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Hotels in Darlinghurst, New South Wales
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- Most bookedibis budget Sydney East
255 people looked at this hotel in the last hour
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"Quite satisfied to the service of this hotel... the location is great and the hotel is very clean!"
A Traveller, Sep 2016, US
- The Kirketon HotelNo Guest Reviews yet
149 people looked at this hotel in the last hour
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"Enjoyed our stay had a late check out. Little bit noisy when guests were returning to their rooms after a night out but settled down quickly. Clean & comfy bed. Everything you need in a compact space. Would stay here again handy for breakie the next day too & convenient affordable parking across ..."
D, Sep 2016, AU
80 people looked at this hotel in the last hour
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"Great access to Oxford Street if that's your sort of party scene. Also close to kings cross station so it's easy to get everywhere. Bed was comfy, room was clean and staff super helpful but decor is tired especially the bathroom."
A Traveller, Sep 2016, GB
- Manor House Boutique HotelNo Guest Reviews yet
39 people looked at this hotel in the last hour
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"Stayed here to go and see Cirque du Soliel were able to get off street parking which was great in this part of town. "
A Traveller, Sep 2016, AU
38 people looked at this hotel in the last hour
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"The customer relations guy was lovely and personable. The room was a tad small but very quiet and comfortable. No complaints "
A Traveller, Aug 2016, AU
37 people looked at this hotel in the last hour
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"I had a lovely time and the hotel was in a perfect location for getting to Bondi beach and exploring Darlinghurst and surrounding areas. Bars, clubs and restaurants on your doorstep."
A Traveller, Apr 2016, GB
33 people looked at this hotel in the last hour
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"This is a hidden Gem; very homely and funky set up and location is great for access to city with bus stop just outside the door. Kitchen has everything you need and the back deck is a great place to unwind. Claire is very accommodating and friendly."
A Traveller, Sep 2016, AU
21 people looked at this hotel in the last hour
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"Great location & friendly staff, but terrible water pressure & insufficient blankets, so over-priced..."
A Traveller, Jul 2016, AU
8 people looked at this hotel in the last hour
- Darlinghurst 103 Far Furnished ApartmentNo Guest Reviews yet
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text:
|Publication number
|Publication type
|Application number
|Publication date
|Apr 17, 2012
|Priority date
|Jul 14, 1997
|Fee status
|Also published as
|Publication number
|Mark F. Pittenger, Stephen L. Gordon, Alastair Morgan Mackay
|Original Assignee
|Osiris Therapeutics, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (12), Non-Patent Citations (65), Referenced by (1), Classifications (21), Legal Events (3)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation of application Ser. No. 12/395,949, filed on Mar. 2, 2009, which is a continuation of application Ser. No. 10/690,435, now U.S. Pat. No. 7,514,074, filed Oct. 21, 2003, which is a continuation-in-part of application Ser. No. 10/278,148, filed Oct. 22, 2002, which is a continuation-in-part of application Ser. No. 10/127,737, filed Apr. 22, 2002, which is a continuation of application Ser. No. 09/446,952, filed Mar. 27, 2000, now U.S. Pat. No. 6,387,369, which is the national phase application of PCT Application No. PCT/US98/14520, filed Jul. 14, 1998, which claims priority of U.S. provisional application Ser. No. 60/052,910, filed Jul. 14, 1997; the contents of each application are incorporated by reference in their entireties.
[Not Applicable]
The present technology relates to the replacement, repair, and/or regeneration of cardiac tissue and muscle.
On a yearly basis, it is estimated over 300,000 Americans will die from congestive heart failure. The ability to augment weakened cardiac muscle would be a major advance in the treatment of cardiomyopathy and heart failure. Despite advances in the medical therapy of heart failure, the mortality due to this disorder remains high, where most patients die within one to five years after diagnosis.
A common heart ailment in the aging population is improper heart valve function, particularly the aortic valve. Mechanical replacement valves are widely used, but require the patient to continually take blood thinners. Valves obtained from cadavers and xenographs (porcine) are also frequently used to replace a patient's own tissue. Such valves are freeze-dried or chemically cross-linked using, for example, glutaraldehyde to stabilize the collagen fibrils and decrease antigenicity and proteolytic degradation. However, these valves remain acellular and often fail after several years due to mechanical strain or calcification. A replacement valve derived from a biocompatible material that would allow ingrowth of the appropriate host cells and renewal of tissue over time would be preferred.
Adult bone marrow is an accessible and renewable source of adult stem cells that can be greatly expanded in culture. For example, mesenchymal stem cells (MSCs) are multipotential cells that have been identified and cultured from avian and mammalian species including mouse, rat, rabbit, dog and human (See Caplan, 1991; Caplan et al. 1993; and U.S. Pat. No. 5,486,359). Isolation, purification and culture expansion of hMSCs is described in detail therein.
In accordance with the present technology, adult bone marrow-derived stem cells are used to regenerate or repair striated cardiac muscle that has been damaged through disease or degeneration. Adult bone marrow-derived stem cells of the present technology have the capacity to differentiate into at least one cell type of each of the mesodermal, ectodermal, and endodermal lineages. For example, the cells can be induced to differentiate into cells of at least osteoblast, chondrocyte, adipocyte, fibroblast, marrow stroma, skeletal muscle, smooth muscle, cardiac muscle, endothelial, epithelial, hematopoietic, glial, neuronal or oligodendrocyte cell types, among others. Adult bone marrow-derived stem cells of the present technology are capable of differentiating into at least one cell type of at least one of the endodermal, ectodermal, or mesodermal embryonic lineages.
The present technology also provides a cell preparation comprising adult bone marrow derived stem cells in a dose effective to stimulate angiogenesis in the heart of a subject in need thereof. The stem cells can be capable of differentiating into at least one cell type of each of the endodermal, ectodermal, and mesodermal embryonic lineages. The effective dose of the cell preparation can comprise about 0.02 to about 150 milliliters of a pharmaceutically acceptable liquid medium. The stem cells can be at a concentration of about 10 to about 40 million cells per milliliter. The stem cells can have an induced expression of at least one cardiac specific marker, which can be, for example, myosin heavy chain, sarcoplasmic reticulum calcium ATPase, or connexin 43. The induced expression of the cardiac specific marker can result from (i) co-culturing the stem cells with cardiac cells, (ii) use of chemical fusigens to create heterokaryons of the stem cells with cardiomyocytes, (iii) incubating the stem cells with extracts of mammalian hearts, (iv) treatment of the stem cells with growth factors, differentiating agents, or both, (v) mechanical or electrical stimulation of the stem cells, or (vi) mechanical or electrical coupling of the stem cells with cardiomyocytes. The subject can be, for example, a myocardial infarction patient, an ischemic heart transplant patient, a congestive heart failure patient, or a coronary artery bypass graft patient. The cell preparation can be capable of improving ventricular ejection fraction (e.g., left ventricular ejection fraction) in the subject.
The present technology further provides a method of improving ventricular ejection fraction in a patient comprising administering to the patient bone marrow-derived stem cells in an amount effective to improve ventricular ejection fraction. Again, the ventricular ejection fraction can be left ventricular ejection fraction. The stem cells can be autologous or allogeneic to the patient. The stem cells can be administered in a pharmaceutically acceptable carrier, such as a liquid carrier. The stem cells can be administered directly to the heart of the patient, such as via a suitable catheter or during an open surgical procedure (e.g., coronary artery bypass graft surgery). The stem cells can be administered systemically, such as intravenously or intraarterially. The stem cells can be mesenchymal stem cells.
The present technology also provides a method of treating a patient having myocardial infarction or congestive heart failure and having reduced cardiac function comprising the step administering to the patient bone marrow-derived stem cells in an amount effective to improve cardiac function. The stem cells can be autologous or allogeneic to the patient. The stem cells can be administered in a pharmaceutically acceptable carrier, such as a liquid carrier. The stem cells can be administered directly to the heart of the patient, such as via a suitable catheter or during an open surgical procedure (e.g., coronary artery bypass graft surgery). The stem cells can be administered systemically, such as intravenously or intraarterially. The stem cells can be mesenchymal stem cells. The method can further comprise assessing cardiac function at a time after administering the stem cells. The assessment can comprise the step of measuring ventricular function, such as by measuring ventricular ejection fraction, ventricular end diastolic pressure, or ventricular wall motion. The method can also comprise measuring left ventricular ejection fraction.
Additionally, the present technology provides a method of augmenting ventricular function in a patient comprising administering to the heart of the patient a cell suspension, wherein the cell suspension comprises bone marrow-derived stem cells and a pharmaceutically acceptable liquid medium. The stem cells can be autologous or allogeneic to the patient. The suspension cells can be administered to the heart of the patient, such as via a suitable catheter or during an open surgical procedure (e.g., coronary artery bypass graft surgery). The stem cells can be mesenchymal stem cells. The patient can have, for example, a left ventricular ejection fraction of less than about 40%, or be suffering from congestive heart failure.
In accordance with the present technology adult bone marrow-derived stem cells, such as mesenchymal stem cells (MSCs), are used to regenerate or repair striated cardiac muscle that has been damaged through disease or degeneration. In certain embodiments, MSCs differentiate into cardiac muscle cells and integrate with the healthy tissue of the recipient to replace the function of the dead or damaged cells, thereby repairing and/or regenerating the cardiac muscle as a whole. Cardiac muscle does not normally have reparative potential. Adult bone marrow-derived stem cells are used, for example, in cardiac muscle repair and/or regeneration for a number of principal indications: (i) ischemic heart transplantations, (ii) therapy for congestive heart failure patients, (iii) prevention of further disease for patients undergoing coronary artery bypass graft, (iv) conductive tissue regeneration, (v) vessel smooth muscle regeneration, or (vi) valve regeneration. Adult bone marrow-derived stem cells are also used to integrate with tissue of a replacement heart valve to be placed into a recipient. For example, MSCs, preferably autologous, repopulate the valve tissue, thereby enabling proper valve function.
Cardiac muscle therapy using adult bone marrow-derived stem cells is based, for example, on the following sequence: harvest of stem cell-containing tissue, isolation/expansion of stem cells, administration by either implantation (including by catheterization) into the damaged heart (with or without a stabilizing matrix and biochemical manipulation) or systemic injection, and in situ formation of myocardium or repair of damaged myocardium. This approach is different from traditional tissue engineering, in which the tissues are grown ex vivo and implanted in their final differentiated form. Biological, bioelectrical and/or biomechanical triggers from the host environment may be sufficient, or under certain circumstances, may be augmented as part of the therapeutic regimen to establish a fully integrated and functional repair or regenerated tissue.
Accordingly, one aspect of the present technology provides a method for producing cardiomyocytes in an individual in need thereof which comprises administering to the individual a myocardium-producing amount of mesenchymal stem cells. The mesenchymal stem cells that are employed may be a homogeneous composition or may be a mixed cell population enriched in MSCs. Homogeneous human mesenchymal stem cell compositions are obtained by culturing adherent marrow or periosteal cells; the mesenchymal stem cells may be identified by specific cell surface markers which are identified with unique monoclonal antibodies. A method for obtaining a cell population enriched in mesenchymal stem cells is described, for example, in U.S. Pat. No. 5,486,359.
Adult stem cells can be obtained from a variety of sources, including bone marrow. For example, human mesenchymal stem cells can be obtained from bone marrow from a number of different sources, including plugs of femoral head cancellous bone pieces, patients with degenerative joint disease during hip or knee replacement surgery, and aspirated marrow from normal donors or oncology patients who have marrow harvested for future bone marrow transplantation. Harvested marrow can be prepared for cell culture by a number of different mechanical isolation processes depending upon the source of the harvested marrow (i.e., the presence of bone chips, peripheral blood, etc.) that are well known in the art. Exemplary culture media and culture conditions are identified in, for example, U.S. Pat. No. 5,486,359 and include media and conditions that allow for expansion, growth, and isolation of mesenchymal stem cells, without differentiation.
Cell preparations having greater than about 95%, usually greater than about 98%, of adult human bone marrow-derived stem cells can be achieved using techniques for isolation, purification, and culture expansion of stem cells. For example, isolated, cultured mesenchymal stem cells can comprise a single phenotypic population (about 95% or about 98% homogeneous) by flow cytometric analysis of expressed surface antigens. The desired cells in such composition can be identified, for example, by expression of a cell surface marker (e.g., CD73 or CD105) specifically bound by an antibody produced from hybridoma cell line SH2, ATCC accession number HB 10743, an antibody produced from hybridoma cell line SH3, ATCC accession number HB 10744, or an antibody produced from hybridoma cell line SH4, ATCC accession number HB 10745. Such antibodies selectively bind bone marrow-derived mesenchymal stem cells and, therefore, can be used to identify, quantify, isolate, or purify mesenchymal stem cells from bone marrow samples.
The administration of the cells can be directed to the heart, by a variety of procedures. Localized administration is preferred. The adult bone-marrow derived stem cells can be from a spectrum of sources including, in order of preference: autologous, allogeneic, or xenogeneic. There are several embodiments to this aspect, including the following.
In one embodiment of this aspect, the adult bone-marrow derived stem cells can be administered as a cell suspension in a pharmaceutically acceptable liquid medium for injection. Injection, in this embodiment, can be local, such as by administration directly into the damaged portion of the myocardium, or systemic, such as intravenously or intraarterially. Here, again, localized administration is preferred.
In another embodiment of this aspect, the adult bone-marrow derived stem cells, such as MSCs, are administered in a biocompatible medium which is, or becomes in situ at the site of myocardial damage, a semi-solid or solid matrix. For example, the matrix may be (i) an injectible liquid which "sets up" (or polymerizes) to a semi-solid gel at the site of the damaged myocardium, such as collagen and its derivatives, polylactic acid or polyglycolic acid, or (ii) one or more layers of a flexible, solid matrix that is implanted in its final form, such as impregnated fibrous matrices. The matrix can be, for example, Gelfoam (Upjohn, Kalamazoo, Mich.). The matrix holds the stem cells in place at the site of injury, and thus serves the function of "scaffolding". This, in turn, enhances the opportunity for the administered the adult bone-marrow derived stem cells to proliferate, differentiate and eventually become fully developed cardiomyocytes. Although not wishing to be bound by any particular theory, it is believed that as a result of their localization in the myocardial environment they then integrate with the recipient's surrounding myocardium. These events likewise occur in the above liquid injectible embodiment, but this embodiment may be preferred where more rigorous therapy is indicated.
In another embodiment of this aspect, the adult bone marrow-derived stem cells are induced or genetically modified or engineered to express proteins of importance for the differentiation and/or maintenance of striated muscle cells. Some examples include growth factors (TGF-β, IGF-1, FGF), myogenic factors (myoD, myogenin, Myf5, MRF), transcription factors (GATA-4), cytokines (cardiotrophin-1), members of the neuregulin family (neuregulin 1, 2 and 3) and homeobox genes (Csx, tinman, NKx family). Also contemplated are genes that encode for factors that stimulate angiogenesis and revascularization (e.g., vascular endothelial growth factor (VEGF)). Any of the known methods for introducing DNA are suitable, however electroporation, retroviral vectors and adeno-associated virus (AAV) vectors are preferred.
Thus, in association with the embodiment of the above aspect using genetically engineered stem cells, the present technology also provides new and unique genetically engineered adult bone marrow-derived stem cells and tissue compositions to treat the above indications. The compositions can include genetically modified cells and unmodified cells in various proportions to regulate the amount of expressed exogenous material in relationship to the total number of cells to be affected.
The present technology also relates to the potential of MSCs to differentiate partially to the cardiomyocyte phenotype using in vitro methods. This technique can under certain circumstances optimize conversion of MSCs to the cardiac lineage by predisposing them thereto. This also has the potential to shorten the time required for complete differentiation once the cells have been administered.
It is believed that he proper environmental stimuli convert MSCs into cardiac myocytes. Differentiation of mesenchymal stem cells to the cardiac lineage is controlled by factors present in the cardiac environment. Exposure of MSCs to a simulated cardiac environment directs these cells to cardiac differentiation as detected by expression of specific cardiac muscle lineage markers. Local chemical, electrical and mechanical environmental influences alter pluripotent MSCs and convert the cells grafted into the heart into the cardiac lineage.
Early in embryonic development following the epithelia-mesenchyme transition, the presumptive heart mesenchyme from the left and right sides of the body migrate to the ventral midline. Here, interaction with other cell types induces continued cardiogenesis. In vitro conversion of MSCs to cardiomyocytes is tested by co-culture or fusion with murine embryonic stem cells or cardiomyocytes, treatment of MSCs with cardiac cell lysates, incubation with specific soluble growth factors, or exposure of MSCs to mechanical stimuli and electrical stimulation.
A series of specific treatments applicable to MSCs to induce expression of cardiac specific genes are disclosed herein. The conditions are effective on rat, canine and human MSCs. Treatments of MSCs include (1) co-culturing MSCs with fetal, neonatal and adult rat cardiac cells, (2) use of chemical fusigens (e.g., polyethylene glycol or sendai virus) to create heterokaryons of MSCs with fetal, neonatal and adult cardiomyocytes, (3) incubating MSCs with extracts of mammalian hearts, including the extracellular matrix and related molecules found in heart tissue, (4) treatment of MSCs with growth factors and differentiating agents, (5) mechanical and/or electrical stimulation of MSCs, and (6) mechanically and/or electrically coupling MSCs with cardiomyocytes. MSCs that progress towards cardiomyocytes first express proteins found in fetal cardiac tissue and then proceed to adult forms. Detection of expression of cardiomyocyte specific proteins is achieved using antibodies to, for example, myosin heavy chain monoclonal antibody MF 20 (MF20), sarcoplasmic reticulum calcium ATPase (SERCA1) (mAb 10D1) or gap junctions using antibodies to connexin 43.
Cardiac injury promotes tissue responses which enhance myogenesis using implanted MSCs. Thus, MSCs are introduced to the infarct zone to reduce the degree of scar formation and to augment ventricular function. New muscle is thereby created within an infarcted myocardial segment. MSCs are directly infiltrated into the zone of infarcted tissue. The integration and subsequent differentiation of these cells is characterized, as described above. Timing of intervention is designed to mimic the clinical setting where patients with acute myocardial infarction would first come to medical attention, receive first-line therapy, followed by stabilization, and then intervention with myocardial replacement therapy if necessary.
Of the four chambers of the heart, the left ventricle is primarily responsible for pumping blood under pressure through the body's circulatory system. It has the thickest myocardial walls and is the most frequent site of myocardial injury resulting from congestive heart failure. The degree of advance or severity of the congestive heart failure ranges from those cases where heart transplantation is indicated as soon as a suitable donor organ becomes available to those where little or no permanent injury is observed and treatment is primarily prophylactic.
The severity of resulting myocardial infarction as determined by the percentage of muscle mass of the left ventricle that is involved can range from about 5 to about 40 percent. This represents affected tissue areas, whether as one contiguous ischemia or the sum of smaller ischemic lesions, having horizontal affected areas from about 2 cm2 to about 6 cm2 and a thickness of from 1-2 mm to 1-1.5 cm. The severity of the infarction is significantly affected by which vessel(s) is involved and how much time has passed before treatment intervention is begun.
The mesenchymal stem cells used in accordance with the present technology are, in order of preference, autologous, allogeneic or xenogeneic, and the choice can largely depend on the urgency of the need for treatment. A patient presenting an imminently life threatening condition may be maintained on a heart/lung machine while sufficient numbers of autologous MSCs are cultured or initial treatment can be provided using other than autologous MSCs.
The MSC therapy of the present technology can be provided by several routes of administration, including the following. First, intracardiac muscle injection, which avoids the need for an open surgical procedure, can be used where the MSCs are in an injectible liquid suspension preparation or where they are in a biocompatible medium which is injectible in liquid form and becomes semi-solid at the site of damaged myocardium. A conventional intracardiac syringe or a controllable arthroscopic delivery device can be used so long as the needle lumen or bore is of sufficient diameter (e.g., 30 gauge or larger) that shear forces will not damage the MSCs. The injectible liquid suspension MSC preparations can also be administered intravenously, either by continuous drip or as a bolus. During open surgical procedures, involving direct physical access to the heart, all of the described forms of MSC delivery preparations are available options.
As a representative example of a dose range is a volume of at least about 20 μl, preferably at least about 500 μl, of injectible suspension containing about 10 to about 40�106 MSCs/ml. The concentration of cells per unit volume, whether the carrier medium is liquid or solid remains within substantially the same range. The amount of MSCs delivered will usually be greater when a solid, "patch" type application is made during an open procedure, but follow-up therapy by injection will be as described above. The frequency and duration of therapy will, however, vary depending on the degree (percentage) of tissue involvement, as already described (e.g., about 5 to about 40% left ventricular mass).
In cases having about 5% to about 10% tissue involvement, it is possible, for example, to treat with as little as a single administration of about one million MSCs in about 20 to about 50 μl of injection preparation. The injection medium can be any pharmaceutically acceptable isotonic liquid. Examples include phosphate buffered saline (PBS), culture media such as Dulbecco's Modified Eagle's Medium (DMEM; preferably serum-free), physiological saline or about 5% dextrose in water (D5W).
In cases having more in a range of about 20% tissue involvement severity level, multiple injections of about 20 to about 50 μl (about 10 to about 40�106 MSCs/ml) are envisioned. Follow-up therapy may involve additional dosings.
In very severe cases, for example in a range of about 40% tissue involvement severity level, multiple equivalent doses for a more extended duration with long term (up to several months) maintenance dose aftercare may well be indicated and are envisaged in accordance with the practice of the present technology.
When given intravenously, the mesenchymal stem cells may be administered in at least about 20 μl, preferably at least about 500 μl, and up to about 150 ml of a suspension containing about 10 to about 40�106 MSCs/ml. In one embodiment, from about 40 ml to about 150 ml of a suspension containing about 10 to about 40�106 MSCs/ml is given intravenously.
Applicants also have discovered that the mesenchymal stem cells may stimulate and/or promote angiogenesis in the heart and/or repair or regenerate blood vessels of the heart. Thus, in accordance with another aspect of the present technology, there is provided a method of stimulating or promoting angiogenesis in the heart, and/or of repairing or regenerating blood vessels of the heart of an individual by administering to the individual mesenchymal stem cells in an amount effective to stimulate or promote angiogenesis, and/or repair or regenerate blood vessels of the heart. The mesenchymal stem cells may be administered as a cell suspension in a pharmaceutically acceptable liquid medium such as described herein, or in a biocompatible medium which is, or becomes in situ at the site of myocardial damage, a semi-solid or solid matrix, also as described herein.
The mesenchymal stem cells may be allogeneic, autologous, or xenogeneic, and may be administered in dosages such as those described herein.
When the mesenchymal stem cells are administered as a cell suspension in a pharmaceutically acceptable liquid medium for injection, they may be administered locally, such as by administration directly into the damaged portion of the heart by, for example, an endocardial catheter, or they may be administered systemically, such as by intravenous or intraarterial administration.
Adult bone marrow-derived stem cells, including mesenchymal stem cells, provide for the repair and/or regeneration of existing blood vessels of the heart, as well as promote angiogenesis (i.e., the formation of new blood vessels of the heart). Blood vessels which may be repaired or regenerated, as well as new blood vessels which may be formed, include arteries (including arterioles), and veins, as well as capillaries.
The present technology is further illustrated, but not limited, by the following examples.
In using MSCs, it is desirable, for example, to maintain cell-cell contact in vivo for the conversion of MSCs to the muscle lineage. Environmental signals identified above act in concert with mechanical and electrical signaling in vivo to lead to cardiac differentiation.
Primary human MSCs (hMSCs) are introduced into athymic rat myocardial tissue by direct injection. The integration of implanted cells, their subsequent differentiation, formation of junctions with cardiac cells, and their long-term survival are characterized with light microscopy, histology, confocal immunofluorescence microscopy, electron microscopy and in situ hybridization.
Whether human MSCs are appropriately grafted into cardiac muscle of athymic rats (strain HSD:RH-RNU/RNU), which lack the immune responses necessary to destroy many foreign cells, is also examined.
Rat MSCs are grafted into the heart muscles of rats. To analyze the injected cells over several weeks and to minimize the possibility of immune system rejection, MSCs are harvested from Fisher 344 rats, the same inbred strain (identical genotype) as the intended MSC recipients.
The MSCs can be marked in a variety of ways prior to their introduction into the recipient. This makes it possible to trace the fate of the MSCs as they proliferate and differentiate in the weeks following the MSC implant. Several methods are utilized to identify positively the injected cells: membrane lipid dyes PKH26 or CM-Dil and genetic marking with adeno-associated virus (AAV) or retroviruses, such as Moloney murine leukemia virus expressing green fluorescent protein (GFP) or galactosidase. PCR also is used to detect the Y chromosome marker of male cells implanted into female animals. The dye-labeled cells are detected readily and offer the simplest method to directly follow the injected cells. This method is reliable for times out to at least 4 weeks. On the day of introduction to recipient animals, MSCs are trypsinized and labeled with CM-Dil according to the recommendations of the manufacturer (Molecular Probes). Subconfluent monolayer cultures of MSCs are incubated with 5 mM CM-Dil in serum-free medium for 20 minutes, trypsinized, washed twice in excess dye-free medium, and utilized for injection.
Alternatively, MSCs are genetically marked prior to injections, such as by using AAV-GFP vector. This vector lacks a selectable marker, but mediates a high-level expression of the transduced genes in a variety of post-mitotic and stem cell types. Recombinant AAV-GFP is added to low density monolayers of MSCs in low serum. Following a four hour incubation at 37� C., the supernatant is removed and replaced with fresh media. At 96 hours after transduction, cells are assayed for green fluorescent protein (GFP) activity. Typically 50% of the cells express the transduced gene. Unselected MSCs on a clonal line, isolated by limiting dilution, are utilized for injection. Cells are collected following trypsin treatment, washed and used at high concentrations for injection (10 to 100 million cells per ml).
To test whether the hMSCs became cardiomyocytes in the heart environment, the hearts of ten week old athymic rats were injected with dye labeled or GFP-labeled human MSCs. All procedures were performed under strict sterile conditions. The animals were placed in a glass jar containing a methoxyflurane anesthesia soaked sponge. Under sterile conditions, a 20 mm anterior thoracotomy was performed, and following visualization of the left ventricle, about 10 μl of the cell suspension, containing about 10,000 to about 100,000 MSCs in serum-free medium were injected into the left ventricular apex using a 30 gauge needle. The procedure was performed rapidly with endotracheal intubation and mechanical ventilation assist. The incision was closed with sutures. Ventilation assist was normally unnecessary after a short period following chest closure.
When sacrificed, the heart is removed, examined by light microscopy for the presence of vascular thrombi or emboli, paraffin-embedded, and sectioned. The histology of serial sections is examined to determine the fate of dye-stained cells. Sections then are tested for immunohistochemical markers of cardiac muscle in the areas of the introduced MSCs to ascertain whether donor MSCs have differentiated into cardiomyocytes in vivo. Implantation surgeries are carried out on animals to be sacrificed at 1, 2, 4, and 6 weeks (4 animals at each time point) and the hearts which received implants are analyzed histologically and immunologically.
For phenotypic characterization, the hearts are removed and processed for histology by immunofluorescence microscopy. Differentiation of MSCs is determined by the immunofluorescence localization of sacomeric myosin heavy chain, SERCA1 and phospholamban. The sequence-specific antibody to gap junction protein connexin 43, which is commercially available (Zymed, San Francisco, Calif.) and detects gap junctions in cardiac tissue is also used.
MSCs are also implanted in biomatrix materials to determine if enhanced grafting would be observed, such as Type I collagen. The MSCs are mixed rapidly with the matrix in a small volume and injected into the ventricle wall. The biomatrices are used at concentrations of 0.1 mg/ml or greater. For example, the biomatrices may be used at concentrations of 1 to 3 mg/ml containing 10 to 100 million cells/ml. The tissue is analyzed at times of 1, 2, 4, and 6 weeks as described above.
Xenograft or homograft valves are made acellular by freeze-drying, which leads to cellular death, or by enzymatic treatment followed by detergent extraction of cells and cell debris. This latter approach was taken by Vesely and coworkers with porcine valves to be repopulated with dermal or aortic fibroblasts. Curtil, et al. 1997 used a freeze-dried porcine valve and attempted repopulation of the valve with human fibroblasts and endothelial cells. These studies were preliminary and limited to short term studies in vitro.
The acellular valve to be populated by autologous hMSCs is incubated with culture expanded hMSCs in a tumbling vessel to ensure loading of cells to all valve surfaces. The valve is then cultured with the hMSCs for 1-2 weeks to allow the hMSCs to infiltrate and repopulate the valve. Within the culture vessel, the valve is then attached to a pump to allow the actuation of the valve leaflets and simulate the pumping motion present in the body. The valve is maintained in the pumping mode for 1-2 weeks to allow cellular remodeling associated with the stresses of the pumping action. Once sufficient cellular remodeling has occurred, the valve is implanted into the body of the patient.
Another embodiment of this aspect of the present technology is to first repopulate the valve with hMSCs and to later incubate the valve tissue during the pumping stage with autologous smooth muscle cells isolated from a vascular graft which will line the lumen of the valve.
Myocardial infarction was produced in Fisher rats as follows:
Fisher rats were given a cocktail of Ketamine/Xylazine/Acepromazine (8.5 mg/1.7 mg/0.3 mg I.P.) The depth of anesthesia was assessed using a toe-pinch and eye-blink reflexes. When a surgical plane of anesthesia was achieved, endotracheal intubation was performed and the animal placed on 1.0% Isoflorane. Positive-pressure breathing was provided throughout the procedure by means of the Engler ADS 1000 small animal ventilator. A left thoracotomy was performed and the pericardium opened. A 6-0 silk ligature snare was then placed around the left anterior descending (LAD) coronary artery at a location distal to the first diagonal branch. A brief (30 sec) LAD test occlusion is performed to insure that a modest region of ischemia is produced, involving a limited region of the anterior free wall and septum. Ischemia is confirmed by characteristic ECG changes, ventricular dyskinesis and regional cyanosis. Myocardial infarction is then produced by occluding the LAD for a period of 45 minutes. At the completion of the 45 minute period, the snare is removed and reperfusion visually confirmed. The chest was then closed by approximating the ribs and all associated musculature. The Isoflurane is turned off, the animal removed from the ventilator and extubated.
Panel A of
Panel B of
These animals received MSCs via the tail vein. Injection of the allogeneic cell suspension occurred when the animals had stabilized, and a normal cardiac rhythm had been reestablished; usually within 15 minutes of reperfusion. At that time approximately 5�106 MSCs in a 0.5 milliliter suspension were injected slowly into the tail vein.
Swine are sedated with 1000 mg ketamine IM and brought into the lab. Intravenous access is established via an ear vein and the animals anesthetized with nembutal. Swine then are intubated, ventilated with 1.0-1.5% isoflurane, and prepped for surgery. ECG leads and rectal temperature probes are placed and the animal is draped to create a sterile field. A midline sternotomy is performed and the heart suspended in a pericardial cradle. A tygon catheter is placed in the apex of the left ventricle and sutured in place to measure ventricular pressure throughout the cardiac cycle. The left anterior descending (LAD) coronary artery is dissected free just distal to the first diagonal branch. A brief (30 sec) occlusion of the coronary artery is performed to identify the regions of ischemia (identified by the extent of cyanosis). Four piezoelectric crystals then are placed within regions destined for infarction.
At the completion of the surgical instrumentation a 15 minute stabilization period is allowed prior to obtaining baseline recordings. Following these recordings, the LAD there is occluded for a period of 60 minutes to produce myocardial infarction. Lidocaine (local anesthetic and antiarrhythmic) is administered at this time to reduce the incidence of ventricular fibrillation (2 mg/kg i.v. bolus plus 0.5 mg/min iv drip). Recordings of left ventricular pressure and regional contractile function are again obtained at 10 and 50 minutes of ischemia. Extensive cyanosis within the ischemic bed was noticed following 50 minutes of ischemia.
At the completion of the 60 minute period of ischemia, the snare is released and reperfusion established. Care is taken to ensure that perfusion is reestablished and that the isolated region of the LAD is not in spasm. At this time the leads (sono leads and LV catheter) are externalized, and the chest closed in layers. A chest tube is placed to reestablish a negative intrapleural pressure (tube is pulled 24 hrs later). The isoflurane is then turned off, and the animal is extubated and allowed to recover.
One set of infarcted swine was treated with allogeneic mesenchymal stem cells and another set (control) did not receive such treatment. The animals were examined using echocardiography. In the mesenchymal stem cell treatment, a 10 ml MSC suspension was drawn up into several 3 cc syringes using an 18 g needle. The needle was switched to a 30 g for delivery. Implantation was accomplished in the open chest setting. The needle was advanced to the mid-wall level, and 0.5 mls of cells were injected. This same procedure was performed approximately 20 times throughout the damaged area. Care was taken to provide cells to the entire apical anterior wall, as well as the septum. At the completion of the implantation procedure, the chest was closed and the animal allowed to recover.
Echocardiography was used to measure the ejection fraction, a measure of global pump efficiency (a normal ejection fraction of 70% indicates that 70% of the LV volume is pumped with each beat of the heart; EF<40% is indicative of heart failure). Ejection fraction data is shown in the upper panels of
A similar graph was used to represent wall motion score index (lower panels of
Further evidence for improved cardiac function with MSC treatment is found when end diastolic pressure (EDP) is examined. When cardiac pump function is reduced following infarction, a pathologic increase in left ventricular EDP is observed. This increase in EDP is a clinically relevant finding that is often predictive of the progression to heart failure following infarction. As shown in
Pathologic ventricular remodeling following myocardial infarction is a major cause of heart failure. It was previously demonstrated that autologous mesenchymal stem cells (MSC) augment local systolic wall thickening and prevent pathologic wall thinning. Based on in vitro studies, it was hypothesized that MSCs may be immuno-privileged, and that implantation of allogeneic MSCs could prevent pathologic remodeling and improve cardiac performance in a swine model of myocardial infarction. Piezoelectric crystals and an LV catheter were implanted in domestic swine prior to a 60′ LAD occlusion to produce infarction. Following reperfusion, treated animals (n=7) were injected with allogeneic Dil-labeled MSCs (2�108 cells in 9 ml) throughout the region of infarction. Control (CON, n=6) received vehicle injections. Allogeneic donor MSCs were previously isolated from swine iliac crest bone marrow, expanded in culture, and cryopreserved until the time of implantation. Hemodynamic parameters and regional wall motion were evaluated in conscious animals bi-weekly using trans-thoracic echocardiography and sonomicrometry. Animals were sacrificed at various time points (6-24 weeks) and tissue harvested for histological examination.
It was observed that implantation of allogeneic MSCs was not associated with ectopic tissue formation, significant inflammatory response or any adverse clinical event. Robust engraftment of allogeneic MSCs was observed in all treated animals. Furthermore, engrafted MSCs were found to express numerous muscle specific proteins, and exhibited morphological changes consistent with myogenesis. A marked improvement in both ejection fraction (55�9.4% vs 32.5�12.5% in CON) and global wall motion score (1.45�0.15 vs 2.1�0.2 in CON) was observed in treated animals at 10 weeks post-MSC implantation. Systolic wall thickening and diastolic wall thickness were also augmented in MSC treated animals. Because no significant difference in infarct size or cardiac loading was noted between groups, improvements in cardiac function are likely attributable to MSC implantation. In conclusion, this example suggests that implantation of allogeneic MSCs at reperfusion may be an effective therapeutic option to prevent or reverse the progression to heart failure following infarction.
The above examples illustrate that mesenchymal stem cells augment ventricular function, as shown, for example by improved cardiac ejection fraction and global wall motion.
A pig was subjected to a 60 minute LAD occlusion to produce infarction as described in Example 5. Three days after the infarction, 200�106 diaminopropidium iodide (DAPI)-labeled allogeneic mesenchymal stem cells were administered to the left venticular wall by endocardial catheter as 20 separate injections of 10�106 cells each in 0.5 ml physiological saline. DAPI is a nuclear stain which emits a strong blue fluorescence and aids in the identification of implanted cells.
Eight weeks after administration of the mesenchymal stem cells, the pig was sacrificed, and the heart was harvested for histological examination. Sections were subjected to hematoxylin and eosin staining, or to fluorescence imaging after being contacted with an FITC-labeled monoclonal antibody against smooth muscle actin.
The hematoxylin and eosin image (
As shown in
As shown in
A pig was subjected to a 60 minute LAD occlusion to produce infarction as described in Example 5. Three days after the infarction; the pig was given 200�106 diaminopropidium iodide (DAPI)-labeled allogeneic mesenchymal stem cells as 20 separate injections of 10�106 cells in 0.5 ml physiological saline into the left ventricular wall by endocardial catheter as described in Example 6.
Twelve weeks after administration of the mesenchymal stem cells, the pig was sacrificed, and the heart was harvested for histological examination. Sections were subjected to hematoxylin and eosin staining, or to fluorescence imaging after being contacted with an FITC-labeled monoclonal antibody against Factor VIII (Von Willebrand Factor) or an FITC-labeled monoclonal antibody against vascular endothelial growth factor (VEGF).
As shown in the hematoxylin and eosin images of
In the fluorescent images of
Thus, it has been shown that the implanted MSCs express Factor VIII and VEGF, which are indicative of angiogenesis. These proteins are not expressed by cultured MSCs, but are expressed only after several weeks in the cardiac environment.
The disclosure of all patents and publications (including published patent applications) are hereby incorporated by reference in their entireties to the same effect as if each patent and publication were individually and specifically incorporated by reference.
It is to be understood, however, that the scope of the present technology is not to be limited to the specific embodiments described above. The technology may be practiced other than as particularly described and still be within the scope of the accompanying claims.
|Cited Patent
|Filing date
|Publication date
|Nov 16, 1990
|Jul 13, 1993
|Caplan Arnold I
|Method for treating connective tissue disorders
|Feb 8, 1994
|Jan 23, 1996
|Osiris Therapeutics, Inc.
|Human mesenchymal stem cells
|Nov 24, 1993
|Jan 7, 1997
|Case Western Reserve University
|Transduced mesenchymal stem cells
|Mar 28, 1995
|Feb 10, 1998
|Thomas Jefferson University
|Isolated stromal cells for treating diseases, disorders or conditions characterized by bone defects
|Jun 7, 1995
|Dec 8, 1998
|The Picower Institute For Medical Research
|Blood-borne mesenchymal cells
|Jun 19, 1998
|Aug 8, 2000
|Genzyme Corporation
|Transplants for myocardial scars
|Jun 22, 1993
|Jan 6, 1994
|Young Henry E
|Scar inhibitory factor and use thereof
|Mar 28, 1996
|Oct 3, 1996
|Thomas Jefferson University
|Isolated stromal cells and methods of using the same
|Jul 14, 1998
|Jan 28, 1999
|Osiris Therapeutics, Inc.
|Cardiac muscle regeneration using mesenchymal stem cells
|Mar 23, 1999
|Sep 30, 1999
|Zymogenetics, Inc.
|Cardiac-derived stem cells
|Jul 29, 1999
|Feb 10, 2000
|Genzyme Corporation
|Improvement of cardiac function by mesenchymal stem cell transplantation
|Sep 21, 1999
|Mar 30, 2000
|Musc Foundation For Research Development
|Non-hematopoietic cells, including cardiomyocytes and skeletal muscle cells, derived from hematopoietic stem cells and methods of making and using them
|Auerbach et al., "Cell surface markers on endothelial cells: a developmental perspective," Biology of Endothelial Cells, ed. Jaffe, E. (Nijhoff, Boston) pp. 393-400 (1984).
|Breitbach et al., Blood, 110:1362-1369 (2007).
|Bruder et al., Mesenchymal Stem Cells in Bone Development, Bone Repair, and Skeletal Regeneration Therapy, J. Cell. Biochem., 56:283-294 (1994).
|Chen et al., Stroke, 32:1005-1011 (2001).
|Chen, J. Neurol. Sci., 189:49-57 (2001).
|Chiu et al., Ann. Therac. Surg., 60:12-18 (1995).
|Chopp et al., The LANCET Neurology, 1:92-100 (2002).
|Dalton et al., J. Cell. Biochem., Supp. 17E, Abstract R 202 (1993).
|Deisher et al., U.S. Appl. No. 60/079,132, filed Mar. 23, 1998.
|European Search Report corresponding to European Patent Application Serial No. 03776501.3-1222, dated Oct. 19, 2009.
|Galinanes et al., Cell Transplantation, 13:7-13 (2004) (Abstract).
|Gerson, Nature Med., 5:262-264 (1999).
|Grigoriadis et al., Differentiation of Muscle, Fat, Cartilage, and Bone from Progenitor Cells Present in a Bone-derived Clonal Cell Population: Effect of Dexamethasone, J. Cell Biol., 106:2139-2151 (1988).
|International Preliminary Examination Report in PCT/US98/14520, dated Sep. 3, 1999.
|International Search Report in PCT/US98/14520, dated Oct. 13, 1998.
|Jorgensen et al., Curr. Opin. Biotechnol., 15:406-410 (2004).
|Kassem, Cloning and Stem Cells, 6(4):369-375 (2004).
|Klug et al., Genetically Selected Cardiomyocytes from Differentiating Embroyonic Stem Cells from Stable Intracardiac Grafts, J. Clin. Invest., 98(1):216-224 (1996).
|Krebsbach et al., Bone Formation In Vivo: Comparison of Osteogenesis by Transplanted Mouse and Human Marrow Stromal Fibroblasts, Transplantation, 63:1059-1069 (1997).
|Lee et al., Ann. Intern. Med., 140:729-737 (2004).
|Li et al., Neurology, 59:514-523 (2002).
|Lin et al., Natural Killer Cell- and Macrophage-Mediated Rejection of Concordant Xenografts in the Absence of T and B Cell Responses, J. Immunol., pp. 5658-5667 (1997).
|Martin et al., "Allogeneic mesenchymal stem cell engraftment in the infracted rat heart: Timing and delivery route." Bone Marrow Transplantation, vol. 29, No. S2, Mar. 2002, XP002549207.
|Martin et al., 1995, Blood, 66: 664-672.
|Minguell et al., Exp. Biol. Med., 226:507-520 (2001).
|Nagaya et al., "Transplantation of Mesenchymal stem cells improves cardiac function in a rat model of dilated cardiomyopathy," Circulation, 112:1128-1135 (2005).
|Office Action in U.S. Appl. No. 09/446,952, dated Jan. 18, 2001.
|Office Action in U.S. Appl. No. 09/446,952, dated Jun. 6, 2000.
|Office Action in U.S. Appl. No. 10/690,435, dated Aug. 18, 2008.
|Office Action in U.S. Appl. No. 10/690,435, dated Jan. 12, 2006.
|Office Action in U.S. Appl. No. 10/690,435, dated Jan. 29, 2008.
|Office Action in U.S. Appl. No. 10/690,435, dated Jul. 17, 2007.
|Office Action in U.S. Appl. No. 10/690,435, dated Mar. 22, 2007.
|Office Action in U.S. Appl. No. 10/690,435, dated Nov. 3, 2005.
|Office Action in U.S. Appl. No. 10/690,435, dated Sep. 22, 2006.
|Office Action in U.S. Appl. No. 11/475,568, dated Apr. 3, 2008.
|Office Action in U.S. Appl. No. 11/475,568, dated Dec. 12, 2007.
|Office Action in U.S. Appl. No. 12/395,949, dated Jan. 12, 2010.
|Office Action in U.S. Appl. No. 12/395,949, dated Jun. 22, 2010.
|Office Action in U.S. Appl. No. 13/012,953, dated Aug. 10, 2011.
|Office Action in U.S. Appl. No. 13/012,953, dated Jun. 28, 2011.
|Office Action in U.S. Appl. No. 13/012,956, dated Jul. 21, 2011.
|Office Action in U.S. Appl. No. 13/012,956, dated Sep. 1, 2011.
|Orkin et al., In Report and Recommendations of the Panel to Assess the NIH Investment in Research on Gene Therapy (1995).
|Ott et al., Basic Res. Cardiol., 100:504-517 (2005).
|Parmley et al., West J. Med., 154:427-441 (1991).
|Pereira et al., Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice, Proc. Natl. Acad. Sci. USA< 92:4857-4861 (1995).
|Pfeffer et al., Circulation, 81:1161-1172 (1990).
|Prockop, Science, 276:71-74 (1997).
|Ramachandran et al., Plasma Homocysteine and Risk for Congestive Heart Failure in Adults Without Prior Myocardial Infarction, JAMA, 289(10):1251-1257 (2003).
|Risau, "Developing brain produces an angiogenesis factor," Proc. Natl. Acad. Sci. USA, 83:3855-3859 (1986).
|Robinson et al., "Implantation of Skeletal Myoblast-Derived Cells," in Cellular Cardiomyoplasty: Myocardial Repair with Cell Implantation, Kao et al., eds., Landes Bioscience, pp. 81-107 (1997).
|Robinson et al., "Implantation of Skeletal Myoblast—Derived Cells," in Cellular Cardiomyoplasty: Myocardial Repair with Cell Implantation, Kao et al., eds., Landes Bioscience, pp. 81-107 (1997).
|Saadi et al., Life Sciences, 62:365-387 (1998).
|Saito et al., Tissue Engineering, 1(4):327-343 (1995).
|Semedo et al., Transplantation Proceedings, 39:421-423 (2007).
|Shake et al., The Annals of Thoracic Surgery, 73:1919-1926 (2002).
|Szabolcs et al., Apoptosis of Cardiac Myocytes During Cardiac Allograft Rejection, Circulation, 94(7):1665-1673 (1996).
|Torre-Amione et al., 1996, JACC, 27: 1201-1206.
|Verma et al., Nature, 389:239-242 (1997).
|Wagers et al., Cell, 116:639-648 (2004).
|Wakitani et al., Myogenic Cells Derived from Rat Bone Marrow Mesenchymal Stem Cells Exposed to 5-Azacytidine, Muscle & Nerve, 18(12):1417-1426 (1995).
|Warejcka et al., A Population of Cells Isolated from Rat Heart Capable of Differentiating into Several Mesodermal Phenotypes, J. Surg. Res., 62:233-242 (1996).
|Written Opinion in PCT/US98/14520, dated Jun. 23, 1999.
|Yanagisawa-Miwa et al., "Salvage of infarcted myocardium by angiogenic action of basic fibroblast growth factor," Science, 257:1401-1403 (1992).
|Citing Patent
|Filing date
|Publication date
|Jan 9, 2014
|Sep 18, 2014
|Agilent Technologies, Inc.
|Transposome tethered to a gene delivery vehicle
|U.S. Classification
|424/93.1, 435/377, 424/93.7
|International Classification
|Cooperative Classification
|European Classification
|Mar 18, 2010
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Capri restaurant in West Allis up for sale
Capri, an Italian restaurant that had been open at 8340 W. Beloit Road, West Allis, for more than four decades, is closed and the business is for sale.
Loyal customers have said recently that the restaurant has been closed for about a month. »Read Full Blog Post(2)
'Malcolm and Teresa' has trouble finding heart of pair's connection
In early 1968, conservative British journalist Malcolm Muggeridge was asked to conduct a televised interview of an Albanian nun. He had never heard of her. The world did not yet know her. It soon would: Her name was Mother Teresa.
That's the setup in Cathal Gallagher's "Malcolm and Teresa," which is receiving its regional premiere at Christian-based Acacia Theatre Company in Mequon, in a production that opened Friday night under Elaine Rewolinski's direction. »Read Full Article
Neko Case's voice shines in an elegant, melancholy set
The human voice can be the most singularly beautiful musical instrument there is, but it's not hard to think of well-compensated pop singers who treat their instruments, or allow them to be treated, as if they were beside the point.
Neko Case is not among them. »Read Full Article(1)


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Spectacular Direct Ocean View in Every Window -Sandpiper Tower
The owner has been emailed.
To book this property, you need to complete your booking through checkout on the VRBO website. We can't protect your payment if we don't know you've booked through us. Learn more
This listing was first published here in 2008.
Date last modified - Tuesday, September 20, 2016


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World's worst public artworks, according to Virtualtourist.com
- From: news.com.au
- September 06, 2011
ART is a very personal thing. One man's Michelangelo is another man's eyesore.
But what about when it's out in the open?
Travel website Virtual Tourist has compiled a list of the world's most terrible pieces of public art, as voted by its members and editors.
Pictures: See the world's worst public artworks
The site's general manager, Giampiero Ambrosi explains: "The problem with public art is that you often can't avoid it."
"At least with art that's housed in a museum you can choose not to go to the museum, but with these pieces you might have to literally take a detour to get away from them."
So what did they come up with?
First on the list is the mammoth Marilyn Monroe in Chicago, Illinois. Created by New-Jersey based artist Steward Johnson, "Forever Marilyn" is an eight-metre tall model of the siren in her classic, wind-blown dress pose.
Not a detail has been spared and tourists who venture beneath Marilyn's billowing dress will catch glimpse of her lace knickers. Now, what would her mother say?
Well, 'been there, seen that' is the sentiment toward these unoriginal critters, who can now be found in just about every nook and cranny.
Where else would you expect to find this nose-twitching curiosity but Salem. Not that locals are too happy about the choice.
Salem residents opposed the monument, saying it wasn't right to erect a statue of a witch in a town where thousands were killed for supposedly practising witchcraft.
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About Kadzidlo
Researching Reading FC for SI since 2006
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Reading FC est.1871
Kadzidlo started following Some quick 'fixes' for defence...anyone?
Kadzidlo started following a good championship striker?
Kadzidlo replied to Gripper's topic in Database and Research IssuesYes, I see the same, it should be Scotland as Primary, and England as his second nation.
Kadzidlo posted a topic in Football Manager General DiscussionDo you have your goalkeeper set to take penalties during games? In my game, I had a testimonial match for Adam Federici (Reading FC Goalkeeper), knowing that he's like most goalkeepers who think they can play as a centre forward, my plan was to stick him up front for the last 15 mins of the match. I also put him as the number one Penalty taker. The game ended 1-1, with Federici scoring the equaliser from open play with about 10mins to go (no pens in the game) 4 Months pass and 18 games into season. The testimonial match is now long forgotten. We are 2 nil up at home to Burnley in an important Premier League game, we win a penalty (first one of the season). I'm looking around on screen to see who's going to take it (normally Pog or Blackman), and then Federici appears next to the ball on the spot and calmly places it in the top corner! Now my dilemma, do I keep him as the Pen taker? I'm very tempted too - how many others use their GK to take Pens? and did you have any success?
Kadzidlo replied to turnip's topic in Football Manager General DiscussionSeason 35/36 A) £0 no players Season 34/35 A) £110k/month loan 2 players (1 Free) B: 20% C: 0 Season 33/34 A) £1.5M on 2 players B: 15% C: 0 Season 32/33 A) £325k 1 player B: 10 % C: 0 Season 31/32 A) £0 no players Normally made my signings before deadline day, or free transfers at the start of the window.
Kadzidlo replied to Jimbokav1971's topic in Football Manager General DiscussionI've been running FM on a Samsung RF711, it easily copes with the demands of FM, on the CPU, memory and graphics card.
Kadzidlo replied to Goldy's topic in FM OnlineDo both the host and client both have to be logged in to steam to play? I ask as I've two laptops on my desk and with last years version FM12 I was able to play a network game using both laptops and my single steam account.
Kadzidlo replied to Apaco's topic in Football Manager General Discussioni'd say so, except Poland were playing at home so that needs to be accounted for too.
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Three models in the Wolverine Ethernet Extender series from Westermo have been enhanced after extensive testing by a third party test house with the Class 1, Division 2 approval. This allows these Ethernet extenders to be installed in hazardous or potentially explosive environments containing volatile, flammable vapors and gases, making them suitable for use in industries such as oil and gas, petrochemical, processing and metalworking. The Wolverine Ethernet extenders have also been ATEX approved according to II 3G EEx nA IIT5 which is the European equivalent to Class 1, Div 2.
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After you have successfully placed your order, your newspaper will be printed in the next print run. We print digital newspapers in batches on Mondays, Tuesdays and Thursdays at 2pm. Traditional newspapers can be printed at any time.
We aim to have all newspapers printed and sent out within 5 working days. The delivery time varies depending on the destination. To find out how long orders take to arrive, see delivery information.
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When was the last time you had tea with a dragon? Folkmanis Puppets have been encouraging young imaginations to blossom for over 25 years. From 4-inch finger puppets to life-sized puppets, these award-winning puppets from Folkmanis are a creative force bringing real life and imagination together. Folkmanis Puppets are award-winners because of one thing: detail! Tongues move, wings flap, legs crawl, eyes open and close! Realistic design even allows first-time puppeteers to become veritable puppet masters in mere minutes.
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Gearing up for school and finding just the right backpack and all the coolest stuff to fill your pencil case, makes this such a fun time of year! Let's get ready for a whole school year's worth of possibilities. Check out our favourite finds ...
We're hiring! For over 30 years Mastermind Toys has been focused on offering the very best in quality toys and books for children in a shopping environment that is engaging, inspiring and fun. Send resumes to email@example.com
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1. Geña: Artful Wednesdays - TODAY!!
2. Fiction Reading with Sjon
3. Pitt Night: The Music of John Williams - is FULL
4. Martha Graham Dance Company - is FULL
5. Letterboxing at The Frick
6. Across the Universe
7. PITT ARTS Notify U
1. Geña: Artful Wednesdays
TODAY! Wednesday, September 28
Nordy's Place – Lower Level William Pitt Union
Enjoy the sounds of Puerto Rico and the Caribbean with this talented artist, as we kick off the 15th anniversary of Artful Wednesdays!
Free Lunch! No need to sign up, just show up and bring all your friends.
Lunch is limited. Valid Pitt Oakland campus ID required.
2. Fiction Reading with Sjón
City of Asylum
Thursday, September 29
Meeting Time: 5:10 PM
Estimated End Time: 9:30 PM
Join us for this unique event featuring Sjón, an award-winning novelist, poet, and playwright born in Iceland. His novels, which include The Whispering Muse, The Blue Fox, and From the Mouth of the Whale, have been translated into thirty-five languages and he is also involved in a wide range of art exhibitions and music events. His longtime collaboration with the Icelandic singer Björk led to an Oscar nomination for his lyrics.
Enjoy dinner with us at Max's Allegheny Tavern before the reading.
3. Pitt Night: The Music of John Williams
Pittsburgh Symphony
Friday, September 30
Meeting Time: 7:00 PM
Estimated End Time: 11:00 PM
This program is FULL.
4. Martha Graham Dance Company
Pittsburgh Dance Council
Saturday, October 1
Meeting Time: 5:40 PM
Estimated End Time: 9:50 PM
This program is FULL.
5. Letterboxing at The Frick
The Frick Pittsburgh
Sunday, October 2
Meeting Time: 11:55 AM
Estimated End Time: 3:00 PM
Like puzzles? Like beautiful Pittsburgh places? Try Letterboxing at the Frick! Letterboxing is an historic pastime where people work together in an activity that combines puzzle-solving, orienteering, and art. After making individual rubber stamps, team up to read maps, solve word puzzle clues and use a compass to find hidden containers, or letterboxes, on the beautifully landscaped historic Frick grounds.
Held rain or shine. Dress for the weather.
Enjoy a Girasole boxed lunch at the Frick before the workshop.
6. Across the Universe
Pittsburgh Filmmakers
Thursday, October 6
Meeting Time: 6:25 PM
Estimated End Time: 9:30 PM
The music of the Beatles and the Vietnam War form the backdrop for this romance between an upper-class American girl (Evan Rachel Wood) and a poor Liverpudlian artist (Jim Sturgess). Golden Globe-nominated for Best Motion Picture and Grammy-nominated for Best Compilation Soundtrack, over 30 Beatles' songs are woven into the plot together with visual allusions to their timeless films.
Sweets at The Screening Room after the film!
7. PITT ARTS Notify U
New this academic year, Pitt student may opt to receive text updates from PITT ARTS through Notify U!
Just send PITTARTS


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2014 Can you snort oxycontin 20 mg ... - read more
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ellasdaddy228 Review
Good hybrid case for the money.
Case works great. The only fault is the headphone jack cover has the hole in the wrong place. It needs to be moved over about 1/4 inch for the connector to fit correctly.
- How did the image on site compare with the actual product? Right on
- Was the product free of any manufacturer defects? Yes
- Please tell us about the quality of the product. Good quality, just has one problem.
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Sustainable cleaning products summit tackles impacts
22 September 2016
The Sustainable Cleaning Products Summit, taking place in Paris from 26-27 October, will discuss practical approaches to addressing the impacts of cleaning products. [Read More]
EDITOR'S PICK - View More Editor's Pick
- REACH after Brexit
- It'll all come out in the wash
- Never run short of industrial-strength wipes
- The smart way to improve performance
- Nursery invests in Miele Professional
- The show must go on
- Accurate dilution of chemical concentrates
- Making recycling cups easier
REACH after Brexit
20 September 2016
Will Brexit really mean an end to EU red tape? Gordon Tranter, lead commentator for Croner-i Environment and Sustainability, discusses how UK-based companies' obligations under the provisions of EC Regulation 1907/2006 on Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) will change after leaving Europe [Read More]
OxyBAC is a new rich-cream foam antibacterial hand wash that combines Deb Foam Technology™ with Accelerated Hydrogen Peroxide antimicrobial agent to provide unique benefits compared with all other antibacterial hand wash products. [Read More]
More Sponsored Content
- The choice is simple...
Recycling and waste management is moving up the agenda in the cleaning sector, with companies increasingly being expected to demonstrate sustainable working practices both in terms of service delivery and supply chain. [Read More]


text: HI I have over 1000 fonts and not the room to store them all on the home drive I use a mac running OSX (snow leopard) so I moved some fonts to an external drive. Now what is the best way to use these fonts with inkscape - currently have 0.48 but use 0.47 now & then with my black cat cougar electronic die cutter A short term solution is I drag the font I want to my fonts folder but it would be nice if I could access the external drive fonts from within inkscape as I don't always know what font I want until I am part way into the design. Can I do this or am I just going to have to either bog down the system with the fonts or drag them over as I need them and re-start inkscape every time? Thanks Gaz


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Last week I had the privilege of being asked to judge Tony Blair's Faith Foundation's Faith Shorts competition, which showcased films by young film makers on the theme of faith. Young people from across the globe submitted films, each telling their own unique story giving a personal interpretation on faith.
I must say it was refreshing to see some positive coverage being given to faith, which so often is misrepresented: on one end of the spectrum it's perceived as being "uncool" or no longer relevant, and the on the other more dangerous end, we see faith hijacked by violent extremists for political ends. These young people have reclaimed faith for today's youth and those who believe in the true tenants of faith: peace, love, forgiveness and hope, all of which could be found within the films submitted.
Apart from the positive messages being espoused through the films, my fellow judges and I were treated to a true carnival of creativity courtesy of a diverse group of talented young artists. No less diverse was the ensemble individuals of who were invited to judge this competition. Just like the entrants, we judges were representative of various countries and from different backgrounds. I found myself judging the work of these young artists in the company of a group of ranging from the movie star and martial artist Jet Li to Her Majesty Queen Raina of Jordan. We all found the films extremely powerful, moving and thought-provoking.
I felt both honored and humbled to be asked to support the work of the Faith Foundation in seeking to bring future generations together through faith and personal expression. The shorts we watched all reminded me of the inexorable optimism of our young people and the ongoing quest for international peace and understanding. If more young people as remarkable as those who were honored last week continue to be given a platform to show us how we can live together, then I think we might have a shot.
For further information visit: www.tonyblairfaithfoundation.org.
More:Interfaith Faith-based Initiatives Tony Blair Faith Shorts Competition Tony Blair Faith Foundation
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Top Definition
adj. Stunning, outstanding, very good, better than any other.
Man, the trees I cop last time from you were unsurpassed.
dari Word in the ass Senin, 13 Juli 2015
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Email dikirim dari email@example.com. Kita nggak bakalan nge-spam kamu kok :).×


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List of ADHD medication for childrenGeneric Ovral do not protect against HIV infection (AIDS) or any other sexually transmitted disease. If there is a danger of infection, use a latex condom and spermicide in addition to the pill.Sumycin 250mg Side Effects
This product is a generic medication. While we strive to keep manufacturers consistent; in order to offer our customers the lowest price, we may purchase from the manufacturers below. warfarin
Diclofenac Sodium EC contains the active ingredient diclofenac sodium, which is a type of medicine called a non-steroidal anti-inflammatory drug (NSAID). All the medicines in the NSAID group reduce inflammation caused by the body\'s own immune system and are effective painkillers. Diclofenac Sodium EC works by blocking the action of a substance in the body called cyclo-oxygenase. Cyclo-oxygenase is involved in the production of various chemicals in the body, some of which are known as prostaglandins. The body in response to injury and certain diseases produces prostaglandins and they go on to cause the pain, swelling and inflammation of these conditions. Arthritic conditions are one example of this. As Diclofenac Sodium EC ss the body producing these inflammatory prostaglandins, it relieves the associated pain and inflammation. Diclofenac Sodium EC is used to relieve pain and inflammation in a wide range of musculoskeletal conditions, including arthritis, gout, sprains, fractures, dislocations, back pain, tendinitis and frozen shoulder. Diclofenac Sodium EC is also used to relieve pain and inflammation following dental, orthopaedic (bone) and other minor surgery. USES: Insulin regular is used with a proper diet and exercise program to control high blood sugar in people with diabetes. Controlling high blood sugar helps prevent kidney damage, blindness, nerve problems, loss of limbs, and sexual function problems. Proper control of diabetes may also lessen your risk of a heart attack or stroke. This man-made insulin product is the same as human insulin. It replaces the insulin that your body would normally make. It is a short-acting insulin. It works by helping blood sugar (glucose) get into cells so your body can use it for energy. This medication is usually used in combination with a medium- or long-acting insulin product. This medication may also be used alone or with other oral diabetes drugs (such as metformin).muscle achesconfusion, hallucinations, depression,...Mental Illness in ChlidrenBuy Arcoxia 120mg
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Top Definition
Slang for smoking crack. Used for saying you smoked crack while not wanting to draw attention to yourself.
Crackhead: I was following the monkeys all night.
Crackwhore: I follow the monkeys everynight!
Crackwhore: I follow the monkeys everynight!
από dustinvk 20 Απρίλιος 2008
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Foreign helmers tell tales
Directors (and one producer) of this year's Academy Award nominees for foreign films gathered Saturday to tell the stories behind their stories.
Directors (and one producer) of this year's Academy Award nominees for foreign films gathered Saturday to tell the stories behind their stories.
Canadian legit and feature helmer Robert Lepage, Gallic thesp Charles Berling and 18-year-old Montreal newcomer Jessica Pare star in "15 Moments," the new pic from Montreal writer-director Denys…
Viacom Inc.'s $162 million offer, unveiled Friday, for the 20% of Spelling Entertainment it doesn't already own likely signals the end of Viacom's long-running attempt to sell the TV production and…
Brazilian distribution veteran Marco Aurelio Marcondes has announced the creation of a distrib with leading producer Luiz Carlos Barreto, plus exhib Art Films and distrib Top Tape.
Canada's ChumCity Intl. has brought its local TV concept to Colombia in the form of CityTV Bogota, which launched at noon on Friday.
British and non-U.K. industryites gathered Saturday for the sixth annual BAFTA/LA pre-Oscars tea, at what British consul general Paul Diamond called "an occasion for tea-bibbers and the nominees."
After hitting an impasse with the filmmaker over the pic's remaining casting, 20th Century Fox has put the supernatural thriller "Sin Eater" in turnaround, allowing writer-director Brian Helgeland to…
Alliance Atlantis Communications has acquired a 47.85% stake in Sportscope Television Network, which operates Canuck specialty channel Headline Sports.
A Cairo court has handed down long terms at hard labor to 14 Islamic militants who were convicted of setting fire to two video shops and a cinema billboard company.
The Dutch government is planning to greenlight later this year the launch of a digital terrestrial platform.
FROM THE 71ST ACADEMY AWARDS The buildup proved bigger than the moment as Elia Kazan received his honorary Oscar with just a few words of thanks and, in his own words, "I think I can just slip away."


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On Location Photography, Part 1
Lesson 1: Introduction and Location Scoutingwith Jeremy Cowart
Duration: 1 hour 32 minutes
Jeremy Cowart has been working as an entertainment photographer for 6 years shooting celebrity portraits, album covers, and concert photography. In the first part of this on location series, he has invited Scott Kelby and his video crew to follow Jeremy around Venice Beach, California as he scouts and chooses locations, lights the scenes, works with his model, and figures out how to get the creative shots he is looking for. All of this is unfolding live in front of the cameras as Jeremy and his crew find random locations and shoot with portable equipment. Scott takes the opportunity to ask questions submitted by viewers and they discuss how Jeremy goes about picking locations, works with clients, and what type of lighting equipment he likes to use.
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Jeremy Cowart
Jeremy Cowart is a professional photographer from Nashville, Tennessee. Beginning his photography career in 2005, Jeremy quickly became a respected artistic voice in the industry. Having shot numerous musicians, entertainers and celebrities, Jeremy is also the founder of Help-Portrait Movement, a worldwide movement of photographers giving free portraits to those less fortunate. As his list of clients continues to grow, so does Jeremy's desire to improve, share, teach, and give back to those around him.


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1-8 of 8 in Krasnoyarsk
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As featured in USA TODAY and recommended by Travel + Leisure in its annual Villa Guide:
FlipKey is a vacation rental marketplace with more than 300,000 homes and rooms around the world. Find the perfect place to stay for your trip, and get great value along with the space, privacy and amenities of home.
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Min comment interval: 14 seconds
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#81 - orangerageMade this gif because of this one
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Might as well be, Voting this as funny is a little offensive don't you think?


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RADical Days: Free admission to Pittsburgh attractions this weekend
Copyright 2014 by WTAE.com All rights reserved. This material may not be published, broadcast, rewritten or redistributed.
Days after patting himself on the back for not invoking them on a debate stage, Donald Trump and his campaign are attempting to use Bill Clinton's sex scandals to cast a shadow on Hillary Clinton.
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Use the tools below to refine your search by only displaying reviews with a certain number of star ratings or to only show reviews from a certain time period. Eg click on '1 star' to just display the reviews we have which received a 1 star rating click or 'Within the last month' to display just reviews posted over the last month.
Written on: 12/02/2008
For the money it has been a truly excellent little car and does everything demanded of it… Read Full Review
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New To This Site
New To This Site
Nov 30, 2004 11:17PM PST
SO All of my firends are on this site so hopefully i can find them... I hear good things about this place..can't wait to find out for myself..I just got DDR for XBox, can't wait to play online..Currently sharing a gamers tag, I think i need to get my own.. I've been trying to learn to play Halo 2..I think im getting the hang of it..but i seem to be the one always dead..I get too excited and im looking all over the place...i think i need to learn to jump more.. If anyone is out there and is willing to train!! PLLEAASEE Help me!! I think i could be pretty good if i wasn't dead all the time...lol.. Well look forward to posting again...
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Albergo Verdi - Padova
Onsite amenities include a bar at guests' disposal and free wireless Internet throughout the hotel.
Albergo Verdi - Padova
Hotel Info: 1-877-214-4288from $82
- Single Room
- Double or Twin Room with Extra Bed
- Double Room
- Single Room in Annex Building (Via San Gregorio Barbarigo 15)
- Single Room, Annex Building
- Double Room in Annex Building (Via San Gregorio Barbarigo 15)
Also popular with travelers from the USA
At a glance
Key facts
Hotel size
- This hotel has 14 rooms
- This hotel is arranged over 3 floors
- Check-in time starts at noon
- Check-out time is 10 AM
- Express check-in/out
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- Children (4 years old and younger) stay free when occupying the parent or guardian's room, using existing bedding
- Pets allowed
- Restrictions apply *
Extra people
- Roll-away beds available, charges may apply*
- Free WiFi in public areas
- Free WiFi in rooms
In the hotel
- Free buffet breakfast daily
- Bar/lounge
- Room service (during limited hours)
- 24-hour front desk
- Concierge services
- Tours/ticket assistance
- Luggage storage
- Multilingual staff
- Number of buildings/towers - 1
- Elevator/lift
- Safe-deposit box at front desk
- Television in common areas
In the room
- In-room climate control (air conditioning)
- Air conditioning
- Iron/ironing board (on request)
- Soundproofed rooms
- Private bathroom
- Shower only
- Free toiletries
- Hair dryer
- Cable/satellite TV channels
- Desk
- Free WiFi
- Phone
- In-room safe
Albergo Verdi - Padova's small print
Also known as
- Albergo Verdi
- Albergo Verdi Hotel
- Albergo Verdi Hotel Padova
- Albergo Verdi Padova
All guests, including children, must be present at check-in and show their government-issued photo ID card or passport.
Cash transactions at this property cannot exceed EUR 2999.99, due to national regulations. For further details, please contact the property using information in the booking confirmation.
Pet-friendly rooms can be requested by contacting the property at the number on the booking confirmation.
Mandatory fees
You'll be asked to pay the following charges at check in or check out:
- A tax is imposed by the city: EUR 2 per person, per night
Optional extras
Parking is available nearby and costs EUR 15 per 24 hours
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Recent reviews
Excellent 4.2 /5 from 58 reviews
Fabulous small hotel right in the centre of Padua, ideal for sight-seeing. The room was small but fine for a couple of nights, lovely firm bed and great shower with plenty of hot water and towels etc. Great breakfast and very friendly, helpful staff Execellent value and highly recommended
Pleasant stay, nice small hotel near the fine Padua squares and Verdi theatre. Good connection with the train station by bus. Fine both for tourists and workers. Very kind staff.


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Brave and astute, David Cameron has regained his authority and kept the coalition and his party together
In all that we do and say about Britain's relationship with our European neighbours, we must never forget that this is really a story about our neighbours' relationships with each other. The chances are that yesterday's new accord between them will fail. In the mayhem that would follow, Britain's particular location outside the stockade would cease to obsess even us British.
But it obsesses us this morning. And it's hard not to feel a sense of relief that we do now know where we are. We're out of it. Whether we jumped or (as I suspect) were pushed, our banishment


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Top Definition
shitabis is the worst weed you can buy its almost like the dealer picked it from his ass crack
damn dog you paid thirty bones for the shitabis
uncle ruckess tarafından 27 Temmuz 2009, Pazartesi
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Damnnn jamal i smoked some dank OG Shitabis smuggled from columbia the other day, damn that was the shit.
Rollsafe_Stormzy tarafından 27 Temmuz 2016, Çarşamba


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The first film to come out of the mysterious and beautiful kingdom of Bhutan, Travellers follows a frustrated and America-obsessed government officer Dondup (Tshewang Dendup) as he tries to leave his village and make contact with a connection who will get him to America, his "dreamland," a place where "you can do anything... wash dishes, pick apples, anything." Clomping through the hillside village with an "I Love NY" t-shirt pulled over his traditional garments, he's extremely impatient to leave. Unfortunately, Dondup misses the infrequent bus to town -- a very unlucky circumstance in transportation-starved Bhutan -- and finds himself walking and hitching with his turquoise suitcase hoping he'll make it in time.
Continue reading: Travellers And Magicians Review
Jackson Millarker will star in episode 'A Stereotypical Day' set to air in the US on Wednesday evening.
The cast had teased something big was coming and all was revealed on Monday night.
The couple have recently found themselves dragged into the Brangelina divorce and even forced to deny split rumours.
The rapper teams up with Apple Music on his latest project.
If you're going to tell a tall tale of the Himalaya, it can't hurt to...


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Concept electro car
This project received 18 bids from talented freelancers with an average bid price of $340 USD.Get free quotes for a project like this
Skills Required
Project Budget$250 - $750 USD
Total Bids18
Project Description
This is a job for the competition. Electric car. In the modern metropolis is very urgent problem parking and air pollution. Requirements for the job:
- Modern design
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- The ability to accommodate two people
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Bellever Youth Hostel
Bellever shown within Devon
|OS grid reference
|West Devon
|Shire county
|South West
|Sovereign state
|United Kingdom
|Post town
|Postcode district
|Dialling code
|Devon and Cornwall
|Devon and Somerset
|South Western
|EU Parliament
|South West England
|UK Parliament
|Torridge and West Devon
Coordinates: 50°34′52″N 3°54′04″W / 50.581°N 03.901°W
Bellever is a hamlet in Dartmoor, Devon, England. It is located on the river East Dart about 2 kilometres (1 mi) south of Postbridge.
The first mention of a settlement at this location is in a Duchy of Cornwall record from 1355 which gives the name of a farm here as Welford, a contraction of wielle (spring) and ford.[1] However, in his High Dartmoor (1983), Eric Hemery proposed that the name comes from bal (mine) and ford, meaning the mine by the ford.[2]
Until the period after World War II the hamlet consisted of Bellever Farm (owned by the Duchy of Cornwall) and its outbuildings, with a small number of cottages for the farm workers. The farm gained a reputation for the introduction of Galloway and Aberdeen Angus cattle onto Dartmoor.[3] However, in 1930 the Forestry Commission bought the farm and began a large planting scheme here; several houses were built in the 1950s to house the forestry workers.[4] The coniferous plantation known as Bellever Forest[4] surrounds the hamlet on its north, west and south sides.
In 1934 some of the barns of Bellever Farm were let to Youth Hostels Association (England & Wales) and now form the oldest, still functioning youth hostel in Devon.[5]
As of 2016 the hamlet is served by a daily bus service that runs between Yelverton, Princetown, Postbridge and Tavistock.[6] During the summer the Forestry Commission run a small visitor centre by the East Dart river.[7]
Bellever Tor lies 1.5 kilometres (1 mi) to the southwest.
- ^ Gover, John; Mawer, Allan; Stenton, Sir Frank Merry (1932). Place-names of Devon. English Place-Name Society. 8. Cambridge University Press. p. 192.
- ^ Hemery, Eric (March 1983). High Dartmoor: Land and People. Robert Hale. p. 486. ISBN 0-7091-8859-5.
- ^ "The Bellever Bulls". Retrieved 2009-04-13.
- ^ a b "Introduction to Bellever Forest" (PDF). Retrieved 2009-04-13.
- ^ Maurice-Jones, Helen; Porter, Lindsey (2008). The Spirit of YHA. YHA. p. 149.
- ^ "98 - Tavistock". Target Travel. Retrieved 3 July 2016.
- ^ "Bellever and Postbridge Trails" (PDF). Moor than meets the eye. Retrieved 3 July 2016.
External links[edit]
Media related to Bellever at Wikimedia Commons