A multicenter prospective study validated a nomogram to predict individual risk of dependence in ambulation after rehabilitation.

OBJECTIVES: To develop the Functional Risk Index for Dependence in Ambulation (FRIDA) score, a nomogram to predict individual risk of dependence in ambulation at discharge from postacute rehabilitation and validate its performance temporally and spatially. STUDY DESIGN AND SETTING: We analyzed the database of a multicenter prospective observational quality cohort study conducted from January 2012 to March 2016, including data from 8,796 consecutive inpatients who underwent rehabilitation after stroke, hip fracture, lower limb joint replacement, debility, and other neurologic, orthopedic, or miscellaneous conditions. RESULTS: A total of 3,026 patients (34.4%) were discharged dependent in ambulation. In the training set of 5,162 patients (58.7%), Lasso-regression selected advanced age, premorbid disability, and eight indicators of medical and functional adverse syndromes at baseline to establish the FRIDA score. At the temporal validation obtained on an external set of 3,234 patients (41.3%), meta-analyses showed that the FRIDA score had good and homogeneous discrimination (summary area under the curve 0.841, 95% confidence interval = 0.826-0.855, I2 = 0.00%) combined with accurate calibration (summary Log O/E ratio 0.017, 95% confidence interval -0.155 to 0.190). These performances remained stable at spatial validation obtained on 3,626 patients, with substantial heterogeneity of estimates across nine facilities. Decision curve analyses showed that a FRIDA score-supported strategy far outperformed the usual "treat all" approach in each impairment categories. CONCLUSION: The FRIDA score is a new clinically useful tool to predict an individual risk for dependence in ambulation at rehabilitation discharge in many different disabilities, and may also reflect well the case-mix composition of the rehabilitation facilities.

Brain sex-dependent alterations after prolonged high fat diet exposure in mice.

We examined effects of exposing female and male mice for 33 weeks to 45% or 60% high fat diet (HFD). Males fed with either diet were more vulnerable than females, displaying higher and faster increase in body weight and more elevated cholesterol and liver enzymes levels. Higher glucose metabolism was revealed by PET in the olfactory bulbs of both sexes. However, males also displayed altered anterior cortex and cerebellum metabolism, accompanied by a more prominent brain inflammation relative to females. Although both sexes displayed reduced transcripts of neuronal and synaptic genes in anterior cortex, only males had decreased protein levels of AMPA and NMDA receptors. Oppositely, to anterior cortex, cerebellum of HFD-exposed mice displayed hypometabolism and transcriptional up-regulation of neuronal and synaptic genes. These results indicate that male brain is more susceptible to metabolic changes induced by HFD and that the anterior cortex versus cerebellum display inverse susceptibility to HFD.

Skeletal Muscle Proteomic Profile Revealed Gender-Related Metabolic Responses in a Diet-Induced Obesity Animal Model.

Obesity is a chronic, complex pathology associated with a risk of developing secondary pathologies, including cardiovascular diseases, cancer, type 2 diabetes (T2DM) and musculoskeletal disorders. Since skeletal muscle accounts for more than 70% of total glucose disposal, metabolic alterations are strictly associated with the onset of insulin resistance and T2DM. The present study relies on the proteomic analysis of gastrocnemius muscle from 15 male and 15 female C56BL/J mice fed for 14 weeks with standard, 45% or 60% high-fat diets (HFD) adopting a label-free LC-MS/MS approach followed by bioinformatic pathway analysis. Results indicate changes in males due to HFD, with increased muscular stiffness (Col1a1, Col1a2, Actb), fiber-type switch from slow/oxidative to fast/glycolytic (decreased Myh7, Myl2, Myl3 and increased Myh2, Mylpf, Mybpc2, Myl1), increased oxidative stress and mitochondrial dysfunction (decreased respiratory chain complex I and V and increased complex III subunits). At variance, females show few alterations and activation of compensatory mechanisms to counteract the increase of fatty acids. Bioinformatics analysis allows identifying upstream molecules involved in regulating pathways identified at variance in our analysis (Ppargc1a, Pparg, Cpt1b, Clpp, Tp53, Kdm5a, Hif1a). These findings underline the presence of a gender-specific response to be considered when approaching obesity and related comorbidities.

Intracerebral Injection of Extracellular Vesicles from Mesenchymal Stem Cells Exerts Reduced Aß Plaque Burden in Early Stages of a Preclinical Model of Alzheimer's Disease.

Bone marrow Mesenchymal Stem Cells (BM-MSCs), due to their strong protective and anti-inflammatory abilities, have been widely investigated in the context of several diseases for their possible therapeutic role, based on the release of a highly proactive secretome composed of soluble factors and Extracellular Vesicles (EVs). BM-MSC-EVs, in particular, convey many of the beneficial features of parental cells, including direct and indirect ß-amyloid degrading-activities, immunoregulatory and neurotrophic abilities. Therefore, EVs represent an extremely attractive tool for therapeutic purposes in neurodegenerative diseases, including Alzheimer's disease (AD). We examined the therapeutic potential of BM-MSC-EVs injected intracerebrally into the neocortex of APPswe/PS1dE9 AD mice at 3 and 5 months of age, a time window in which the cognitive behavioral phenotype is not yet detectable or has just started to appear. We demonstrate that BM-MSC-EVs are effective at reducing the Aß plaque burden and the amount of dystrophic neurites in both the cortex and hippocampus. The presence of Neprilysin on BM-MSC-EVs, opens the possibility of a direct ß-amyloid degrading action. Our results indicate a potential role for BM-MSC-EVs already in the early stages of AD, suggesting the possibility of intervening before overt clinical manifestations.

Microstructural characterization of corticospinal tract in subacute and chronic stroke patients with distal lesions by means of advanced diffusion MRI.

PURPOSE: The aim of the paper is to evaluate if advanced dMRI techniques, including diffusion kurtosis imaging (DKI) and neurite orientation dispersion and density imaging (NODDI), could provide novel insights into the subtle microarchitectural modifications occurring in the corticospinal tract (CST) of stroke patients in subacute and chronic phases. METHODS: Seventeen subjects (age 68 ± 11 years) in the subacute phase (14 ± 3 days post-stroke), 10 of whom rescanned in the chronic phase (231 ± 36 days post-stroke), were enrolled. Images were acquired using a 3-T MRI scanner with a two-shell EPI protocol (20 gradient directions, b = 700 s/mm2, 3 b = 0; 64 gradient directions, b = 2000 s/mm2, 9 b = 0). DTI-, DKI-, and NODDI-derived parameters were calculated in the posterior limb of the internal capsule (PLIC) and in the cerebral peduncle (CP). RESULTS: In the subacute phase, a reduction of FA, AD, and KA values was correlated with an increase of ODI, RD, and AK parameters, in both the ipsilesional PLIC and CP, suggesting that increased fiber dispersion can be the main structural factor. In the chronic phase, a reduction of FA and an increase of ODI persisted in the ipsilesional areas. This was associated with reduced Fic and increased MD, with a concomitant reduction of MK and increase of RD, suggesting that fiber reduction, possibly due to nerve degeneration, could play an important role. CONCLUSIONS: This study shows that advanced dMRI approaches can help elucidate the underpinning architectural modifications occurring in the CST after stroke. Further follow-up studies on bigger cohorts are needed to evaluate if DKI- and NODDI-derived parameters might be proposed as complementary biomarkers of brain microstructural alterations.

Epigenomics of Neural Cells: REST-Induced Down- and Upregulation of Gene Expression in a Two-Clone PC12 Cell Model.

Cell epigenomics depends on the marks released by transcription factors operating via the assembly of complexes that induce focal changes of DNA and histone structure. Among these factors is REST, a repressor that, via its strong decrease, governs both neuronal and neural cell differentiation and specificity. REST operation on thousands of possible genes can occur directly or via indirect mechanisms including repression of other factors. In previous studies of gene down- and upregulation, processes had been only partially investigated in neural cells. PC12 are well-known neural cells sharing properties with neurons. In the widely used PC12 populations, low-REST cells coexist with few, spontaneous high-REST PC12 cells. High- and low-REST PC12 clones were employed to investigate the role and the mechanisms of the repressor action. Among 15,500 expressed genes we identified 1,770 target and nontarget, REST-dependent genes. Functionally, these genes were found to operate in many pathways, from synaptic function to extracellular matrix. Mechanistically, downregulated genes were predominantly repressed directly by REST; upregulated genes were mostly governed indirectly. Among other factors, Polycomb complexes cooperated with REST for downregulation, and Smad3 and Myod1 participated in upregulation. In conclusion, we have highlighted that PC12 clones are a useful model to investigate REST, opening opportunities to development of epigenomic investigation.

MR imaging monitoring of iron-labeled pancreatic islets in a small series of patients: islet fate in successful, unsuccessful, and autotransplantation.

Islet transplantation is one of the most promising and effective therapies for restoring normoglycemia in type 1 diabetes (T1D) patients, but islet engraftment is one of the main obstacles hampering long-term success. Monitoring graft loss, caused either by immunological or nonimmunological events, occurring in the first phase after transplantation and at later stages of a patient's life is a very important issue. Among the imaging approaches previously applied, magnetic resonance imaging (MRI) monitoring of islet fate following labeling with superparamagnetic iron oxide agents yielded promising results. The aim of this study was to translate into patients the method of islet labeling and MRI monitoring developed in our preclinical setting and to compare imaging results with graft clinical outcome.Three T1D patients and one nondiabetic patient undergoing autotransplantation following subtotal pancreatectomy received Endorem(®)-labeled islets. Patients were monitored by MRI and metabolically (HbA1c, exogenous insulin requirement, and C-peptide, TEF) at 1, 3, and 7 days following transplantation and once a month up to 10 months. Labeled transplanted islets appeared as hypointense areas scattered within the liver parenchyma, whose absolute number at 24 h after transplantation reflected the labeling efficiency. In patients #1 and #3 with good midterm graft function, MRI follow-up showed an important early loss of hypointense spots followed by a slow and progressive disappearance at later timepoints. Graft loss of function in patient #2 4 weeks after transplantation was associated with the complete disappearance of all hypointense signals. The autotransplanted patient, stably insulin free, showed no significant signal reduction during the first 3 days, followed by loss of spots similar to a patient with good midterm graft function. These results suggest that MRI monitoring of islet transplantation at early time points could represent a meaningful readout for helping in predicting transplant failure or success, but its relevance for mid/long-term islet function assessment appears evanescent.

Tyrosine kinase signal modulation: a matter of H2O2 membrane permeability?

Abstract H2O2 produced by extracellular NADPH oxidases regulates tyrosine kinase signaling inhibiting phosphatases. How does it cross the membrane to reach its cytosolic targets? Silencing aquaporin-8 (AQP8), but not AQP3 or AQP4, inhibited H2O2 entry into HeLa cells. Re-expression of AQP8 with silencing-resistant vectors rescued H2O2 transport, whereas a C173A-AQP8 mutant failed to do so. Lowering AQP8 levels affected H2O2 entry into the endoplasmic reticulum, but not into mitochondria. AQP8 silencing also inhibited the H2O2 spikes and phosphorylation of downstream proteins induced by epidermal growth factor. These observations lead to the hypothesis that H2O2 does not freely diffuse across the plasma membrane and AQP8 and other H2O2 transporters are potential targets for manipulating key signaling pathways in cancer and degenerative diseases.

On/off TLR signaling decides proinflammatory or tolerogenic dendritic cell maturation upon CD1d-mediated interaction with invariant NKT cells.

Invariant NKT (iNKT) cells play an effector/adjuvant function during antimicrobial and antitumoral immunity and a regulatory role to induce immune tolerance and prevent autoimmunity. iNKT cells that differentially modulate adaptive immunity do not bear a unique phenotype and/or specific cytokine secretion profile, thus opening questions on how a single T cell subset can exert opposite immunological tasks. In this study, we show that iNKT cells perform their dual roles through a single mechanism of action relying on the cognate interaction with myeloid dendritic cells (DCs) and leading to opposite effects depending on the presence of other maturation stimuli simultaneously acting on DCs. The contact of murine purified iNKT cells with immature autologous DCs directly triggers the tolerogenic maturation of DCs, rendering them able to induce regulatory T cell differentiation and prevent autoimmune diabetes in vivo. Conversely, the interaction of the same purified iNKT cells with DCs, in the presence of simultaneous TLR4 stimulation, significantly enhances proinflammatory DC maturation and IL-12 secretion. The different iNKT cell effects are mediated through distinct mechanisms and activation of different molecular pathways within the DC: CD1d signaling and activation of the ERK1/2 pathway for the tolerogenic action, and CD40-CD40L interaction and NF-κB activation for the adjuvant effect. Our data suggest that the DC decision to undergo proinflammatory or tolerogenic maturation results from the integration of different signals received at the time of iNKT cell contact and could have important therapeutic implications for exploiting iNKT cell adjuvant/regulatory properties in autoimmune diseases, infections, and cancer.

Expression of the neurosecretory process in PC12 cells is governed by REST.

The neurosecretory process is acquired during differentiation and can be lost en block by differentiated cells. To investigate the role of REST/NRSF, a transcription repressor, in the maintenance of the process we studied two PC12 clones, one wt and one defective, expressing low and high levels of endogenous RE-1 silencing transcription (factor) (REST), respectively. Stable transfection of constructs demonstrated that REST represses 10 genes coding for proteins of neurosecretory vesicles and their exocytosis, eight including and two lacking the REST-binding sequence, RE-1. Of these genes, those of chromogranins were strongly repressed by fewfold increases of REST, those of VAMP2 and syntaxin1a required much higher levels. Moreover, in wt cells transfected with an active construct the dense-core vesicles, still competent for regulated exocytosis, were much smaller, with lighter cores; in defective cells, the dominant-negative construct induced the rescue of many vesicle/exocytosis genes but not of those of chromogranins. Small dense-core vesicles, exocytized upon stimulation, were rescued when the construct-transfected defective cells were transfected also with chromograninA or treated with trichostatinA, a blocker of histone deacetylases. Our results identify REST, working by direct and indirect mechanisms, as the factor governing the maintenance of the neurosecretory process and the properties of dense-core vesicles in PC12 cells.

Monoclonal antibody 76F distinguishes IA-2 from IA-2beta and overlaps an autoantibody epitope.

IA-2 and IA-2beta are highly related proteins that are autoantigens in type 1 diabetes, and provide a model for developing reagents and assays that distinguish similar proteins with unique autoantibody epitopes. Monoclonal antibodies (mAb) to IA-2 and IA-2beta were prepared and tested for their ability to bind to the related proteins and their ability to compete for specific autoantibody epitope binding by sera from patients with type 1 diabetes. Monoclonal antibodies that specifically bound IA-2 (76F) or bound both IA-2 and IA-2beta (A9) were isolated and characterized. 76F mAb recognized IA-2 of human, rat and mouse origin in native and denatured forms and had an epitope specificity for residues 626-630 (FEYQD) which are found in the juxtamembrane (JM) region of human and mouse IA-2, but not IA-2beta. This region overlaps with the autoantibody epitope JM2. Binding to the 76F monoclonal antibody was specifically inhibited by sera with antibodies to the JM2 epitope but not with antibodies to the adjacent JM1 epitope, indicating that unique epitopes can be distinguished by this approach. 76F mAb has the unique property to distinguish between the two closely related autoantigens IA-2 and IA-2beta by targeting an IA-2 specific epitope of the juxtamembrane region. The findings define an approach to develop assays for specific antibody epitope measurements which may be relevant for disease prognosis and monitoring intervention therapies.

Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are stromal cells with the ability to proliferate and differentiate into many tissues. Although they represent powerful tools for several therapeutic settings, mechanisms regulating their migration to peripheral tissues are still unknown. Here, we report chemokine receptor expression on human BM-MSCs and their role in mediating migration to tissues. A minority of BM-MSCs (2% to 25%) expressed a restricted set of chemokine receptors (CXC receptor 4 [CXCR4], CX3C receptor 1 [CX3CR1], CXCR6, CC chemokine receptor 1 [CCR1], CCR7) and, accordingly, showed appreciable chemotactic migration in response to the chemokines CXC ligand 12 (CXCL12), CX3CL1, CXCL16, CC chemokine ligand 3 (CCL3), and CCL19. Using human pancreatic islets as an in vitro model of peripheral tissue, we showed that islet supernatants released factors able to attract BM-MSCs in vitro, and this attraction was principally mediated by CX3CL1 and CXCL12. Moreover, cells with features of BM-MSCs were detected within the pancreatic islets of mice injected with green fluorescent protein (GFP)-positive BM. A population of bona fide MSCs that also expressed CXCR4, CXCR6, CCR1, and CCR7 could be isolated from normal adult human pancreas. This study defines the chemokine receptor repertoire of human BM-MSCs that determines their migratory activity. Modulation of homing capacity may be instrumental for harnessing the therapeutic potential of BM-MSCs.

Dense-core granules: a specific hallmark of the neuronal/neurosecretory cell phenotype.

Expression of dense-core granules, a typical exocytic organelle, is widely believed to be controlled by coordinate gene expression mechanisms specific to neurones and neurosecretory cells. Recent studies in PC12 cells, however, have suggested the number of granules/cells depends on the levels of only one of their cargo proteins, chromogranin A, regulating the metabolism of the other proteins, and thus the composition of the organelles, by an on/off switch mechanism. In addition, transfection of chromogranin A was reported to induce appearance of dense-core granules in the non-neurosecretory fibroblasts of the CV-1 line. Here the role of chromogranin A has been reinvestigated using not the heterogeneous PC12 line but several clones isolated therefrom. In these clones, investigated as such or after transfection with chromogranin A antisense sequences, the ratio between chromogranin A and its secretory protein mate, chromogranin B, was not constant but highly and apparently randomly variable. Variability of the chromogranin A/chromogranin B ratio was seen by confocal immunofluorescence also among the cells of single clones and subclones and among the granules of single cells. Moreover, stable and transient transfections of chromogranin A in a PC12 clone characterised by a low number of dense-core granules (one fifth of the reference clone) failed to modify significantly the number of the organelles, despite the several-fold increase of the granin. Finally, in three types of non-neurosecretory cells (CV-1, adenocarcinoma TS/A and a clone of PC12 incompetent for secretion) the transfected chromogranin A accumulated mostly in the Golgi/transGolgi area and was released rapidly from resting cells (constitutive secretion) as revealed by both immunofluorescence during cycloheximide treatment and pulse-chase experiments. Only a minor fraction was sorted to discrete organelles that were not dense-core granules, but primarily lysosomes because they contained no chromogranin B, and were largely positive for the late endosomal-lysosomal markers, lamp1 and lamp3. Dense-core granules are therefore true hallmarks of neurones and neurosecretory cells. Their number/cell appears independent of chromogranin A and their composition does not appear to be constant; in particular, they exhibit considerable, and so far unexplained variability in the chromogranin A/chromogranin B ratio.

Neurosecretion competence. A comprehensive gene expression program identified in PC12 cells.

The phenotype of neurosecretory cells is characterized by clear vesicles and dense granules, both discharged by regulated exocytosis. However, these organelles are lacking completely in a few neurosecretion-incompetent clones of the pheochromocytoma PC12 line, in which other specific features are maintained (incompetent clones). In view of the heterogeneity of PC12 cells, a differential characterization of the incompetent phenotype based on the comparison of a single incompetent and a single wild-type clone would have been inconclusive. Therefore, we have compared two pairs of PC12 clones, studying in parallel the transcript levels of 4,200 genes and 19,000 express sequence tags (ESTs) by high density oligonucleotide arrays. After accurate data processing for quality control and filtration, a total of 755 transcripts, corresponding to 448 genes and 307 ESTs, was found consistently changed, with 46% up-regulated and 54% down-regulated in incompetent versus wild-type clones. Many but not all neurosecretion genes were profoundly down-regulated in incompetent cells. Expression of endocytosis genes was normal, whereas that of many nuclear and transcription factors, including some previously shown to play key roles in neurogenesis, was profoundly changed. Additional differences appeared in genes involved in signaling and metabolism. Taken together these results demonstrate for the first time that expression of neurosecretory vesicles and granules is part of a complex gene expression program that includes many other features that so far have not been recognized.