PPARγ Controls Ectopic Adipogenesis and Cross-Talks with Myogenesis During Skeletal Muscle Regeneration.

Skeletal muscle is a regenerative tissue which can repair damaged myofibers through the activation of tissue-resident muscle stem cells (MuSCs). Many muscle diseases with impaired regeneration cause excessive adipose tissue accumulation in muscle, alter the myogenic fate of MuSCs, and deregulate the cross-talk between MuSCs and fibro/adipogenic progenitors (FAPs), a bi-potent cell population which supports myogenesis and controls intra-muscular fibrosis and adipocyte formation. In order to better characterize the interaction between adipogenesis and myogenesis, we studied muscle regeneration and MuSC function in whole body <i>Pparg</i> null mice generated by epiblast-specific Cre/lox deletion ( <i>Pparg <sup>Δ/Δ</sup></i> ). We demonstrate that deletion of PPARγ completely abolishes ectopic muscle adipogenesis during regeneration and impairs MuSC expansion and myogenesis after injury. Ex vivo assays revealed that perturbed myogenesis in <i>Pparg <sup>Δ/Δ</sup></i> mice does not primarily result from intrinsic defects of MuSCs or from perturbed myogenic support from FAPs. The immune transition from a pro- to anti-inflammatory MuSC niche during regeneration is perturbed in <i>Pparg <sup>Δ/Δ</sup></i> mice and suggests that PPARγ signaling in macrophages can interact with ectopic adipogenesis and influence muscle regeneration. Altogether, our study demonstrates that a PPARγ-dependent adipogenic response regulates muscle fat infiltration during regeneration and that PPARγ is required for MuSC function and efficient muscle repair

Nuclear Proteomics Uncovers Diurnal Regulatory Landscapes in Mouse Liver.

Diurnal oscillations of gene expression controlled by the circadian clock and its connected feeding rhythm enable organisms to coordinate their physiologies with daily environmental cycles. While available techniques yielded crucial insights into regulation at the transcriptional level, much less is known about temporally controlled functions within the nucleus and their regulation at the protein level. Here, we quantified the temporal nuclear accumulation of proteins and phosphoproteins from mouse liver by SILAC proteomics. We identified around 5,000 nuclear proteins, over 500 of which showed a diurnal accumulation. Parallel analysis of the nuclear phosphoproteome enabled the inference of the temporal activity of kinases accounting for rhythmic phosphorylation. Many identified rhythmic proteins were parts of nuclear complexes involved in transcriptional regulation, ribosome biogenesis, DNA repair, and the cell cycle and its potentially associated diurnal rhythm of hepatocyte polyploidy. Taken together, these findings provide unprecedented insights into the diurnal regulatory landscape of the mouse liver nucleus

ENHANCED ALLOREACTIVITY TO BI-DIRECTIONAL NON-PERMISSIVE HLA-DPB1 MISMATCHES SUPPORTS NON-HIERARCHICAL T-CELL EPITOPE GROUP DIVERSITY AS UNDERLYING BIOLOGICAL MECHANISM

Transplantation and autoimmunit

A comparison of cytotoxic T lymphocyte precursor frequencies in responder/stimulator pairs with increasing degrees of mismatch

Limiting dilution assays of helper or cytotoxic T cell precursor (HTL-p and CTL-p) frequencies have been developed to measure, with high sensitivity and specificity, donor/recipient alloreactivity before bone marrow transplantation (BMT). Most studies demonstrated a significant correlation between the frequency of donor host-specific CTL-p before transplant and the incidence of severe (≥ grade II) acute graft-versus-host disease (aGvHD) in unrelated donor BMT. Recently we demonstrated that the CTL-p assay was sensitive enough to provide similar predictive information also in HLA-identical sibling BMT. In the present study, the CTL-p frequencies were compared in four groups of responder/stimulator pairs with different degree of mismatch: (1) monozygotic twins (MZ) (n=7), (2) HLA-identical sibling pairs (SIB) (n=67), (3) HLA-matched unrelated pairs (HLA-A, B serologically and HLA-DRBI allele-matched) (MUP) (n=59), (4) 2-4 antigen HLA-mismatched pairs (MM) (n=27). Logistic regression analysis showed that as mismatch increased, the probability of a high (>1:100.000) CTL-p frequency (HF) significantly increased. Then, MUP group was divided in two subgroups: (3a) well-matched pairs (WM) (n=20): matched for HLA-DRB3, B4, B5 alleles and, by DNA heteroduplex analysis, for HLA-A, B genes; (3b) matched pairs (M) (n=39): with either a single HLA-A, B mismatch as revealed by DNA heteroduplex analysis, or a single mismatch in HLA-DRB3, B4, B5 alleles. The CTL-p frequencies were analysed again in the five groups, re-using a logistic regression model. The probability of a HF response was significantly higher in the M than in SIB group. Otherwise, WM and SIB groups had the same probability of a HF response. The probability of a HF response in the M group was significantly higher than in WM group, and lower, though not significantly, than in MM group. The results confirmed not only the power of CTL-p assay in the identification of functionally significant polymorphisms, but also in measuring the degree of mismatch in responder/stimulator pairs. They also showed that when unrelated pairs were more closely matched, CTL-p frequencies approached those of SIB. In conclusion, the CTL-p assay may be an important "in vitro" tool in BMT helping also to predict alloreactivity due to minor histocompatibility antigen differences, which still remain difficult to define despite the improvements in unrelated donor matching. © 2001 Blackwell Science Ltd,

Circulating CD14+ and CD14highCD16− classical monocytes are reduced in patients with signs of plaque neovascularization in the carotid artery

Background and aims: Monocytes are known to play a key role in the initiation and progression of atherosclerosis and contribute to plaque destabilization through the generation of signals that promote inflammation and neoangiogenesis. In humans, studies investigating the features of circulating monocytes in advanced atherosclerotic lesions are lacking. Methods: Patients (mean age 69 years, 56% males) with intermediate asymptomatic carotid stenosis (40–70% in diameter) were evaluated for maximal stenosis in common carotid artery, carotid bulb and internal carotid artery, overall disease burden as estimated with total plaque area (TPA), greyscale and neovascularization in 244 advanced carotid plaques. Absolute counts of circulating CD14+ monocytes, of classical (CD14highCD16-), intermediate (CD14highCD16+) and non-classical (CD14lowCD16+) monocytes and HLA-DR+ median fluorescence intensity for each subset were evaluated with flow cytometry. Results: No correlation was found between monocytes and overall atherosclerotic burden, nor with high sensitivity C-reactive protein (hsCRP) or interleukin-6 (IL-6). In contrast, plaque signs of neovascularization were associated with significantly lower counts of circulating CD14+ monocytes (297 versus 350 cells/mm3, p = 0.039) and of classical monocytes (255 versus 310 cells/mm3, p = 0.029). Conclusions: Neovascularized atherosclerotic lesions selectively associate with lower blood levels of CD14+ and CD14highCD16- monocytes independently of systemic inflammatory activity, as indicated by normal hsCRP levels. Whether the reduction of circulating CD14+ and CD14highCD16- monocytes is due to a potential redistribution of these cell types into active lesions remains to be explored

Loss of fibronectin from the aged stem cell niche affects the regenerative capacity of skeletal muscle in mice

Age-related changes in the niche have long been postulated to impair the function of somatic stem cells. Here we demonstrate that the aged stem cell niche in skeletal muscle contains substantially reduced levels of fibronectin (FN), leading to detrimental consequences for the function and maintenance of muscle stem cells (MuSCs). Deletion of the gene encoding FN from young regenerating muscles replicates the aging phenotype and leads to a loss of MuSC numbers. By using an extracellular matrix (ECM) library screen and pathway profiling, we characterize FN as a preferred adhesion substrate for MuSCs and demonstrate that integrin-mediated signaling through focal adhesion kinase and the p38 mitogen-activated protein kinase pathway is strongly de-regulated in MuSCs from aged mice because of insufficient attachment to the niche. Reconstitution of FN levels in the aged niche remobilizes stem cells and restores youth-like muscle regeneration. Taken together, we identify the loss of stem cell adhesion to FN in the niche ECM as a previously unknown aging mechanism

Loss of fibronectin from the aged stem cell niche affects the regenerative capacity of skeletal muscle in mice

Age-related changes in the niche have long been postulated to impair the function of somatic stem cells. Here we demonstrate that the aged stem cell niche in skeletal muscle contains substantially reduced levels of fibronectin (FN), leading to detrimental consequences for the function and maintenance of muscle stem cells (MuSCs). Deletion of the gene encoding FN from young regenerating muscles replicates the aging phenotype and leads to a loss of MuSC numbers. By using an extracellular matrix (ECM) library screen and pathway profiling, we characterize FN as a preferred adhesion substrate for MuSCs and demonstrate that integrin-mediated signaling through focal adhesion kinase and the p38 mitogen-activated protein kinase pathway is strongly de-regulated in MuSCs from aged mice because of insufficient attachment to the niche. Reconstitution of FN levels in the aged niche remobilizes stem cells and restores youth-like muscle regeneration. Taken together, we identify the loss of stem cell adhesion to FN in the niche ECM as a previously unknown aging mechanism