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Functional vascular smooth muscle-like cells derived from adult mouse uterine mesothelial cells.
In mammalian visceral organs, vascular smooth muscle cells (VSMCs) originate from an epithelial-to-mesenchymal transition (EMT) of embryonic mesothelial cells (MCs). The ability of adult MCs to recapitulate EMT and to acquire smooth muscle (SM) markers upon provasculogenic culture suggested they might retain embryonic vasculogenic differentiation potential. However, it remains unknown whether adult MCs-derived SM-like cells may acquire specific vascular SM lineage markers and the functionality of differentiated contractile VSMCs. Here, we describe how a gentle trypsinization of adult mouse uterine cords could selectively detach their outermost uterine mesothelial layer cells. As other MCs; uterine MCs (UtMCs) uniformly expressed the epithelial markers β-catenin, ZO-1, E-cadherin, CD54, CD29, and CK18. When cultured in a modified SM differentiation media (SMDM) UtMCs initiated a loss of epithelial characteristics and gained markers expression of EMT (Twist, Snail, and Slug), stem and progenitor (Nanog, Sox2, C-kit, Gata-4, Isl-1, and nestin), SM (α-SMA, calponin, caldesmon, SM22α, desmin, SM-MHC, and smoothelin-B) and cardiac (BMP2, BMP4, ACTC1, sACTN, cTnI, cTnT, ANF, Cx43, and MLC2a). UtMCs repeatedly subcultured in SMDM acquired differentiated VSM-like characteristics and expressed smoothelin-B in the typical stress-fiber pattern expression of contractile VSMCs. Relevantly, UtMCs-derived VSM-like cells could generate "mechanical force" to compact collagen lattices and displayed in diverse degree voltage (K(+)) and receptor (endothelin-1, oxytocin, norepinephrine, carbachol and vasopressin)-induced [Ca(2+)](i) rises and contraction. Thus, we show for the first time that UtMCs could recapitulate in vitro differentiative events of early cardiovascular differentiation and transdifferentiate in cells exhibiting molecular and functional characteristics of VSMCs.
Generation of Pancreatic Islets from Stem Cells
Diabetes mellitus is one of the most prevalent chronic diseases. Glucose homeostasis disruption occurs when β-cells fail to secrete the insulin necessary to maintain the homeostasis of glucose in the blood flow. Over time, diabetes can lead to the rise of different long-term complications, such as diabetic foot, retinopathy, neuropathy, nephropathy and arteriosclerosis. Nowadays, the only treatments for diabetes consist of exogenous insulin supply or pancreas/islet transplantation, but the inability to achieve a tight control over glucose regulation by exogenous insulin administration and the shortage of pancreatic islets donors have motivated recent efforts to develop renewable sources and protocols for effective β-cell replacement.Embryonic stem cells are non-specialized cells that share two important characteristics: self -renewal, which allows them to expand indefinitely while maintaining the undifferentiated state; and pluripotency, which is the capacity to differentiate into almost all specialized cell types. Proof-of-concept experiments demonstrate that embryonic stem cells have the ability to differentiate into insulin-producing cells, even if at a very low frequency.In this chapter, we review the attempts that have been made thus far to convert embryonic stem cells into pancreatic endocrine cell types of potential use in the treatment of type I diabetes. © 2014 Elsevier Inc. All rights reserved.
On reducing vibration transmission in a two-dimensional cantilever truss structure using geometric optimization and active vibration control techniques
Four optimization strategies were used to improve the average vibration isolation between the base and the end of a 10-m long two-dimensional (2D) cantilever truss structure. These were combinations of optimizing the structure geometry and the application of active vibration control (AVC) with optimal actuator positions. A power distribution analysis to investigate the mechanisms by which each strategy achieves reductions in the vibration transmission is reported. The trade-off is also explored between the freedom allowed in the size of the geometric changes and the number of actuators used in an AVC system to achieve a given level of vibration attenuation. © 2001 Acoustical Society of America.
Effect of methylprednisolone on the ulceration, matrix metalloproteinase distribution and eicosanoid production in a model of colitis in the rabbit.
This study has examined the response of a rabbit model of inflammatory bowel disease to methylprednisolone. Colitis was induced in the colon of rabbits with 40 mg trinitrobenzenesulphonic acid in 25% ethanol (TNBS). The effect of methylprednisolone (0.5 mg/kg/day) on the development of colitis was determined at one week, by examining the colon's macroscopic and microscopic appearance, the distribution of matrix metalloproteinases (MMPs) and by measuring eicosanoid production. Although there was no difference in the area of ulcerated colonic tissue in the treated and untreated TNBS animals, the increase in polymorphonuclear leucocytes was significantly reduced in TNBS rabbits given methylprednisolone. The only difference in the distribution of MMPs was a reduction in the number of polymorphonuclear leucocytes containing gelatinase B. The release of immunoreactive PGE2 and LTB4, but not 6-keto PGF1 alpha, was increased in the TNBS animals and was unchanged by methylprednisolone. These results show that methylprednisolone does not modify the injury produced by TNBS in this model despite reducing the infiltration of polymorphonuclear leucocytes. Hence it suggests that these cells do not contribute to the injury observed, are not the source of the eicosanoids and that gelatinase B is not required in the healing process in this model.
NEUROBID--an EU-funded project to study the developing brain barriers.
Brain diseases are one of the most prevalent groups of diseases in Europe with estimated annual costs amounting to euro386 billion. Data collected by the WHO suggest that brain diseases are responsible for 35% of Europe's total disease burden. In the treatment of neurological disease, the blood brain barrier (BBB) still represents an obstacle for the delivery of drugs to the brain and thus a major challenge for the development of therapeutic regimens. Understanding the molecular basis and functioning of the BBB in health and disease, including transport mechanisms across the BBB, therefore holds significant potential for future strategies to prevent and ameliorate neurological disease. Recent research indicates that some neurological disorders have a developmental etiologic component. The major goal of the NEUROBID project is thus to understand the molecular mechanisms and function of the BBB in health and disease both in the developing brain and the adult central nervous system. With an interdisciplinary consortium from the fields of developmental neurobiology and BBB research, NEUROBID aims to (i) understand the involvement of normal and disturbed BBB function in normal and abnormal brain development and (ii) to develop novel strategies for drug delivery to the brain. Unique transport mechanisms across the BBB will be used to target potential therapeutic macromolecular and cellular agents specifically to the brain barriers and transport them into the brain. The main target disorders of NEUROBID are non-inherited neurodevelopmental disorders arising from perinatal adverse exposure, such as cerebral palsy, and classic adult neurological disorders such as multiple sclerosis and stroke. In the long term, NEUROBID hopes to pave the way for new treatment strategies and thus reduce the economic and social burden of neurological disease.
Modulation of human neutrophil function by fibronectin degradation products isolated from cryoglobulins.
The proteolytic fragments of native fibronectin (cryopeptides) complexed to pathological immunoglobulins in cryoglobulins were isolated from the serum of six different patients. Two peptides of 117,000 daltons and 70,000 daltons were purified and characterized by N-terminal amino acid sequencing. The priming effect of the mixture of these two peptides, referred to as cryopeptides, on neutrophil functions, namely the respiratory burst, exocytosis, and Ca2+ release, was investigated. Cryopeptides and cryoglobulins assayed separately did not increase neutrophil respiration or cytosol free calcium concentration; however, both of them induced granule enzyme secretion. Sequential exposure of neutrophils to either cryopeptides or the respective cryoglobulins, and then to N-formyl-methionyl-leucyl-phenylalanine or serum opsonized zymosan, resulted in a synergistic stimulation of O2 uptake compared to the effect of N-formyl-peptides or opsonized zymosan tested separately; Ca2+ release was significantly enhanced by the pretreatment of neutrophils with cryopeptides. These data suggest that cryopeptides and cryoglobulins may play a role in host defense against bacterial infections through neutrophil activation.
The contribution of the acute phase response to the pathogenesis of relapse in chronic-relapsing experimental autoimmune encephalitis models of multiple sclerosis
Background: Increased relapse rates in multiple sclerosis (MS) as a consequence of peripheral immune system activation, owing to infection for example, have been widely reported, but the mechanism remains unclear. Acute brain injury models can be exacerbated by augmenting the hepatic acute phase response (APR). Here, we explored the contribution of the hepatic APR to relapse in two rodent models of MS. Methods: Mice with MOG-CFA-induced chronic relapsing experimental autoimmune encephalitis (CR-EAE) were killed before, during and after the first phase of disease, and the brain and liver chemokine, cytokine and acute phase protein (APP) mRNA expression profile was determined. During remission, the APR was reactivated with an intraperitoneal lipopolysaccharide (LPS) and clinical score was monitored throughout. To explore the downstream mediators, CXCL-1, which is induced as part of the APR, was injected into animals with a focal, cytokine/MOG-induced EAE lesion (fEAE) and the cellularity of the lesions was assessed. Results: Compared to CFA control, in a rodent CR-EAE model, an hepatic APR preceded clinical signs and central cytokine production in the initial phase of disease. Compared to administration in naïve animals, an LPS challenge during the asymptomatic remission phase of CR-EAE rodents provoked relapse and resulted in the increased and extended expression of specific peripheral hepatic chemokines. CXCL-1 and several other APPs were markedly elevated. A single intravenous administration of the highly induced chemokine, CXCL-1, was found to be sufficient to reactivate the lesions by increasing microglial activation and the recruitment of T cells in fEAE lesions. Conclusions: The APR plays a contributing role to the pathology seen in models of chronic brain injury and in translating the effects of peripheral immune system stimulation secondary to trauma or infection into central pathology and behavioural signs. Further elucidation of the exact mechanisms in this process will inform development of more effective, selective therapies in MS that, by suppressing the hepatic chemokine response, may prevent relapse.
The characterization of a rabbit model of inflammatory bowel disease.
The absence of a simple, clinically relevant, animal model of inflammatory bowel disease (IBD) hampers research into this disease. In this study, colitis was induced in rabbits by intracolonic installation of 2,4,6-trinitrobenzene sulphonic acid (TNB) in 25% ethanol. Rabbits were killed from zero hours to 6 weeks and their colons examined. Rabbits were examined by endoscopy at weekly intervals. A single dose of TNB in ethanol produced dose dependent inflammation and ulceration, which at its optimum (40 mg) resulted in cobblestoning, strictures, and bowel wall thickening. The damage score at endoscopy was consistent with the score on macroscopic examination of the colon. Histopathological features of inflammation and ulceration observed in all animals that received 40 mg TNB included crypt abscesses, ulceration, crypt architectural distortion and, occasionally, granulomas and pseudopolyps. These changes, which are similar to those observed in IBD, persisted for 6 weeks. No lasting abnormalities were observed in control animals treated with TNB in saline, with ethanol alone, or with saline only. Histopathological similarity and the prolonged duration of inflammation, compared to other models, make this a suitable model for investigating inflammation in the colon. Furthermore, the model is accessible to endoscopy which adds to its value in experimental studies.
Special issue commentary: the changing face of inflammation in the brain.
The study of inflammation in the brain has been extended to include a wide range of conditions, but there remains plenty of argument over semantics and the precise definition of what constitutes inflammation in these pathologies. In this special issue, we sought to highlight the diversity of what is considered to be inflammation in the brain, and we have accepted that the presence of microglia cells with altered morphology remains a useful starting point. However, it is clear that whatever is the molecular expression profile that accompanies an activated microglial cell, it is not static and it is influenced by factors both intrinsic and extrinsic to the brain. This article is part of a Special Issue entitled 'Neuroinflammation in neurodegeneration and neurodysfunction'.
The contribution of the acute phase response to the pathogenesis of relapse in chronic-relapsing experimental autoimmune encephalitis models of multiple sclerosis.
BACKGROUND: Increased relapse rates in multiple sclerosis (MS) as a consequence of peripheral immune system activation, owing to infection for example, have been widely reported, but the mechanism remains unclear. Acute brain injury models can be exacerbated by augmenting the hepatic acute phase response (APR). Here, we explored the contribution of the hepatic APR to relapse in two rodent models of MS. METHODS: Mice with MOG-CFA-induced chronic relapsing experimental autoimmune encephalitis (CR-EAE) were killed before, during and after the first phase of disease, and the brain and liver chemokine, cytokine and acute phase protein (APP) mRNA expression profile was determined. During remission, the APR was reactivated with an intraperitoneal lipopolysaccharide (LPS) and clinical score was monitored throughout. To explore the downstream mediators, CXCL-1, which is induced as part of the APR, was injected into animals with a focal, cytokine/MOG-induced EAE lesion (fEAE) and the cellularity of the lesions was assessed. RESULTS: Compared to CFA control, in a rodent CR-EAE model, an hepatic APR preceded clinical signs and central cytokine production in the initial phase of disease. Compared to administration in naïve animals, an LPS challenge during the asymptomatic remission phase of CR-EAE rodents provoked relapse and resulted in the increased and extended expression of specific peripheral hepatic chemokines. CXCL-1 and several other APPs were markedly elevated. A single intravenous administration of the highly induced chemokine, CXCL-1, was found to be sufficient to reactivate the lesions by increasing microglial activation and the recruitment of T cells in fEAE lesions. CONCLUSIONS: The APR plays a contributing role to the pathology seen in models of chronic brain injury and in translating the effects of peripheral immune system stimulation secondary to trauma or infection into central pathology and behavioural signs. Further elucidation of the exact mechanisms in this process will inform development of more effective, selective therapies in MS that, by suppressing the hepatic chemokine response, may prevent relapse.
Circulating endothelial cell-derived extracellular vesicles mediate the acute phase response and sickness behaviour associated with CNS inflammation.
Brain injury elicits a systemic acute-phase response (APR), which is responsible for co-ordinating the peripheral immunological response to injury. To date, the mechanisms responsible for signalling the presence of injury or disease to selectively activate responses in distant organs were unclear. Circulating endogenous extracellular vesicles (EVs) are increased after brain injury and have the potential to carry targeted injury signals around the body. Here, we examined the potential of EVs, isolated from rats after focal inflammatory brain lesions using IL-1β, to activate a systemic APR in recipient naïve rats, as well as the behavioural consequences of EV transfer. Focal brain lesions increased EV release, and, following isolation and transfer, the EVs were sequestered by the liver where they initiated an APR. Transfer of blood-borne EVs from brain-injured animals was also enough to suppress exploratory behaviours in recipient naïve animals. EVs derived from brain endothelial cell cultures treated with IL-1β also activated an APR and altered behaviour in recipient animals. These experiments reveal that inflammation-induced circulating EVs derived from endothelial cells are able to initiate the APR to brain injury and are sufficient to generate the associated sickness behaviours, and are the first demonstration that EVs are capable of modifying behavioural responses.
Endothelium-derived extracellular vesicles promote splenic monocyte mobilization in myocardial infarction.
Transcriptionally activated monocytes are recruited to the heart after acute myocardial infarction (AMI). After AMI in mice and humans, the number of extracellular vesicles (EVs) increased acutely. In humans, EV number correlated closely with the extent of myocardial injury. We hypothesized that EVs mediate splenic monocyte mobilization and program transcription following AMI. Some plasma EVs bear endothelial cell (EC) integrins, and both proinflammatory stimulation of ECs and AMI significantly increased VCAM-1-positive EV release. Injected EC-EVs localized to the spleen and interacted with, and mobilized, splenic monocytes in otherwise naive, healthy animals. Analysis of human plasma EV-associated miRNA showed 12 markedly enriched miRNAs after AMI; functional enrichment analyses identified 1,869 putative mRNA targets, which regulate relevant cellular functions (e.g., proliferation and cell movement). Furthermore, gene ontology termed positive chemotaxis as the most enriched pathway for the miRNA-mRNA targets. Among the identified EV miRNAs, EC-associated miRNA-126-3p and -5p were highly regulated after AMI. miRNA-126-3p and -5p regulate cell adhesion- and chemotaxis-associated genes, including the negative regulator of cell motility, plexin-B2. EC-EV exposure significantly downregulated plexin-B2 mRNA in monocytes and upregulated motility integrin ITGB2. These findings identify EVs as a possible novel signaling pathway by linking ischemic myocardium with monocyte mobilization and transcriptional activation following AMI.