Circadian control of tissue homeostasis and adult stem cells.

The circadian timekeeping mechanism adapts physiology to the 24-hour light/dark cycle. However, how the outputs of the circadian clock in different peripheral tissues communicate and synchronize each other is still not fully understood. The circadian clock has been implicated in the regulation of numerous processes, including metabolism, the cell cycle, cell differentiation, immune responses, redox homeostasis, and tissue repair. Accordingly, perturbation of the machinery that generates circadian rhythms is associated with metabolic disorders, premature ageing, and various diseases including cancer. Importantly, it is now possible to target circadian rhythms through systemic or local delivery of time cues or compounds. Here, we summarize recent findings in peripheral tissues that link the circadian clock machinery to tissue-specific functions and diseases.

Neural differentiation and therapeutic potential of adipose tissue derived stem cells.

Neural tissue has historically been regarded as having poor regenerative capacity but recent advances in the growing fields of tissue engineering and regenerative medicine have opened new hopes for the treatment of nerve injuries and neurodegenerative disorders. Adipose tissue has been shown to contain a large quantity of adult stem cells (ASC). These cells can be easily harvested with low associated morbidity and because of their potential to differentiate into multiple cell types, their use has been suggested for a wide variety of therapeutic applications. In this review we examine the evidence indicating that ASC can stimulate nerve regeneration by both undergoing neural differentiation and through the release of a range of growth factors. We also discuss some of the issues that need to be addressed before ASC can be developed as an effective cellular therapy for the treatment of neural tissue disorders.

A role for altered TLR gene expression in association with increased expression of CD200R in the induction of mucosal tissue CD4(+) Treg in aged mice following gavage with a liver extract along with intramuscular monophosphoryl lipid A (MPLA) injection.

Previous studies showed a fetal sheep liver extract (FSLE), in association with LPS, injected into aged (>20 months) mice reversed the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFN-gamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. Aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+)Treg and so-called Tr3 (CD4(+)TGFbeta(+)). Their number/function is restored to levels seen in control (8-week-old) mice by FSLE. We have reported at length on the ability of a novel pair of immunoregulatory molecules, members of the TREM family, namely CD200:CD200R, to control development of dendritic cells (DCs) which themselves regulate production of Foxp3(+) Treg. The latter express a distinct subset of TLRs which control their function. We report that a feature of the altered Treg expression following combined treatment with FSLE and monophosphoryl lipid A, MPLA (a bioactive component of lipid A of LPS) is the altered gene expression both of distinct subsets of TLRs and of CD200Rs. We speculate that this may represent one of the mechanisms by which FSLE and MPLA alter immunity in aged mice.

Integrative molecular bioinformatics study of human adrenocortical tumors : microRNA, tissue-specific target prediction, and pathway analysis.

MicroRNAs (miRs) are involved in the pathogenesis of several neoplasms; however, there are no data on their expression patterns and possible roles in adrenocortical tumors. Our objective was to study adrenocortical tumors by an integrative bioinformatics analysis involving miR and transcriptomics profiling, pathway analysis, and a novel, tissue-specific miR target prediction approach. Thirty-six tissue samples including normal adrenocortical tissues, benign adenomas, and adrenocortical carcinomas (ACC) were studied by simultaneous miR and mRNA profiling. A novel data-processing software was used to identify all predicted miR-mRNA interactions retrieved from PicTar, TargetScan, and miRBase. Tissue-specific target prediction was achieved by filtering out mRNAs with undetectable expression and searching for mRNA targets with inverse expression alterations as their regulatory miRs. Target sets and significant microarray data were subjected to Ingenuity Pathway Analysis. Six miRs with significantly different expression were found. miR-184 and miR-503 showed significantly higher, whereas miR-511 and miR-214 showed significantly lower expression in ACCs than in other groups. Expression of miR-210 was significantly lower in cortisol-secreting adenomas than in ACCs. By calculating the difference between dCT(miR-511) and dCT(miR-503) (delta cycle threshold), ACCs could be distinguished from benign adenomas with high sensitivity and specificity. Pathway analysis revealed the possible involvement of G2/M checkpoint damage in ACC pathogenesis. To our knowledge, this is the first report describing miR expression patterns and pathway analysis in sporadic adrenocortical tumors. miR biomarkers may be helpful for the diagnosis of adrenocortical malignancy. This tissue-specific target prediction approach may be used in other tumors too.

Isolated limb perfusion with tumor necrosis factor and melphalan for non-resectable soft tissue sarcomas: long-term results on efficacy and limb salvage in a selected group of patients

Background And Objectives: Isolated limb perfusion with TNF-alpha and melphalan (TM-ILP) is a limb salvage therapy for non-resectable soft tissue sarcomas (STS) of the extremities. It is indicated for patients for whom amputation or debilitating surgery is the only alternative. It can be used either as an exclusive therapy (in palliation) or as a neo-adjuvant treatment, followed by marginal resection of tumor remnants with minimal functional impairment. Methods: Between February 1992 and March 2006, 57 TM-ILPs were performed on 51 patients with 88% high grade and 84% advanced stage tumors. Results: Mean follow-up is 38.9 months (4-159, median 22 months). Twenty-one percent patients had significant early complications, with 3 major re-operations, and 23% suffered long-lasting complications. Complete response was observed in 25%, partial response in 42%, stable disease in 14% and progressive disease in 14%. Resection of the tumor remnants was possible in 65%. A complementary treatment was necessary in 31%, mostly radiation therapy. A local recurrence was observed in 35%, after a mean of 20.3 months (2-78), and distant relapse was seen in 45%, after a mean of 13.4 months (5-196). Mean Disease-free survival was 14.9 months, and overall 5-year-survival 43.5%. Amputation rate at 5 years was 24%. Conclusions: TM-ILP is a conservative treatment with a high complications rate, but it can be successful even for the most severe STS of extremities. As a consequence the limb can be spared from amputation or debilitating surgery on the long term in about 75% of patients

The structure of nervous tissue and of Gram-positive bacteria studied by cryo-electron microscopy of vitreous sections

Résumé La structure, ou l'architecture, des êtres vivants définit le cadre dans lequel la physique de la vie s'accomplit. La connaissance de cette structure dans ses moindres détails est un but essentiel de la biologie. Son étude est toutefois entravée par des limitations techniques. Malgré son potentiel théorique, la microscopie électronique n'atteint pas une résolution atomique lorsqu'elle est appliquée ä la matièxe biologique. Cela est dû en grande partie au fait qu'elle contient beaucoup d'eau qui ne résiste pas au vide du microscope. Elle doit donc être déshydratée avant d'être introduite dans un microscope conventionnel. Des artéfacts d'agrégation en découlent inévitablement. La cryo-microscopie électronique des sections vitreuses (CEMOVIS) a ëté développée afin de résoudre cela. Les spécimens sont vitrifiés, c.-à-d. que leur eau est immobilisée sans cristalliser par le froid. Ils sont ensuite coupés en sections ultrafines et celles-ci sont observées à basse température. Les spécimens sont donc observés sous forme hydratée et non fixée; ils sont proches de leur état natif. Durant longtemps, CEMOVIS était très difficile à exécuter mais ce n'est plus le cas. Durant cette thèse, CEMOVIS a été appliqué à différents spécimens. La synapse du système nerveux central a été étudiée. La présence dans la fente synaptique d'une forte densité de molécules organisées de manière périodique a été démontrée. Des particules luminales ont été trouvées dans Ies microtubules cérébraux. Les microtubules ont servi d'objets-test et ont permis de démontrer que des détails moléculaires de l'ordre du nm sont préservés. La compréhension de la structure de l'enveloppe cellulaire des bactéries Grampositives aété améliorée. Nos observations ont abouti à l'élaboration d'un nouveau modèle hypothétique de la synthèse de la paroi. Nous avons aussi focalisé notre attention sur le nucléoïde bactérien et cela a suscité un modèle de la fonction des différents états structuraux du nucléoïde. En conclusion, cette thèse a démontré que CEMOVIS est une excellente méthode poux étudier la structure d'échantillons biologiques à haute résolution. L'étude de la structure de divers aspects des êtres vivants a évoqué des hypothèses quant à la compréhension de leur fonctionnement. Summary The structure, or the architecture, of living beings defines the framework in which the physics of life takes place. Understanding it in its finest details is an essential goal of biology. Its study is however hampered by technical limitations. Despite its theoretical potential, electron microscopy cannot resolve individual atoms in biological matter. This is in great part due to the fact. that it contains a lot of water that cannot stand the vacuum of the microscope. It must therefore be dehydrated before being introduced in a conventional mìcroscope. Aggregation artefacts unavoidably happen. Cryo-electron microscopy of vitreous sections (CEMOVIS) has been developed to solve this problem. Specimens are vitrified, i.e. they are rapidly cooled and their water is immobilised without crystallising by the cold. They are then. sectioned in ultrathin slices, which are observed at low temperatures. Specimens are therefore observed in hydrated and unfixed form; they are close to their native state. For a long time, CEMOVIS was extremely tedious but this is not the case anymore. During this thesis, CEMOVIS was applied to different specimens. Synapse of central nervous system was studied. A high density of periodically-organised molecules was shown in the synaptic cleft. Luminal particles were found in brain microtubules. Microtubules, used as test specimen, permitted to demonstrate that molecular details of the order of nm .are preserved. The understanding of the structure of cell envelope of Gram-positive bacteria was improved. Our observations led to the elaboration of a new hypothetic model of cell wall synthesis. We also focused our attention on bacterial nucleoids and this also gave rise to a functional model of nucleoid structural states. In conclusion, this thesis demonstrated that CEMOVIS is an excellent method for studying the structure of bìologìcal specimens at high resolution. The study of the structure of various aspects of living beings evoked hypothesis for their functioning.

Restoration of lymphoid organ integrity through the interaction of lymphoid tissue-inducer cells with stroma of the T cell zone.

The generation of lymphoid microenvironments in early life depends on the interaction of lymphoid tissue-inducer cells with stromal lymphoid tissue-organizer cells. Whether this cellular interface stays operational in adult secondary lymphoid organs has remained elusive. We show here that during acute infection with lymphocytic choriomeningitis virus, antiviral cytotoxic T cells destroyed infected T cell zone stromal cells, which led to profound disruption of secondary lymphoid organ integrity. Furthermore, the ability of the host to respond to secondary antigens was lost. Restoration of the lymphoid microanatomy was dependent on the proliferative accumulation of lymphoid tissue-inducer cells in secondary lymphoid organs during the acute phase of infection and lymphotoxin alpha(1)beta(2) signaling. Thus, crosstalk between lymphoid tissue-inducer cells and stromal cells is reactivated in adults to maintain secondary lymphoid organ integrity and thereby contributes to the preservation of immunocompetence.

A new technique to improve tissue grip and contact force in arthroscopic capsulolabral repair: the MIBA stitch.

The success of anatomic repair of Bankart lesion diminishes in the presence of a capsule stretching and/or attenuation is reported in a variable percentage of patients with a chronic gleno-humeral instability. We introduce a new arthroscopic stitch, the MIBA stitch, designed with a twofold aim: to improve tissue grip to reduce the risk of soft tissue tear, particularly cutting through capsular-labral tissue, to and address capsule-labral detachment and capsular attenuation using a double loaded suture anchor. This stitch is a combination of horizontal mattress stitch passing through the capsular-labral complex in a "south-to-north" direction and an overlapping single vertical suture passing through the capsule and labrum in a "east-to-west" direction. The mattress stitch is tied before the vertical stitch in order to reinforce the simple vertical stitch, improving grip and contact force between capsular-labral tissue and glenoid bone.

Coagulation Factor Xa Activates Thrombin and Signals Ischemic Neuronal Death Via Par-1 and JNK in Ischemic Neural Tissue

Background: The coagulation factor thrombin mediates ischemic neuronal deathand, at a low concentration, induces tolerance to ischemia.We investigated its modeof activation in ischemic neural tissue using an in vitro approach to distinguish therole of circulating coagulation factors from endogenous cerebral mechanisms. Wealso studied the signalling pathway downstream of thrombin in ischemia and afterthrombin preconditioning.Methods: Rat organotypic hippocampal slice cultures to 30 minute oxygen (5%)and glucose (1 mmol/L) deprivation (OGD).Results: Selective factor Xa (FXa) inhibition by fondaparinux during and afterOGD significantly reduced neuronal death in the CA1 after 48 hours. Thrombinactivity was increased in the medium 24 hours after OGD and this increasewas prevented by fondaparinux suggesting that FXa catalyzes the conversion ofprothrombin to thrombin in neural tissue after ischemia in vitro. Treatment withSCH79797, a selective antagonist of the thrombin receptor protease activatedreceptor-1 (PAR-1), significantly decreased neuronal cell death indicating thatthrombin signals ischemic damage via PAR-1. The JNK pathway plays an importantrole in cerebral ischemia and we observed activation of the JNK substrate,c-Jun in our model. Both the FXa inhibitor, fondaparinux and the PAR-1 antagonistSCH79797, decreased the level of phospho-c-Jun Ser73. After thrombin preconditioningc-Jun was activated by phosphorylation in the nuclei of neurons of the CA1.Treatment with a synthetic thrombin receptor agonist resulted in the same c-Junactivation profile and protection against subsequent OGD indicating that thrombinalso signals via PAR-1 and c-Jun in cell protection.Conclusion: These results indicate that FXa activates thrombin in cerebral ischemia,leading via PAR-1 to the activation of the JNK pathway resulting in neuronal death.Thrombin induced tolerance also involves PAR-1 and JNK, revealing commonfeatures in cell death and survival signalling.

Transscleral Coulomb-controlled iontophoresis of methylprednisolone into the rabbit eye: influence of duration of treatment, current intensity and drug concentration on ocular tissue and fluid levels.

The major problems associated with the use of corticosteroids for the treatment of ocular diseases are their poor intraocular penetration to the posterior segment when administered locally and their secondary side effects when given systemically. To circumvent these problems more efficient methods and techniques of local delivery are being developed. The purposes of this study were: (1) to investigate the pharmacokinetics of intraocular penetration of hemisuccinate methyl prednisolone (HMP) after its delivery using the transscleral Coulomb controlled iontophoresis (CCI) system applied to the eye or after intravenous (i.v.) injection in the rabbit, (2) to test the safety of the CCI system for the treated eyes and (3) to compare the pharmacokinetic profiles of HMP intraocular distribution after CCI delivery to i.v. injection. For each parameter evaluated, six rabbit eyes were used. For the CCI system, two concentrations of HMP (62.5 and 150mg ml(-1)), various intensities of current and duration of treatment were analyzed. In rabbits serving as controls the HMP was infused in the CCI device but without applied electric current. For the i.v. delivery, HMP at 10mg kg(-1)as a 62.5mg ml(-1)solution was used. The rabbits were observed clinically for evidence of ocular toxicity. At various time points after the administration of drug, rabbits were killed and intraocular fluids and tissues were sampled for methylprednisolone (MP) concentrations by high pressure liquid chromatography (HPLC). Histology examinations were performed on six eyes of each group. Among groups that received CCI, the concentrations of MP increased in all ocular tissues and fluids in relation to the intensities of current used (0.4, 1.0 and 2.0mA/0.5cm(2)) and its duration (4 and 10min). Sustained and highest levels of MP were achieved in the choroid and the retina of rabbit eyes treated with the highest current and 10min duration of CCI. No clinical toxicity or histological lesions were observed following CCI. Negligible amounts of MP were found in ocular tissues in the CCI control group without application of current. Compared to i.v. administration, CCI achieved higher and more sustained tissue concentrations with negligible systemic absorption. These data demonstrate that high levels of MP can be safely achieved in intraocular tissues and fluids of the rabbit eye, using CCI. With this system, intraocular tissues levels of MP are higher than those achieved after i.v. injection. Furthermore, if needed, the drug levels achieved with CCI can be modulated as a function of current intensity and duration of treatment. CCI could therefore be used as an alternative method for the delivery of high levels of MP to the intraocular tissues of both the anterior and posterior segments.

Specific delineation of BK polyomavirus in kidney tissue with a digoxigenin-labeled DNA probe.

The detection of BK polyomavirus (BK virus, BKV) in kidney tissue is hampered by nonspecificity of antibodies suited to immunohistochemistry, and nonspecific background with in situ hybridization. The biotin-labeled DNA probe that is commercially available (Enzo Life Sciences, Inc.) shows good signal, but the intrinsic background in kidney tissue is high. We determined that the intrinsic background is due to endogenous biotin or biotin-binding activity in the renal tubular epithelium. Neither antibody blocking procedures nor an avidin/biotin block were entirely satisfactory for eliminating this background staining. We developed a digoxigenin-labeled DNA probe, and protocol, for detecting BK virus in formalin-fixed, paraffin embedded, kidney tissue obtained at autopsy. The hybridization signal is strong and there is no perceptible background staining. Eleven negative control kidneys all failed to hybridize. Conditions for low stringency hybridization may be employed, detecting both the related JC polyomavirus and BKV. Alternatively, high stringency hybridization conditions may be utilized, detecting BKV only. BK associated tubular necrosis is clearly demonstrated in two cases of BK nephritis.

p53 status correlates with histopathological response in patients with soft tissue sarcomas treated using isolated limb perfusion with TNF-alpha and melphalan.

BACKGROUND: Recombinant tumor necrosis factor-alpha (TNF-alpha) combined to melphalan is clinically administered through isolated limb perfusion (ILP) for regionally advanced soft tissue sarcomas of the limbs. In preclinical studies, wild-type p53 gene is involved in the regulation of cytotoxic action of TNF-alpha and loss of p53 function contributes to the resistance of tumour cells to TNF-alpha. The relationship between p53 status and response to TNF-alpha and melphalan in patients undergoing ILP is unknown. PATIENTS AND METHODS: We studied 110 cases of unresectable limbs sarcomas treated by ILP. Immunohistochemistry was carried out using DO7mAb, which reacts with an antigenic determinant from the N-terminal region of both the wild-type and mutant forms of the p53 protein, and PAb1620mAb, which reacts with the 1620 epitope characteristic of the wild-type native conformation of the p53 protein. The immunohistochemistry data were then correlated with various clinical parameters. RESULTS: P53DO7 was found expressed at high levels in 28 patients, whereas PAb1620 was negative in 20. The tumours with poor histological response to ILP with TNF-alpha and melphalan showed significantly higher levels of p53-mutated protein. CONCLUSIONS: Our results might be a clue to a role of p53 protein status in TNF-alpha and melphalan response in clinical use.

Assessment of vascular invasion by bone and soft tissue tumours of the limbs: usefulness of MDCT angiography.

OBJECTIVE: To evaluate the accuracy of computed tomography angiography (CTA) in predicting arterial encasement by limb tumours, by comparing CTA with surgical findings (gold standard). METHODS: Preoperative CTA images of 55 arteries in 48 patients were assessed for arterial status: cross-sectional CTA images were scored as showing a fat plane between artery and tumour (score 0), slight contact between artery and tumour (score 1), partial arterial encasement (score 2) or total arterial encasement (score 3). Reformatted CTA images were assessed for arterial displacement, rigid wall, stenosis or occlusion. At surgery, arteries were classified as free or surgically encased; 45 arteries were free and 10 were surgically encased. RESULTS: Multivariate logistic regression identified the axial CTA score as a relevant predictor for arterial encasement and subsequent vascular intervention during surgery. All sites where CTA showed a fat plane between the tumour and the artery were classified as free at surgery (n = 28/28). The sensitivity of total arterial encasement on CTA (score 3) was 90%, specificity 93%, accuracy 93% and positive likelihood ratio 13.5. CONCLUSION: CTA evidence of total arterial encasement is a highly specific indication of arterial encasement. The presence of fat between the tumour and the artery on CTA rules out arterial involvement at surgery.

The long-term survival of in vitro engineered nervous tissue derived from the specific neural differentiation of mouse embryonic stem cells.

Embryonic stem cells (ESCs) offer attractive prospective as potential source of neurons for cell replacement therapy in human neurodegenerative diseases. Besides, ESCs neural differentiation enables in vitro tissue engineering for fundamental research and drug discovery aimed at the nervous system. We have established stable and long-term three-dimensional (3D) culture conditions which can be used to model long latency and complex neurodegenerative diseases. Mouse ESCs-derived neural progenitor cells generated by MS5 stromal cells induction, result in strictly neural 3D cultures of about 120-mum thick, whose cells expressed mature neuronal, astrocytes and myelin markers. Neurons were from the glutamatergic and gabaergic lineages. This nervous tissue was spatially organized in specific layers resembling brain sub-ependymal (SE) nervous tissue, and was maintained in vitro for at least 3.5 months with great stability. Electron microscopy showed the presence of mature synapses and myelinated axons, suggesting functional maturation. Electrophysiological activity revealed biological signals involving action potential propagation along neuronal fibres and synaptic-like release of neurotransmitters. The rapid development and stabilization of this 3D cultures model result in an abundant and long-lasting production that is compatible with multiple and productive investigations for neurodegenerative diseases modeling, drug and toxicology screening, stress and aging research.