text:
Ferguson not looking to sell
Sat, 04 May 2013 07:10:00
Manchester United manager Sir Alex Ferguson insists he has no plans to offload any of his star men.
Ferguson has already confirmed he has a few ideas in mind for the summer, even though executive vice-chairman Ed Woodward claimed on Thursday there was no need for "major retooling" of the Red Devils squad.
There are some players who appear surplus to requirements though, including midfielder Anderson and wide-man Nani, both of whom have failed to match expectations since their arrivals in 2007.
Nani has made more of an impact but his contract is due to expire next year and so far, the respective parties are poles apart in their valuations. However, Ferguson insists no one is going anywhere.
"We have no plans to sell anyone," said Ferguson.
"That's a fact. There is no need for us to sell anyone. Nani played last week at Arsenal and he did well.
"When he is in form there are very few better than him. So I think we will bring him back in pre-season training and he will be all right."
Ferguson did confirm there had been no progress with the Portugal international's contract negotiations though and it is highly unlikely United would allow Nani to simply run his contract down.
Strangely enough, if Southampton had responded to United's inquiry about Gareth Bale, Nani might never have been signed from Sporting Lisbon in the first place.
Instead, Bale went to Tottenham, since when he has gone on to become one of the Premier League's star names, pipping Robin van Persie for both the PFA and FWA Player of the Year awards over the past week.
"We were disappointed in Southampton because we were first there but they never came back to us," said Ferguson.
"He was a left back when we tried to sign him from Southampton.
"He reminds me a bit of when we signed Lee Sharpe. He was a six foot, gangly slim boy but all of a sudden he was built like a light-heavyweight American boxer.
"Bale is the same given the way he has developed physically in the last two years. He has matured very well."
Van Persie had to be content with the his second Premier League player of the month prize. However, as the Dutchman's 10-match barren streak coincided with Bale's best form of the campaign, and voting for those individual prizes, Ferguson has few complaints.
"You can't dispute it," he said.
"Some of the goals Bale has scored this year have been fantastic.
"Robin is a little bit unlucky in that he hit a quiet spell and didn't score at a time Bale was bashing them in from all sorts of distances.
"There is not much between the two of them in terms of performances this season and that was reflected in the percentage of the votes."
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US 20030228638 A1 Abstract The detection of endothelial cell antibodies has been proven clinically important for successful organ transplantation. Disclosed are methods of isolating endothelial cell antibodies for donor-specific crossmatching.
Claims(28) 1. A method of isolating an endothelial cell from a biological sample comprising:
(a) providing a sample containing, or suspected of containing, an endothelial cell; (b) contacting the sample with a detection reagent to form a detection reagent-endothelial cell complex; and separating the detection reagent-endothelial cell complex from the biological sample thereby isolating the endothelial cell. 2. The method of
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13. A method of cross-matching a donor and a recipient comprising:
(a) providing a donor sample containing, or suspected of containing, an endothelial cell; (b) contacting the donor sample with a detection reagent to isolate the endothelial cell; and (c) screening a recipient sample for a reactivity with the isolated endothelial cell wherein no reactivity of the recipient sample with the isolated endothelial cell indicates compatibility between the donor and recipient. 14. The method of
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25. A method of diagnosing of an immune related or vascular disorder in a subject, comprising
(a) providing a first sample containing, or suspected of containing, an endothelial cell from the subject; (b) providing a second sample containing, or suspected of containing an autoantibody from the subject; (c) contacting the first sample with the second sample; and (d) identifying an autoantibody-endothelial cell precursor complex, if present wherein the presence of the complex indicates the presence of the disorder in the subject. 26. The method of
27. A method of determining the efficacy of treatment, or assessing the prognosis, of an immune related or vascular disorder in a subject, comprising:
(a) providing a first sample containing, or suspected of containing, an endothelial cell from the subject; (b) providing a second sample containing, or suspected of containing, an autoantibody from the subject; (c) contacting the first sample with the second sample; (d) measuring an autoantibody-endothelial cell precursor complex if present to yield a subject profile; and (e) comparing the subject profile with a reference profile, wherein a similarity between the subject profile and reference profile indicates the treatment is efficacious and a favorable prognosis for the subject. 28. The method of
Description [0001] This application claims priority to U.S. Ser. No. 60/381,033, filed May 16, 2002, the content of which is incorporated herein by reference in its entirety. [0002] The invention relates to a method for the direct isolation of endothelial cells from whole blood for routine donor-specific crossmatching to detect anti-endothelial cell antibodies prior to organ transplantation. [0003] The presence of donor lymphocyte-reactive Human Leukocyte Antigen (HLA)-specific antibodies either before and/or after renal allograft transplantation has been associated with hyperacute rejections, early acute rejections, and poor graft survival. However, rejections may occur in the absence of detectable lymphocytotoxic antibodies, suggesting that non-HLA antigenic systems may also play a role in renal allograft hyperacute and acute rejections. Antibodies reactive with endothelial cells and monocytes (also called the EM-antigenic system), or only with endothelial cells, have been described and reported to have a deleterious effect in several organ transplantations. [0004] Recently, the major histocompatibility class I-related chain A antigen (MICA) expressed on endothelial cells was identified as one of the target antigens of humoral immunity associated with irreversible rejections of kidney allografts. Studies of HLA-identical living-related donor allografts showed that the presence of endothelial cell/monocyte reactive antibodies correlated with rejection, graft loss, and poor allograft function. It was reported that this reactivity could be responsible for up to 80% of irreversible rejections in this group of patients. However, the routinely used lymphocyte cross-match (LXM) does not permit detection of the clinically relevant HLA class-I, class-II, endothelial/monocyte-reactive and endothelial cell-specific antibodies. Although the presence of circulating endothelial cells in whole blood has been a subject of debate for many years, the existence of circulating precursor endothelial cells in adult humans has recently been reported by some investigators. However, there is currently no suitable method available to perform a routine donor-specific endothelial cell-crossmatch (ECXM). [0005] Therefore, there is a need to efficiently perform routine donor-specific endothelial cell cross-matching to aid in the identification of better donor-recipient combinations, which will thereby have a greater impact on transplant survival than the current method of lymphocyte cross-match. [0006] The invention pertains to a method of isolating endothelial cells which are useful in donor specific crossmatching prior to transplantation. The isolated endothelial cells are also useful in diagnosing various vascular and immune related disorders. [0007] The invention includes a method of isolating an endothelial cell from a biological sample by providing a sample known to contain, or suspected of containing, an endothelial cell. The endothelial cell is Tie-2 positive. Alternatively, the isolated endothelial cell is VEGFR positive. The isolated endothelial cell is a mature cell. Alternatively, the isolated endothelial cell is an endothelial cell precursor. The sample is contacted with a detection reagent to form a detection reagent-endothelial cell complex. The endothelial cell is isolated by separating the complex form the sample. Separation is accomplished, for example, by flow cytometry or use of a magnetic field. [0008] The detection reagent is a ligand for an endothelial sell surface receptor such as angiopoietin or vascular endothelial growth factor (VEGF). Alternatively the detection reagent is an antibody specific for an endothelial cell surface receptor such as Tie-2 or the vascular endothelial growth factor receptor (VEGFR). The antibody is an intact antibody or an antibody fragment (e.g. Fab, or Fv). The detection reagent is attached to a solid support, such as a non-magnetic, magnetic, or paramagnetic bead. [0009] The biological sample is whole blood, sera, tissue homogenate, peripheral blood mononuclear cells (PBMCs) or leucapherisate. [0010] The invention further provides a method of cross-matching a donor and a recipient by providing a biological sample from a donor that is known to contain an endothelial cell, contacting the donor sample with detection reagent to isolate an endothelial cell. A recipient biological sample is screened for reactivity with the donor isolated endothelial cell. No reactivity between the recipient sample and the isolated endothelial cell indicates compatibility between the donor and recipient and a higher probability that the organ transplant will be successful. [0011] In a further aspect, the invention includes a method of diagnosing an immune related or vascular disorder in a subject by providing a first sample from a subject known to contain, or suspected of containing, an endothelial cell. The first sample is contacted with a second sample. The second sample is from a subject known to contain, or suspected of containing, an auto antibody. Alternatively the second sample contains reagents (e.g. antibodies) that recognize endothelial cell surface markers associated with the particular disorder. Complex formation between the endothelial cell and the second sample is determined. The presence of the complex indicates the disorder in the subject. [0012] In yet another aspect, the invention includes a method of determining the efficacy of treatment or prognosis of an immune related or vascular disorder in a subject by providing a first sample from a subject known to contain, or suspected of containing, an endothelial cell, contacting the endothelial cell with a second sample from a subject known to contain, or suspected of containing, an autoantibody, measuring any autoantibody-endothelial cell precursor complex present to yield a subject profile. The subject profile is compared with a reference profile, wherein a similarity between the subject profile and reference profile indicates that the treatment is efficacious or a favorable prognosis. [0013] Immune related or vascular disorders include vasculitis, atherosclerosis, bleeding disorders, angiogenesis, thrombosis, defective wound healing, and transplantation. [0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. [0015] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. [0016]FIG. 1 shows dot plots representing forward and side scatter profile as well as fluorescence of peripheral blood mononuclear cells (PRMCs) stained with anti-Tie-2+ monoclonal antibodies (Mabs). Tie-2+ cells (grey dots) appeared in the lymphocyte gate. histograms show the percentage of Tie2+ cells (app. 2=3%) in PBMCs stained with negative control abs and Tie-2-Mabs. [0017]FIGS. 2a-c are photographs of the morphology of Tie-2+ cells under light microscope at various time points after isolation from peripheral blood. Initially, these cells appeared either as single cells or as clusters of round cells (a), which after few days of culture converted into adherent cells with extended cytoplasm, (the dark objects are beads) (b). After 7-12 days in culture the cells gradually developed a spindle shape (c). [0018]FIG. 3a is a series of photographs showing Tie-2+ cells stained positive for the endothelial associated markers, acetylated low density lipoprotein (Ac-LDL), von Willebrand factor (vWF), vascular endothe helial growth factor receptor-1 (VEGFR-1) and the vascular cell adhesion molecule (VCAM)(on activated Tie-2+ cells). Tie-2+ cells also constitutively expressed the human leukocyte antigen class I (HLA class I) and the monocyte/macrophage marker CD68. [0019]FIG. 3b shows that quiescent Tie-2+ cells expressed small amounts of the clinically important MICA antigens (dark grey), and HLA class II (light grey), control antibodies (black). [0020]FIG. 4a is a dot plot that represents the forward and side scatter of rosettes of Tie-2+ cells and paramagnetic beads (gate R1) and paramagnetic beads only (gate R2). [0021]FIGS. 4b-g are histograms showing that pre-transplant sera from two kidney-transplanted patients with hyperacute rejections reacted strongly with their respective donor-specific Tie-2+ cells (b & e), while their Tie-2− fractions, which included lymphocytes did not (d & g). Histograms c & f show that the paramagnetic beads alone did not react with the sera non-specifically. The grey lines represent control or negative sera, and the black lines represent reactivity with patient's sera. [0022] The invention is based in part on the discovery that targeting a single specific cell population allows for the detection of clinically relevant donor-specific human leukocyte antigens(HLA) class I, class II, endothelial-monocytes, or endothelial cell-specific antibodies prior to transplantation of organs. Routine use of the endothelial cell cross matching will aid in identifying better donor-recipient combinations and thus have a greater impact on transplant survival as compared to traditional lymphocyte cross matching. [0023] The clinical importance of endothelial cell (EC) antibodies in allo-transplantation has been reported. However, lack of a suitable method for isolation of donor-specific ECs has prevented routine detection of these antibodies prior to transplantation. The invention provides a quick and simple method for the direct isolation of ECs from whole blood, for routine cross-matching to detect anti-EC antibodies. The presence of endothelial cell-reactive antibodies has previously been detected using human umbilical vein endothelial cell (HUVEC) lines, keratinocyte cell lines, or monocytes as targets, or by immunohistochemistry. However, these methods are cumbersome because endothelial cell culturing is tedious and a generally large panel of donors of HUVEC lines/keratinocytes is required to represent all the known polymorphic alleles for screening of endothelial cell-reactive antibodies. Moreover, these methods do not permit the detection of donor-specific endothelial cell antibodies. Thus, use of an endothelial cell cross-match is advantageous over use of lymphocyte cross-match because lymphocyte cross-match does not allow for such detection or isolation of donor-specific endothelial cell-reactive antibodies. [0024] ECs were isolated using magnetic beads coated with antibodies against the angiopoietin receptor, Tie-2 that is expressed on EC precursors. Tie genes play an important role in renal vascular development, and based on transplantation experiments these precursors have been shown to contribute to the generation of glomerular maturation. A retrospective analysis of 50 previously well characterized crossmatch sera taken immediately prior to transplantation from patients with end-stage kidney disease were tested. Tie-2+ cells expressed HLA class I, class II and other endothelial cell markers. Sera known to contain only EC specific or EC and monocyte (EM) reactive antibodies reacted positively with Tie-2+ cells, but not with Tie-2− cells from the same individual. In addition, the Tie-2+ cells reacted with sera containing only HLA class I or class II abs. In all, 3/25 sera from patients with stable graft outcome and no rejections reacted with Tie-2+ cells. This antigen-antibody interaction is relevant to the pathogenesis of rejection, since in many studies these antibodies are not detected in the serum of patients with good graft function or in non-transplantation patients. [0025] The invention includes methods of isolating an endothelial cell from a mixture of cells by contacting the mixture of cells with a detection reagent to form a endothelial cell-detection reagent complex. The complex is formed via a specific affinity interaction between the detection reagent and the cell. The complex is separated from the mixture to isolate the endothelial cell. The complex is separated from the mixture using techniques known in the art, such as by, e.g., liquid chromatography (e.g., HPLC or FPLC), High Performance Membrane Chromatography (HPMC), flow cytometry or use of a magnetic field. Alternatively, the complex is separated from the mixture by attaching the detection reagent to a solid support. A washing step may be employed by re-suspending the complex in a biologically compatible solution. The complex can be re-suspended, i.e., washed as many times as desired. Typically the particles are washed three times. A biologically compatible solution include biological buffers known in the art such as phosphate buffer saline (PBS). [0026] The detection reagent is an endothelial cell detection reagent. An endothelial cell detection reagent is any reagent that specifically identifies an endothelial cell. For example, the detection reagent is a ligand for a endothelial cell surface receptor. The endothelial cell surface receptors include, for example, the EC-specific tyrosine kinase receptor Tie-2 or VEGFR. The ligand is for example angiopoietin, VEGF or an antibody specific for the cell surface receptor. The antibody is a monoclonal antibody or a polyclonal antibody. The term antibody encompasses not only an intact antibody, but also an immunologically-active antibody fragment, e.g., a Fab or (Fab)2 fragment; an engineered single chain Fv molecule; or a chimeric molecule, e.g., an antibody which contains the binding specificity of one antibody, e.g., of murine origin, and the remaining portions of another antibody, e.g., of human origin. For example the detection reagent is monoclonal antibody Tie-2 or VEGFR-1 [0027] The detection reagent is attached to a solid support. The solid support is a particle, a polymer (e.g., polystyrene, polyethylene), a vessel, a chamber, a dipstick, beads, particles, membranes (e.g., nylon, nitrocellulose or polyvinylidenedifluoride (PVDF)), or other forms known in the art. [0028] The solid support may carry functional groups such as hydroxyl, carboxyl, aldehyde or amino groups. The solid support may be positively charged, negatively charged or hydrophobic. Functionalized coated supports for use in the present invention may be prepared by modification of the support. For example, uncoated support is treated with a polymer carrying one or such functional groups, such as polyurethane together with a polyglycol to provide hydroxyl groups, or a cellulose derivative to provide hydroxyl groups, a polymer or copolymer of acrylic acid or methacrylic acid to provide carboxyl groups, or an aminoalkylated polymer to provide amino groups. U.S. Pat. No. 4,654,267 describes the introduction of many surface coatings. [0029] The particle is made of metal compounds, silica, latex, polymeric material, or a silica, latex or polymer nuclei coated with a metal or metal compound. Preferably, the particle is made of a metal compound, such as iron, gadolinium, zinc, indium, gold, silver, cobalt, copper, or magnesium. Most preferably, the particle is magnetizable or magnetic. By �magnetizable or magnetic� is meant that the particle is capable of having a magnetic moment impaired to it when it is placed in a magnetic field. [0030] The detection reagent is labeled with a detectable marker. For example the detection reagent to labeled with a radioactive isotopes (e.g., 125I, and 131I), enzymes (e.g., peroxidase, beta.-galactosidase, alkaline phosphatase) or fluorescent substances (e.g., fluorescein isothiocyanate (FITC). The labels are quantified by the conventional methods well-known in the art, thereby the formed immune complex is quantified. The mixture of cells is any sample known to or suspected of containing an endothelial cell. For example the mixture is a biological sample such as whole blood, sera, leucapherisate, bone marrow, peripheral blood mononuclear cells or a tissue homogenate. [0031] An endothelial cell is any cell derived from any part of the vascular tree. For example the endothelial cell is from large and small veins and arteries capillaries, the umbilical vein of newborns, blood vessels in the brain or from vascularized solid tumors. The endothelial cell is a mature cell. Alternatively, the endothelial cell endothelial cell precursor. Preferably, the endothelial cell is Tie-2 positive. [0032] Cross-matching detects those antigenic differences to which the recipient is already sensitized. A donor is crossmatched to a recipient by contacting a donor sample with a detection reagent to isolate an endothelial cell. The recipient sample is contacted with the isolated endothelial cell and reactivity of the recipient sample with the isolated endothelial cell is determined. By �reactivity� it is meant that that a complex is formed via a specific affinity interaction between the recipient sample and the cell. No reactivity of the recipient sample with the isolated endothelial cell indicates compatibility between the donor and recipient sample. In contrast, reactivity of the of the recipient sample with the isolated endothelial cell indicates non-compatibility between the donor and recipient sample. Compatibility is measured by no or low hyperacute rejection of the donor transplant by the recipient. [0033] The donor and the recipient are , e.g., any mammal, e.g., a human, a pig, a cow, or a horse. The donor and the recipient are the same species. Alternatively, the donor and the recipient are of different species. [0034] The donor and recipient sample is, for example, whole blood, sera, leucapherisate, bone marrow, peripheral blood mononuclear cells or a tissue homogenate. Optionally, the samples are subjected to a pre-purification step prior to crossmatching. [0035] Reactivity is determined my methods known in the art. For example reactivity is measured by an ELISA assay, flow cytometry (e.g. flow cytometric cross-match), or complement-dependent lymphotoxicity cross-match. [0036] The invention also provides methods of diagnosing, accessing the prognosis or monitoring the course of treatment of vascular and immune disorders. [0037] In these methods a first test sample is provided from a subject The sample is known to or suspected of containing an endothelial cell. Optionally, the endothelial cell is isolated from the first sample. [0038] An immune disorder is diagnosed by contacting the first sample to a second sample from the subject known to contain, or suspected of containing, an auto-antibody and identifying an autoantibody-endothelial cell complex. The presence of an autoantibody-endothelial cell complex indicates the subject is suffering from or predisposed to an immune disorder. In contrast, the absence of an autoantibody-endothelial cell complex indicates the subject is not suffering from or predisposed to an immune disorder. [0039] A vascular disorder is diagnosed by contacting the first sample to a second sample. The second sample is derived from the subject. Alternatively, the second sample comprises antibodies to cell surface markers know to be associated with a particular vascular disorder The presence of a second sample-endothelial cell complex indicates the subject is suffering from or predisposed to a vascular disorder. In contrast, the absence of a second sample-endothelial cell complex indicates the subject is not suffering from or predisposed to a vascular disorder. [0040] The methods allow the course of treatment of a vascular or immune to be monitored or the prognosis of the subject to be determined. In this method, a test sample is provided from a subject undergoing treatment for the disorder. If desired, test samples are obtained from the subject at various time points before, during, or after treatment. The presence of an endothelial cell complex is then determined to create a subject profile. The subject profile is compared to a reference profile whose vascular disorder or immune disorder state is known. The reference profile has not been exposed to the treatment. The reference profile is derived from a sample type as similar to test sample. Optionally, the reference profile is derived from a database of molecular information derived from samples for which the assayed parameter or condition is known. [0041] If the reference profile contains no autoantibody-endothelial cell complexes, a similarity in the amount of complexes between the subject profile and the reference profile indicates that the treatment is efficacious (e.g., that one or more symptoms of the immune disorder are alleviated or that the severity of the disorder is reduced), and thus, a favorable prognosis for the subject. However, a shift in the amount of complexes between the subject profile and the reference profile indicates that the treatment is not efficacious, and thus, an unfavorable prognosis for the subject. [0042] When the reference profile contains autoantibody-endothelial cell complexes, e.g., when the reference profile includes complexes taken from the subject at the time of diagnosis but prior to beginning treatment, a similarity in the expression of complexes pattern between the subject profile and the reference profile indicates the treatment is not efficacious. In contrast, a shift in expression complexes in the subject profile and this reference profile indicates the treatment is efficacious. [0043] By �efficacious� it is meant that the treatment leads to a decrease in any of the symptoms of an autoimmune disorder in a subject previously noted. When treatment is applied prophylactically, �efficacious� means that the treatment retards or prevents an immune related or vascular disorder. [0044] An immune disorder includes disorders mediated by an immune mechanism such as deposition of immune complexes, inflammation, direct attack by circulating antibodies (e.g., autoimmune disorders). An autoimmune disorder or an autoimmune related disorder includes those disorders caused by an immune response against the body's own tissues. Autoimmune disorders result in destruction of one or more types of body tissues, abnormal growth of an organ, or changes in organ function. The disorder may affect only one organ or tissue type or may affect multiple organs and tissues. Organs and tissues commonly affected by autoimmune disorders include blood components such as red blood cells, blood vessels, connective tissues, endocrine glands such as the thyroid or pancreas, muscles, joints, and skin. Autoimmune disorders include for example, autoimmune hemolytic anemia, autoimmune hepatitis, Berger's disease, chronic fatigue syndrome, Crohn's disease, Hashimoto's thyroiditis, fibromyalgia, systemic lupus erthyematosus, Graves' disease, idiopathic thrombocytopenia purpura, multiple sclerosis, psoriasis, rheumatic fever, rheumatoid arthritis, [0045] Vascular disorders include disease associated with the vascular system. For example vasculitis, atherosclerosis, bleeding disorders, defective wound healing. [0046] Symptoms of an autoimmune disorder depend on the specific disease and the organ or tissue that is affected. For example, systemic lupus erythematosus may cause kidney failure, arthritis, and a skin rash on the face. Autoimmune hemolytic anemia causes anemia, or low red blood cell counts. Generally symptoms of autoimmune disorders may include: low-grade fever, malaise, which is a vague feeling of illness, fatigue, or tiring easily. Autoimmune disorders are diagnosed based on symptoms, a physical exam, and the results of blood tests. [0047] Treatments to reduce symptoms may include: nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin or ibuprofen, to relieve fever, joint pain, and muscle aches corticosteroids, or steroids, help reduce inflammation. These medications are often used on a short-term basis to get a person through a sudden episode or flare-up, medications to suppress the immune system, such as methotrexate, azathioprine, and cyclophosphamide, which help to reduce inflammation and organ damage. In some cases, other treatments may be needed. For example, surgery may be needed for blockage of the bowels, which may occur in Crohn's disease. Blood transfusions may be needed in severe cases of autoimmune hemolytic anemia. Insulin is given to individuals with type 1 diabetes to control blood glucose levels. [0048] The subject is preferably a mammal. The mammal can be, e.g., a human, non-human primate, mouse, rat, dog, cat, horse, or cow. [0049] The data described herein was generated using the following reagents and methods. [0050] Mouse anti-human Tie-2 monoclonal abs. (Mabs) (BD Pharmingen, Oxford, UK) were first coupled to pan-mouse Dynabeads with a DNA-linker (cat. no. 115.19)(DYNAL, Oslo, Norway). For this purpose, 10 μg of Tie-2 Mabs were added to 500 μls of pan-mouse Dynabeads. The bead-Mab suspension was rotated on a rock n' roller for 24 hrs at 4� C. Excess Mab was removed and the Tie-2 Mab coated beads were blocked with 2 ml phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) on a rock n' roller at 4� C. for 10 min. The blocking step was repeated six times after which, the beads were resuspended in original volume (500 μls) of PBS/0.1% BSA. [0051] Due to the large numbers of cells required to establish the specificity of the Tie-2+ cells, in the initial experiments PBMCs were isolated from leucapherisate of healthy blood donors by gradient centrifugation with lymphoprep (Nycomed-Oslo, Norway). ECs were isolated from PBMCs using anti-Tie-2 Mabs coated magnetic beads. PBMCs were first distributed into several tubes each containing 40�106 cells. Fifteen μls of pre-coated Tie-2 magnetic beads were added to each tube and incubated in a volume of 500 μls RPMI medium (GIBCO, Paisley, UK) supplemented with 2 mM L-glutamine and 10% heat inactivated fetal calf serum. Cells+beads were incubated on rock 'n roller at 4� C. for 30 min. Tie-2+ cells were separated after extensive washing (5-6 times) with PBS using a magnet. Tie-2+ cells from all tubes were pooled together and rosettes were counted under a light microscope. [0052] 40 ml-heparinized blood was obtained from normal healthy donors. The blood was washed once as follows: 10 ml blood was diluted with 40 ml PBS/0.1%BSA. The blood was centrifuged at 800 g for 10 min. The supernatant was discarded and the blood cells were resuspended in 10 ml of PBS containing 0.6% sodium citrate. Fifty His of Tie-2 Mabs coated magnetic beads were added to each tube and incubated for 30 min at 4� C. on a rock 'n roller. Tie-2+ cells were collected by a magnet and washed once with PBS-Na-citrate. After 4-5 washes with PBS, Tie-2+ cells were collected and the concentration of the cells adjusted to approximately 3-4�106 cells/ml. These cells surrounded by beads were used either in a microcytotoxicity assay or the flow cytometer. [0053] The Mabs used for immunocytochemistry and FACs analysis are given in Table 1. Tie-2+ cells obtained from leucapherisate of normal volunteers gave sufficient numbers of cells to perform various immunocytochemical analysis. Tie-2+ cells were grown on fibronectin-coated tissue culture plates. Cells were allowed to attach (24 hrs) prior to staining for various EC-specific markers. Cells were left either untreated or stimulated with TNF-α and IFN-γ for 14 hrs. For immunocytochemistry, cells were fixed using 30% acetone in methanol for 1 min. After two washes with PBS, cells were blocked using 1% bovine serum albumin (BSA) in PBS for 1 hr at room temperature. The cells were washed twice with PBS and incubated with the above mentioned primary antibodies diluted 1:100 (in PBS) at 4� C. for 2 hrs. The secondary antibody was a goat anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC) diluted 1:500 (in PBS). After incubation for 1 hr at 4� C., the cells were washed twice and analysed under a fluorescence microscope. [0054] A total of 50 sera from kidney-transplanted patients were studied. Over the past years (1988-2001) the pre-transplant sera from kidney patients has been meticulously characterized, using various methods, for the presence of endothelial cell specific, endothelial-monocyte reactive, and HLA antibodies found to be associated with rejections. During these years 15 pre-tranplantation sera, characterized as having either anti-EM or EC specific antibodies from non-alloimmunized patients with rejections, have been collected. [0055] The general methods described herein are based on these 15 sera. Previous characterization showed that ten sera gave positive reactions with human umbilical vein endothelial cell (HUVECs) lines and monocytes (Table 2, pt. nos. 1-10), and five sera with only ECs (Table 2, pt. nos. 11-15). Since none of the fifteen patients with EM or EC-specific antibodies had been alloimmunized, no detectable HLA alloantibodies were found in these sera. In addition, ten well-characterized sera from alloimmunized patients known to contain only HLA class I or only class II alloantibodies were selected (Table 2). As controls, 25 sera from patients with no graft rejections were also tested. [0056] Using these sera, it was determined whether the isolated Tie-2+ cells could be suitable targets for detection of clinically relevant antibodies in kidney organ transplantation. A pool of sera from patients who had formed alloantibodies as a result of multiple blood transfusions or organ transplantations was used as a positive control. Sera from healthy non-transfused blood group AB males served as negative controls. The following antibodies, FITC-conjugated F(ab′)2 fragments of goat anti-human IgG (Fc specific), or IgM (Immunotech, USA) were used. For the flow cytometric ECXM 100,000 Tie-2+ cells coupled to beads were used and the procedure carried out as described earlier. The cells were analyzed on a Becton Dickinson flow cytometer (FACSorter). A shift in the mean fluorescence of 10 channels in the test sample as compared to negative control was considered as positive, determined as described before. This value is arbitrary and should be determined by each transplant laboratory. The Tie-2+ cells were also used in the routinely performed immuno-magnetic microcytotoxicity assay as described elsewhere. In addition, Tie-2+ cells immediately after isolation were tested in the flow cytometer for all the endothelial cell surface markers (Table 1). [0057] Tie-2+ ECs were isolated from human peripheral blood by magnetic bead selection. FACS analysis showed that approximately 3�4% of PBMCs were Tie-2+ (FIG. 1). After 24 hours in culture, the majority of Tie-2+ cells attached to fibronectin-coated 24 well plates and became adherent. Initially, these cells appeared either as single cells or as clusters of round cells, which after few days of culture converted into adherent cells with extended cytoplasm. After 4 days in culture the cells gradually developed spindle shape (FIGS. 2a-c). [0058]FIG. 3 and Table 1 summarize the results of the immunocytochemical analyses using antibodies to known antigens of endothelial cells. Already at 0 hours, FACS analysis indicated that Tie-2+ cells expressed the acLDL-receptor, vWF, VEGFR-1, and strong expression of HLA class I and HLA class II antigens. Importantly, the cells expressed the clinically important MICA antigen, though weakly. Upon cytokine activation, the cells expressed both the endothelial cell specific adhesion molecules CD62 E (E-selectin) and CD 106 (VCAM), as well as increased expression of HLA class II. Thus, both the immunocytochemical and FACS analysis indicated that Tie-2+ cells expressed majority of the endothelial cell markers. [0059] On an average, approximately, 3�4�104 Tie-2+ cells/106 PBMCs can be obtained from single donors. Since a large number of sera were analyzed herein Tie-2+ cells from leucapherisate of healthy blood donors were isolated. The results are shown in Table 2. All the 10 sera known to have endothelial-monocyte antibodies and 5 sera with endothelial cell-specific antibodies showed varying patterns of reactivity with the panel of Tie-2+ cells from six different donors. However, the Tie-2− fractions, which included the lymphocytes from the same donors, did not react with any of the sera. The reactivity pattern with the Tie-2+ cell panel of both the endothelial-monocyte and endothelial cell-specific antibodies as seen in Table 2, indicates the existence of polymorphism in these antigenic systems or alternatively presence of antibodies with varied specificities. [0060] Sera known to contain only HLA class I broadly reactive alloantibodies also gave positive reactions in every instance with Tie-2+ cells from single donors. Reactivity of HLA class II alloantibodies (limited specificities) was also observed with unstimulated Tie-2+ cells. 3/25 (12%) sera from control patients with no rejections and with stable graft functions gave positive reactions with four of the Tie-2 panel donors (Table 2). Results obtained using the immuno-magnetic microcytotoxicity assay were in agreement with the flow cytometric analysis (Table 3). [0061] Donor-specific endothelial cell crossmatches were retrospectively performed in two cases where frozen donor peripheral blood mononuclear cells were available. The first kidney grafts of both patients were lost in hyperacute rejections in the absence of demonstrable donor-specific HLA antibodies. Cross-match sera from one of these patients had been previously well characterized as having anti-endothelial cell-specific antibodies using HUVECs. [0062] In this instance, Tie-2+ cells from the peripheral blood mononuclear cells of this patient's (point. no.11, Table 2) first donor (father) were isolated and kept frozen in liquid N2. A cross-match with the serum of the patient taken immediately prior to the first transplant was retrospectively performed. The results are shown in FIGS. 4b-d. The results from a second case (point. no. 12, Table 2) are very similar to point no. 11, and are shown in FIGS. 4e-g. As seen, in Table 2, the sera from both these points gave similar pattern of reactivity with Tie-2+ cell panel. No reactivity with the Tie-2− fractions were observed.
[0063] [0064] [0065] Kissmeyer-Nielsen F, Olsen S, Posborg-Petersen V, Fjeldborg O. Hyperacute rejection of kidney allografts, associated with pre-existing humoral antibodies against donor cells. Lancet 1966; 2: 662. [0066] Ting A. Positive crossmatches�when is it safe to transplant? Transplant Int. 1989; 2:2. [0067] Sumitran-Karuppan S. The clinical importance of choosing the right assay for detection of HLA-specific donor-reactive antibodies. Transplantation 1999; 68: 502. [0068] Chapman J R, Taylor C J, Ting A, Morris P J. Immunoglobulin class and specificity of antibodies causing positive T cell crossmatches. Relationship to renal transplant outcome. Transplantation 1989; 42: 608. [0069] Brasile L, Rodman E, Shield CFd, Clarke J, Cerilli J. The association of antivascular endothelial cell antibody with hyperacute rejection: a case report. Surgery 1986; 99:637. [0070] Sumitran-Karuppan S, Tyden G, Reinholt F, Berg U, Moller E. Hyperacute rejections of two consecutive renal allografts and early loss of the third transplant caused by non-HLA antibodies specific for endothelial cells. Transplant Lmmunol. 1997; 5: 321. [0071] Cerilli J, Brasile L. Endothelial cell alloantigens. Transplant. Proc. 1980; 12 (3 Suppl 1): 37. [0072] Stastny P. Endothelial-monocyte antigens. Transplant. Proc. 1980; 12 (3 Suppl 1): 32. [0073] Pierce J C, Waller M, Phibbs M. A mixed antiglobulin test with kidney cells in suspension for IgG antibody in human allograft recipients. Transplantation 1975; 19: 343. [0074] Wilson C B. Individual and strain differences in renal basement membrane antigens. Transplant. Proc. 1980; 12 (3 Suppl 1): 69. [0075] Paul L C, Carpenter C B. Antibodies against renal endothelial alloantigens. Transplant. Proc. 1980; 12 (3 Suppl 1): 43. [0076] Mohanakumar T, Waldrep J C, Phibbs M, Mendez-Picon G, Kaplan A M, Lee H M. Serological characterization of antibodies eluted from chronically rejected human renal allografts. Transplantation 1981; 32: 61. [0077] Hosenpud J D, Everett J P, Morris T E, Mauck K A, Shipley G D, Wagner C R. Cardiac allograft vasculopathy. Association with cell-mediated but not humoral alloimmunity to donor-specific vascular endothelium. Circulation 1995; 92: 205. [0078] Perrey C, Brenchley P E, Johnson R W, Martin S. An association between antibodies specific for endothelial cells and renal transplant failure. Transplant Immunol. 1998; 6: 101. [0079] Kalil J, Guilherme L, Neumann J, et al. Humoral rejection in two HLA identical living related donor kidney transplants. Transplant. Proc. 1989; 21 (1 Pt 1): 711. [0080] Sumitran-Holgersson S, Wilczeck H, Holgersson J and Soderstrom K: Identification of the non-classical HLA molecules,MICA, as targets for humoral immunity associated with irreversible rejections of kidney allografts. Accepted. Transplantation. [0081] Cerilli J, Bay W, Brasile L: The significance of the monocyte crossmatch in recipients of living-related HLA identical kidney graft. Hum Immunol. 1983; 7: 45. [0082] Ianhez L, Saldanha L B, Paula F J et al: Humoral rejection with negative crossmatches. Transplant Proc. 1989; 21: 720. [0083] Asahara T, Murohara T, Sullivan A. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997; 275: 964. [0084] Peichev M, Naiyer A J, Pereira D et al. Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of functional endothelial precursors. Blood 2000; 95: 952. [0085] Vartdal F, Gaudernack G, Funderud S, Brattie A, Lea T, Ugerstad S and Thorsby E: HLA class I and II typing using cells positively selected from blood by immunomagnetic isolation�a fast and reliable technique. Tissue Antigens 1986; 28: 301. [0086] Sumitran-Karuppan S, Lindholm A, M�ller E: Fewer acute rejection episodes and improved outcome in kidney transplanted patients with changed selection of criteria based on cross-matching? Transplantation 1992; 53: 666. [0087] Sumitran-Karuppan S, Moller E: Specific inhibition of HLA class I and II antibodies by soluble antigens�A method for the identification of antibody specificity in sera from alloimmunized individuals. Transplantation 1994; 58: 713. [0088] Sumitran-Karuppan S, Moller E. The use of magnetic beads coated with soluble HLA class I or class II proteins in antibody screening and for specificity determinations of donorreactive antibodies. Transplantation 1996; 61: 1539. [0089] Moraes J R, Moraes M E, Luo Y, Stastny P: Alloantibodies against donor epidermis and early kidney transplant rejection. Transplantation 1991; 51: 370. [0090] Schnurch H, Risau W. Expression of Tie-2, a member of a novel family of receptor tyrosine kinases, in the endothelial cell lineage. Development 1993; 119: 957. [0091] Woolf A S, Yuan H T. Angiopoietin growth factors and Tie receptor tyrosine kinases in renal vascular development. Pediatr Nephrol. 2001; 16: 177. [0092] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Classifications
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Most Recent Articles In Retailing
Latest Retailing Articles
- Rituals Adds Makeup to Its U.S. Lineup
- Beauty Brands Gets Modern Overhaul
- Body Central Said to Prepare to File for Bankruptcy Within Week
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The next floor is the mezzanine, a 2,000-square-foot area called the Color Library, that features mass market brands, including Revlon, Neutrogena, L'Oréal, Maybelline, as well as Ulta's private label brand. Here, Ulta is merchandising products by usage (i.e., lip, eye, nail, face) rather than by brand, complete with testers. The mezzanine looks over the entire first floor. This new merchandising system is now in about six Ulta stores.
"Mass is self service," said Ulta's Kirby in a recent interview. "This will be a more fun experience." She said the aim was to reinvent mass with the four "E's": esthetics, education, entertainment and escape.
The second floor is dedicated to professional hair care and appliances, such as Redken, Paul Mitchell, Matrix, Tigi, PureOlogy, T-3, Hot Tools and Conair. What Ulta refers to as "hair artistry" brands are also here and include Frédéric Fekkai and Warren-Tricomi. Styling tools are displayed as testers so guests can touch and feel them, and feel "the force of a blow-dryer," said Buhler-Brandon.
The third, and top floor, is where the salon is located compete with 12 chairs and a spa. It is reached by a spiral staircase that allows a brief glimpse of the salon.
"We believe that if she can't see what's up the stairs, she won't go," said Kirby.
All types of treatments are available at the salon, including cut, color, facials and waxing.
Kirby is not arrogant to believe Ulta will not be up against a challenging holiday period. "Gas prices are playing into the shopping trip," she said.
For the second quarter ended Aug. 2, Ulta's sales grew 24.3 percent to $249.1 million. Operating income grew 46.2 percent to $7.2 million, which includes incremental preopening expenses of $1.2 million. Net income increased 67.2 percent to $3.7 million. Income per diluted share was 6 cents, compared with a loss per diluted share of 23 cents in the second quarter of fiscal 2007. Ulta opened 18 new stores during the quarter.
Jason Gere, a senior analyst at Wachovia, views Ulta's new South State Street store as a way for the chain to test out its successful merchandising strategy in a bigger box and to not be limited to its current 10,000-square-foot store average. It seems Ulta may need more real estate options as it embarks on opening 81 new stores next year and another 100 units in 2010.
"This is a way to see if they can make the model work with any size," he said. "It also may represent more real estate opportunities. I really think the success of the format has been what is in the store not the 10,000 square feet. It gives them more options to pick more real estate sites and to do more demographics research in their due diligence."
Kirby admitted that she "wants to learn with this one," referring to the store, which she will not refer to as a flagship since they "usually are expensive but don't produce."
Gere said that Ulta is not exempt from what the economy can do to a business, but it is well poised to tackle hardship: "They are not recession proof but they are recession resilient, like the beauty category. Having the one-stop shopping concept has been a help and also being away from the malls — the outlets that are closer to home as opposed to making separate trips help."
Gere is also impressed by Ulta's comps and he believes the chain's promotions are helping to drive sales.
In a recent proprietary survey of 25 retail locations (10 Ulta, 10 Sephora, and five freestanding boutiques), Gere learned that the distribution of Bare Escentuals Getting Started kits "could modestly benefit sales at Ulta locations more immediately" since most consultants there indicated that the sampling initiative has been very popular to date, with consumers rarely leaving the store "empty handed."


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Founder: Len Mullenger | Classical Editor: Rob
Barnett |
ROSSINI (1792-1868) Il Turco in Italia - dramma buffo in two acts (1814) Libretto by Felice Romani based on an earlier libretto by Mazzola written for Seydelmann, a contemporary of Mozart, and performed in Dresden in 1788. First performed at the Teatro alla Scala Milan, 14 August 1814. Selim, womanising Turkish Prince captivated by Fiorilla, Nicola Rossi-Lemeni (bass); Fiorilla, capricious wife of Don Geronio, Maria Callas (sop); Geronio, elderly husband of Fiorilla, Franco Calabrese (bass); Don Narciso, servant and lover of Fiorilla, Nicolai Gedda (ten); Prosdocimo, a poet and friend of Geronio, Mario Stabile (bar); Zaida, enamoured of Selim, Jolanda Gardino (mezzo) Chorus and orchestra of La Scala, Milan/Gianandrea Gavazzeni rec. La Scala, Milan. August, September 1953. MONO APPENDIX: Coloratura arias sung by Maria Callas. MONO Il barbiere di Siviglia, Una voce poco fa Giacomo MEYERBEER (1791-1864) Dinorah, Ombre légère (Shadow Song) Léo DELIBES (1836-1891) Lakme, Où va la jeune Hindoue? (Bell Song) Giuseppe VERDI (1813-1901) I vespri siciliani, Mercé dilette amiche (Bolero. Act 5)) rec. 21 September 1954, Watford Town Hall. Philharmonia Orchestra /Tullio Serafin Restoration Engineer: Mark Obert-Thorn. From English Columbia label LPs NAXOS HISTORICAL GREAT OPERA RECORDINGS 8.111028-29 [76.55 + 60.31] |
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The Woods Inn offers 20 uniquely different rooms, Adironack Guide Tents, a handicapped-accessible room, and two bedroom Lakeview cottage. All are inviting, comfortable and woodsy. The down comforters, the quiet of no phones or TV in the rooms and the […]
The main Dining Room is lakeside with an unparalleled view of Fourth Lake and the mountains. A Breakfast Buffet featuring bagels, english muffins, cereal, yogurt, milk, scrambled eggs, a meat selection, and either hash browns or french toast is served […]
Lake camera coming soon


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Texans Talk Football talk only please. Keep it to the game, the players, the coaches and management. |
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12-09-2008 | #1 |
Hall of Shame
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Free agents
I have been thinking about the disparity between how great the last few drafts have been and how awful the free agent signings have been and come to the conclusion that maybe Smith/Kubiak KNEW we were not going to be contending for anything and didn't want to waste any real money on top tier FA's until they thought we were ready to go to the next level. I am probably giving them too much credit, but hopefully we can improve in this area and get a star or two instead of other peoples garbage.
12-09-2008 | #2 |
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Re: Free agents
Anyone know how much cap space the Texans will have this offseason?
12-09-2008 | #3 | |
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Re: Free agents
Of course, if one ignores contradictory observations, one can claim to have an elegant or robust theory. But it isnt science. Halton Arp |
12-09-2008 | #4 | |
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Re: Free agents
Balls on that free agent crap. Build through the draft, and re-sign your own team's players that are performing at a high level. But signing other teams' "star players" become your team's overpaid "garbage". |
12-09-2008 | #5 | |
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Re: Free agents
Being a D-bag and being factually correct are not in any way mutually exclusive! |
12-09-2008 | #6 | |
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Re: Free agents
And that is what I was trying to say. For some reason we seem to get all the great guys in the draft but end up signing Roosevelt Colvin and Chris Brown in Free Agency. If you add up what they waste on the sorry guys you could get one or two impact players. The trick is figuring out who is going to stay an impact player and who is looking for the final payday. The Texans just sign has been's and ne'er will be's. |
12-09-2008 | #7 |
Bucco Bruce
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Re: Free agents
Mostly it's because impact free agents want to go to a team that WINS. Until the Texans can prove to be a consistent winner and a playoff caliber team, the only free agents that will be interested in signing with the Texans are players that are desperate for money, or players that cannot find a better team to go to.
I can't wait for the day that star players are going to want to play for the Texans. All it takes is a winning program, and we're heading that way. |
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12-09-2008 | #8 |
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Re: Free agents
The list will start with Fat Al.
I suspect his price tag will be $35M guaranteed and based on his work the last 2 years, I suspect he will have little problem getting a bidding war, and I think you guys are going to take a run at him.
When I lost you honey sometimes, I think I lost my guts too.... Follow me @blakegk on twitter...the entertainment never stops... |
12-09-2008 | #9 | |
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Now, if they want to pursue low profile blue collars like Kevin Walter, I have no problem with that. |
12-09-2008 | #10 | |
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Re: Free agents
He'll pocket that money . . . and coast. No, thank you. |
12-09-2008 | #11 |
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Re: Free agents
It isn't the Roosevelt Colvins or Chris Browns that are the problem. Its the Morlon Greenwood's and Anthony Weaver's that are the problem. Sure Colvin and Brown didn't pan out for the Texans this year but their contracts were structured such that cutting them wouldn't result in a huge cap hit. The same cannot be said about Greenwood's and Weaver's contracts. Cutting them early on would have resulted in huge cap hits.
"Hey fool, this ain't no football game!" - Mr. T |
12-09-2008 | #12 |
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Re: Free agents
I think we've done a decent enough job in free agency. Sure there are a couple of exceptions, but for the most part I think they were grabbing guys to be more of a stop gap/foundation builder while the young guys are developing. A lot of these FA's have special teams ability that we as fans rarely notice. I think not signing Charlie Anderson was a mistake (possibly von Hutchins) but what can you do when another team is willing to pay more than you. Reeves was a necessary evil. Yall can make all your jokes if you want about how a 3 year old could cover better... but if we went into the season with only Bennett, Faggins and Molden - we'd be giving up a lot more points per game than we are now.
"But make no mistake about it, my friends..." we've been hitting homeruns in the draft left and right and that's the best way to build a team. Hopefully this year we can get one more key FA on the D.
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12-09-2008 | #13 |
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Re: Free agents
I think you're right, which was why I was fine with the club franchising him last year. Titans are in a bad spot on him - he's their MVP, but who the hell knows if he invades the all you can eats after signing the contract and closes down the restaurant.
When I lost you honey sometimes, I think I lost my guts too.... Follow me @blakegk on twitter...the entertainment never stops... |
12-09-2008 | #14 |
hipster elite
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Re: Free agents
FA with a team should be more like a hired gun after you've established your team.
The winning teams know that you draft well and intelligently plug those guys in. Our problem sometimes is evaluating NFL talent outside our team. Weaver and Greenwood are just bad contracts period and they don't tell the whole story about what a good FA can do for your team. Steve Hutchinson and Alan Faneca are two pretty recent examples of how a guy can impact one side of the ball. I hate to say this but TO has altered two offenses into more explosive ones. Reggie White's move to GB proved to be franchise altering. I don't think we NEED any of these high priced guys myself but it couldn't hurt on defense to shore up a spot or two we won't be able to fill on draft day alone. |
12-09-2008 | #15 |
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Re: Free agents
I'm probably in the minority on this but my gut is telling me he is still going to be a great player even after he gets paid. I'd love for the Texans to sign him but its a long shot so I'm just hoping he gets the heck out of the AFC South.
"If the bra don't fit, you must acquit" |
12-09-2008 | #16 |
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Re: Free agents
I think Vinny has been spotlighting Bobby Grier as the root of the FA problem. He is the lead scout for pro personell, and if Vinny is right, which I believe he is - then he needs to go.
Your local CruiseOne Vacation specialist. www.firstclasscruising.biz Visit me on Facebook. |
12-09-2008 | #17 | |
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Re: Free agents
I 100% agree. The guy made some bone headed decisions early in his career but seems to have figured it out. He is still young and is playing at an unbelievable level. |
12-09-2008 | #18 | |
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Re: Free agents
☻/ Here's 2 u & here's 2 me & I /▌hope we never disagree. But / \ if that should eva be, 2 HELL with u, here's 2 ME!! |
12-09-2008 | #19 | |
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Re: Free agents
The Art of War Last edited by infantrycak; 12-09-2008 at 10:34 PM. |
12-09-2008 | #20 | |
Join Date: Mar 2005
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Re: Free agents
Sometimes "BIG" free agents choose their location on more than omney. Houston is a place where players traditionally have seen their careers die. Not a good motivation
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A scenic view, an established upick farm and farm stand, and an idea that would not go away, convinced this farmer to do it. Who would have thought it would become the larger income source on her farm!
We Built It: The Myth of the Self Made Man
The Republican National Convention theme "We Built It" is a beautifully crafted slogan; if it only provided the full story.
Capturing the True Truffle Flavor In a Bottle
It's not so easy to deliver nature in a bottle.
A Sustainable Dairyman Talks about the Milk Pasteurization Process
Sustainable dairyman Garry Hansen explains the different types of milk pasteurization in the dairy industry.
The Albion Strawberry: Everbearing, June through October (video)
Kathy Unger, of Unger Farms, talks about her Albion strawberries, and their amazing longevity for reliably producing fruit from June through October. Their flavor, is perhaps second only to the (unbelievably great) Hood River variety in the region.
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Tuesday, 15 May 2007 00:03
Amazon buys dpreview.com
Say cheese and cash in on your hobby
Monday, 14 May 2007 23:01
A4 color e-paper developed by LG Philips
More screen news from South Korea
Thursday, 10 May 2007 16:20
Canon summer collection
S5 IS and IXUS 950 IS coming soon
Thursday, 10 May 2007 14:45
New DVD anti-theft technology
Five finger discount no more ?
Tuesday, 08 May 2007 16:29
New used CD control legislation in the US
Wait for 30 days before you can sell it
Tuesday, 08 May 2007 16:04
Samsung HD Blu ray gets Simplay certificate
Compatibility not an issue they claim
Saturday, 05 May 2007 16:29
Pirate Bay guys will be prosecuted after all
MPAA and publicity behind the decision ?
Wednesday, 02 May 2007 21:22
Sony announces Cyber-Shot S800
6x zoom meets compact Cyber-Shot
Monday, 30 April 2007 21:30
Google Earth software engineer tells the tale
The people and technology behind Google Earth
Friday, 27 April 2007 21:59
India to offer free nationwide broadband
I'm moving down there in 2009


text:
I've read several mentions of using iodine for a starch test. Will Iodophor work?
Yes it works fine.
Yep. Just iodine with a something to keep it soluble. One of my go to chemicals. I keep 2 or 3 bottles around the process area (house). Scrapes and cuts, sanitizing, starch test... Good stuff. Cheers!


text:
Latest Stories
Angry Birds Dev making The Croods for iOS, Android
CommentsMarch 1, 2013
About This Game
- Summary
- Specifications
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The Croods finds the family breaking out of the Stone Age with the use of a new r-evolutionary tool: the IDEA! The Croods will Hunt & Gather their way through landscapes as they tame never-seen-before creatures. Help The Croods survive the wild and each other!
Release Date: March 14, 2013
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Sign in to digital edition
Thursday 29 Jan 2015
By Hanne Cecilie Gulstad. Web onlyPublished online: 19 November 2013
Eight international artists and architects have been shortlisted to design two memorials to commemorate the 2011 attacks in Oslo and on the island of Utoya.
After the attacks in July 2011, the Norwegian government decided to establish two memorial sites, one in the Hole district near Utoya—the island where Anders Behring Breivik shot dead 69 people and injured 110 more—and one in the government quarter in Oslo where eight people were killed and 209 injured by an exploding car bomb planted by Breivik.The competition has attracted a great deal of attention, receiving more than 300 submissions from artists, architects, poets and musicians from more than 40 countries. The competition is now in its second phase and the remaining contestants must submit their proposals by 15 February 2014. The eight shortlisted candidates are: the Swedish artist Jonas Dahlberg; the British artist Jeremy Deller; the Spanish architects Estudio SIC; the Norwegian artists Goksoyr & Martens with Snohetta Architects; the Norwegian Olav Christopher Jenssen with LPO architects; the Norwegian architects Haugen/Zohar; Paul Murdoch Architects from the US and the Nigerian architect Kunlé Adeyemi.The Norwegian ministry of culture is overseeing the project, with help from the Art Committee, which includes members of Koro (the government body responsible for public art in Norway) and the Labour Party's youth association. To engage the public, a committee conducted a survey earlier this spring, asking the Norwegian people if there were any words they would like to see inscribed on the memorial sculptures. The results were collected and will be forwarded to the candidates. An exhibition of the proposals will also be held to encourage a dialogue. According to the project manager, Harald Fenn, the names of all the victims of the attacks will be included at the memorial sites.
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Also in News
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InnoCept Marketing
InnoCept Marketing Interview Questions
Interview Experience
Interview Experience
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Interviews for Top Jobs at InnoCept Marketing
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I applied online. The process took a day – interviewed at InnoCept Marketing in June 2010.
Interview DetailsNOT LEGIT! Easy interview, which kind of tipped me off to the idea that they may be a scam. Went home to do some more research. Came across Indeed blog/discussion thing. 3 people had negative things to say-- one said he worked for them for 3 weeks and didn't average out to minimum hourly wage. Another guy had an even worse experience. Some of the people got really nasty emails and got hassled when they tried to leave.
Interview Questions- Which is stronger for you: Love to win or Hate to lose? Are you a competetive person? View Answers (2)
- How'd you hear about us? View Answer
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I applied online. The process took 2 days – interviewed at InnoCept Marketing in October 2009.
Interview DetailsI received a call informing me that I had been selected for an interview
I visited their office in North Dallas and had a 15-20 minute interview with the hiring manager
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We drove about 45 minutes outside of Dallas to some small town where we went door to door to businesses trying to get them to buy office supplies.Interview Questions- What would you say is your weakness? Answer Question
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Reasons for DecliningI decided to to decline offer because I am not moving at this time. Innocept was a goor experience.
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The Magazine
November/December 2011
50th Anniversary
The Moments
Fifty years of media culture, as captured by Magnum photographers
By The Editors Dec 13, 2011 at 06:00 AM
Magnum Photos, founded during the most glorious age of photojournalism, has always represented a dream of how journalism can be... More
Power of Dispassion
Alan Schwarz changed football
By Greg Marx Dec 2, 2011 at 12:45 PM
On October 17, 2010, the Philadelphia Eagles hosted the Atlanta Falcons before a crowd of nearly 70,000. The game... More
Immediate Returns
Ben Smith is not an old-school political reporter
By Liz Cox Barrett Dec 2, 2011 at 06:00 AM
Thirty-five-year-old Ben Smith reports on national politics for Politico from a rent-a-desk writers' workspace on the first floor of... More
A Reporter in Full
Isabel Wilkerson listens
By Pamela Newkirk Dec 1, 2011 at 06:00 AM
Isabel Wilkerson spent most of her journalism career at The New York Times where, as Chicago bureau chief, she... More
Dana Priest wants to show you how the world works
By Jill Drew Nov 30, 2011 at 06:00 AM
Washington Post reporter Dana Priest says she has always had an insatiable curiosity. At age six, she liked climbing... More
What He Knew
Anthony Shadid saw the deeper story in Iraq
By Terry McDermott Nov 29, 2011 at 06:00 AM
Anthony Shadid is the most honored foreign correspondent of his generation: two Pulitzer Prizes, a George Polk Award, an... More
Sustained Outrage
Ken Ward Jr. stayed home to make a difference
By Brent Cunningham Nov 28, 2011 at 10:00 AM
Since he began reporting full-time, in 1991, Ken Ward Jr. has embodied the credo of Ned Chilton III, The... More
Just Ask Questions
Stanley Nelson searches for truth in the past
By Hank Klibanoff Nov 22, 2011 at 09:00 AM
Stanley Nelson is the editor of the weekly Concordia Sentinel, a 5,000-circulation newspaper in Ferriday, Louisiana. Nelson, head of... More
A Different Life
Andrea Bruce was a community journalist in Iraq
By Michael Kamber Nov 21, 2011 at 06:00 AM
Andrea Bruce is a freelance photojournalist, currently based in Afghanistan, whose powerful documentary work attempts to connect people across... More
The Reporter's Voice
Seven accomplished reporters and one great photographer talk about what they do, how they do it, and why.
By The Editors Nov 21, 2011 at 06:00 AM
Since 1961, when CJR was born, journalism has undergone all manner of seismic shifts, from hot type to wireless... More
In Our Time
CJR's editor takes stock
By Mike Hoyt Nov 15, 2011 at 06:00 AM
On my first day at the Columbia Journalism Review, the editors were reading page proofs for an upcoming issue, and... More
The Newspaper That Almost Seized the Future
The San Jose Mercury News, Silicon Valley's own daily, was poised to ride the digital whirlwind. What happened?
By Michael Shapiro Nov 10, 2011 at 06:00 AM
1. 'It Was Written' Randall Keith and I are talking about the past when his boss, Dave Butler, slides... More
Pulitzer's Magazine?
Our founder reflects on CJR's roots
By James Boylan Nov 9, 2011 at 09:00 AM
Here is the best and here is the worst story of the day. . . . Here is the wrong of the day; here... More
Fiftieth Anniversary
A Plea for the Polls
'The press seems to behave as if it were operating in a simpler yesterday'
By Elmo Roper Nov 8, 2011 at 05:54 PM
Elmo Roper was one of the early giants of American opinion polling. His survey work for Fortune magazine, beginning in... More
Fiftieth Anniversary
Why a Review of Journalism?
The arguments for a critical journal far outweigh the hazards
By The Editors Nov 1, 2011 at 01:53 PM
What journalism needs, it has been said time and again, is more and better criticism. There have been abundant... More
Through the Years
Five decades of journalism, from the pages of CJR
By Clint Hendler Oct 27, 2011 at 06:15 PM
1961 • Walter Lippmann writes three columns based on more than four hours of interviews with Soviet Premier Nikita Khrushchev.... More
The Moments
Fifty years of media culture, as captured by Magnum photographers
By The Editors Oct 27, 2011 at 10:40 AM
Magnum Photos, founded during the most glorious age of photojournalism, has always represented a dream of how journalism can be... More
Language Corner
The living language
By Merrill Perlman Dec 7, 2011 at 10:00 AM
To look back at the early years of the Columbia Journalism Review is to look at how we used... More
Letters to the Editor
Letters to the Editor
Reader's congratulations, and reactions from our September/October issue
By The Editors Nov 28, 2011 at 06:00 AM
Fifty Candles Journalism the world over is in the midst of profound, transformative change, and it is not yet clear... More
Opening Shot
Opening Shot
Here's to another fifty
By The Editors Nov 28, 2011 at 06:00 AM
C JR's debut was mostly greeted with "bouquets," though a few readers, our second issue noted, "reacted with unblemished hostility."... More
Letters to the Editor
Notes From Our Online Readers
Readers respond to Erika Fry's "Escape from Thailand"
By The Editors Nov 28, 2011 at 06:00 AM
In September, Erika Fry, a CJR assistant editor, wrote of her "Escape from Thailand," an ordeal that began when she... More
Chairman's Note
By Victor Navasky Nov 22, 2011 at 09:00 AM
As I write this, every day seems to yield a new story about something called Occupy Wall Street. I have... More
Editor's Note
By Mike Hoyt Nov 22, 2011 at 09:00 AM
This is a handsome issue, no? Two entities are responsible for that. The first is Point Five Design, our art... More
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Markers in a changing news landscape, from sourcing to salaries to cyberspace
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An accounting of fifty years' worth of Darts is hardly a balm for an industry careening through a wrenching transition.... More
The Complications of our Age
What we want is a journalism to match them
By The Editors Nov 1, 2011 at 01:53 PM
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The Lower Case
The Lower Case
Bad News!
By The Editors Dec 8, 2011 at 06:00 AM
Editorial Page Almost a Garbage Dump— Delta (BC) Optimist 3/11/81 Newsmen Threaten Exposure— The Guild Reporter 7/24/70 Readers: We invent... More
A Mad Libs Keynote
The future of journalism? Just fill in the blanks.
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Independent journalism can't lean on a few rich donors
By Tom McGeveran Nov 16, 2011 at 06:00 AM
In lower Manhattan as I write, thousands of protesters, recently joined by some unions, local New York politicians, and a... More
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The digital-news parade threatens to pass some communities by
By The Editors Nov 14, 2011 at 06:00 AM
In Modesto, California, the need for news far exceeds the current supply. A city of 200,000 with one midsized... More
Modesto, California
By the numbers
By The Editors Nov 14, 2011 at 05:00 AM
Population 201,165 Eighteenth-largest city in California; 107th-largest city in the US, between Des Moines, Iowa, and Fayetteville, North Carolina... More
School's Out
A lost generation of journalists
By Laura Paull Nov 14, 2011 at 05:00 AM
A journalist walks across the Modesto Junior College campus in the mid-1990s and peeks in the newspaper office, where dedicated... More
Class Struggle
Tech won't end the digital divide
By Jen Schradie Nov 14, 2011 at 05:00 AM
Like many American cities, Modesto has been decimated by local media layoffs and cutbacks in recent years. Journalists have more... More
Just Press On
Templates for Anytown, USA
By Michael Stoll Nov 14, 2011 at 05:00 AM
Nic Roethlisberger and Dhyana Levey now live in the foggy Richmond District of San Francisco, flanked by the Pacific Ocean... More
Plowing Ahead
A farm newspaper's future
By Kristin Platts Nov 14, 2011 at 05:00 AM
Agriculture is and always has been the backbone of the California economy. Last year, Stanislaus County exported agriculture products to... More
A Paperless Bee
Making the future online
By Rusty Coats Nov 14, 2011 at 05:00 AM
In 1993, I was driving home to Modesto after covering a Bay Area conference on cryptography, having spent the past... More
Second Read
How the Past Saw the Present
The future of journalism has always been on journalism's mind
By Megan Garber Nov 11, 2011 at 06:00 AM
CJR knew about the iPad a good fifteen years before there was an iPad to know about. In a... More
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The limited vision of the news gurus
By Dean Starkman Nov 8, 2011 at 06:00 AM
"The question that mass amateurization poses to traditional media is 'What happens when the costs of reproduction and distribution go... More
A Reading List for Future Journalists
By The Editors Nov 3, 2011 at 12:09 AM
We asked some of our favorite journalists, scholars, and critics to recommend books and other works that could help... More
It's a Rall World
A series of thoughts on our media future from cartoonist Ted Rall
By Ted Rall Oct 27, 2011 at 01:28 PM
. More
- « September / October 2011
- « July / August 2011
- « May / June 2011
- « March / April 2011
- « January / February 2011
- « November / December 2010
- « September / October 2010
- « July / August 2010
- « May / June 2010
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- « January / February 2010
- « November / December 2009
- « September / October 2009
- « More Back Issues
Virginian-Pilot journalists: Corporate management pressure is stifling coverage - "Lovers of journalism in this newsroom are pissed. It's bad."
Paper files public records request—and city's response is a lawsuit - Local officials argue Montana courts should strike balance between privacy and disclosure
BBC Pop-Up reports from small town America - A small team is traveling across the United States for six months in hopes of finding underreported local stories
What game design can do for journalism - Three newly selected fellows at American University talk about the medium's future
Timeline, an app based on 'the history of…' - But chronology doesn't reveal everything
Email blasts from CJR writers and editors
Photojournalism while pregnant (NYT Mag)
"Momentarily forgetting I was pregnant, I jockeyed for a position close enough to capture the initial moments of euphoria, hurling myself into the mix of hundreds of frenzied relatives. As the weight of men started to close in on me, I realized how vulnerable I was and started to panic."
We should all step back from security journalism (Medium)
"It should be made clear, in law, that the tasks security reseachers do to make the net more secure and journalists do to understand and contextualize the truth for the public are not crimes"
Trust In Business And Media Is Declining, But People Have Faith In Search Engines (Buzzfeed)
People have become less trusting of major institutions, according to the annual Edelman Trust Barometer. And large majorities doubt that businesses want to make the world a better place.
With New Charlie Hebdo Cover, News Value Should Have Prevailed (NYT)
Public editor Margaret Sullivan on why the paper should have published the images.
Greg Marx discusses democracy and news with Tom Rosenstiel of the American Press Institute
CJR's Guide to Online News Startups
ACEsTooHigh.com – Reporting on the science, education, and policy surrounding childhood trauma
Who Owns What
The Business of Digital Journalism
A report from the Columbia University Graduate School of Journalism
Questions and exercises for journalism students.


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Ice Climbing World Championship 2015 at Corvara
The final stages of the Ice Climbing World Championship 2015 are currently taking place in Corvara / Rabenstein, Italy, today. The Speed discipline was won last night by the Russians Vladimir Kartashev and Maria Krasavina. At present the Men's Lead Semifinal is being battled out, while Italian hopes are high for locals Angelika Rainer and Barbara Zwerger who will compete in the finals as of 13:30. The entire event is being streamed live now.


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Props to English riders I must give props to all you English riders lol.. I am a western rider and I have recently started jumping lessons and I honestly don't no how you guys do it first of all I can't even get my foot in the stirrup cuz there really short lol but I was wondering what kind of exercises you can do that Would be benificial especially for your thighs and abs cuz I don't have alot of muscle.. Any tips to improve my balance and posture in the saddle? |
Try Pilates. :) |
Lots of two pointing, with and without stirrups. It really builds up leg muscles, and helps your body figure out how to balance in that position |
^Def! Also trot sets in two point! Also, crunches and just doing barn chores have helped me tons! |
lots of stretching...you will build up plenty of muscle riding...but you need to stretch those thighs as much as possible...I do yoga on my Wii fit :-P |
Pilates helps your core a lot. You could also do forward and side lunges. There's also this video that has lots of good exercises to do under saddle. How To Improve Your Classical Seat In Horse Riding (Sports & Outdoors: Horse Riding) |
everything everyone has mentioned is right on: working in a 2 point, stirrupless, etc. i'd also like to add that i've noticed that western riders who switch to english often have a tendancy to start off wanting to sit in a chair seat. so make sure that your leg is always correct so you are using and building up the correct muscles. There should be a vertical line from your hip down to your heel with your stirrup on the balls of your feet (outside branch should be slightly angled out so it's perpendicular to the horse). Your heel should be stretched down with more weight on the inside (big toe side) if your foot so your ankle also flexes slightly out. |
It is a little different than western huh...lol...works totally different muscles when you jump! I have found NO exercises that help the EXACT muscles you use when riding. Sorry!! =) You can certainly do all of the above mentioned exercises that will make you a stronger person and help with your endurance, but the best way to work the muscles that you need to use while riding is to ride! Good luck! I hope you have fun! |
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Based on 4 reviews
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Huntsman Innovation Wins Top Award at Paris JEC Show
...e. Huntsman received the award for the innovative use of its composites in fuel cell applications developed in cooperation with its US-based customer graftech International. Andre Genton, President of Huntsman's Advanced Materials division, commented: "At Huntsman, our commitment to innovation provides ...GrafTech International - PRODUCTS
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GrafTech Reports Second Quarter 2009 Results. Jul 30, 2009. 44.9 KB ... GrafTech To Announce Fourth Quarter and Full Year 2008 Results. Feb 3, 2009. 10.8 KB ...


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Double Gourd Lucite Table Lamp Large in Egg Blue -Open BoxPin It Houzz |
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The Official Skepchick & Mad Art Lab Gift Guide
Here's something so incredibly cool — a Gift Guide that has been sourced and curated and compiled and coded by the fabulous Courtney Caldwell. She's done a ton of work on this so you don't have to. And, best of all, this seasonal listy-ness will actually make shopping fun! …………. Are you a holiday procrastinator (like me)? Never fear, the Official Skepchick... »


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Research Article
Theoretical Restrictions on Farm-Retail Price Transmission Elasticities: A Note
Article first published online: 30 OCT 2013
DOI: 10.1002/agr.21362
© 2013 Wiley Periodicals, Inc.
Additional Information
How to Cite
Kinnucan, H. W. and Tadjion, O. (2014), Theoretical Restrictions on Farm-Retail Price Transmission Elasticities: A Note. Agribusiness, 30: 278–289. doi: 10.1002/agr.21362
Publication History
- Issue published online: 15 JUL 2014
- Article first published online: 30 OCT 2013
Funded by
- Alabama Agricultural Experiment Station
- Abstract
- Article
- References
- Cited By
Zero restrictions implied by Gardner's (1975) model are exploited to develop a simple test for perfect price transmission. Applying the test to the domestic marketing channels for U.S. beef and pork, we reject the hypothesis of competitive market clearing for pork, but not for beef. A retest using the four-firm concentration ratio in U.S. meat packing as a proxy for market power affirms results from the simple test. [JEL Classification: Q11, Q13].


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- Features: Male-to-Male, Data Transfer Capabilities
- Cable/Cord Length: 10.0 ' L
- Connection 1 Type: USB
- Connection 1 Gender: Male
- Connection 2 Type: USB
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See the Target return policy for complete information.


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Fierce tiger heads adorn the front shoulders of a textured sweater topped with trendy faux-leather trim at the neckline.