinity labelling, site-directed mutagenesis, the construction of receptor chimeras and molecular modelling. Major results of these studies will be summarized. The N-terminus of the OTR is mainly involved in agonist binding. Notably, antagonists that are derived from the ground structure of oxytocin, bind the receptor at distinct sites partly non-overlapping with the agonist binding site. OTRs are able to couple to different G proteins, with a subsequent stimulation of phospholipase C-beta isoforms. In dependence on G protein coupling, OTRs can transduce growth-inhibitory or proliferatory signals. Some evidence is provided that OTRs are also present in form of dimeric or oligomeric complexes at the cell surface. The affinity of the receptor for ligands is strongly dependent on the presence of divalent cations (Mg(2+)) and cholesterol that both act like positive allosteric modulators. While the high-affinity state of the receptor for agonists requires divalent cations and cholesterol, the high-affinity state for antagonists is only dependent on a sufficient amount of cholesterol. Cholesterol affects ligand-binding affinity, receptor signalling and stability. Since the purification of the OTR has never been achieved, alternative methods to study the receptor in its native environment are necessary. Promising strategies for the site-specific labelling of the OTR will be presented. The employment of diverse reporter molecules introduced at different positions within the OTR might allow us in the near future to measure conformational changes of the receptor in its native lipid environment.