text:
|Publication number
|Publication type
|Application number
|Publication date
|Dec 19, 1995
|Filing date
|Feb 22, 1993
|Priority date
|Feb 22, 1993
|Fee status
|Publication number
|Thomas J. Reid, Barbara Alving
|Original Assignee
|The United States Of America As Represented By The Secretary Of The Army
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (16), Non-Patent Citations (14), Referenced by (6), Classifications (13), Legal Events (11)
|External Links: USPTO, USPTO Assignment, Espacenet
This invention relates to laboratory testing useful in monitoring of anticoagulant therapy. More specifically, this invention relates to assays for the anticoagulants that are known to inhibit the enzyme thrombin.
Presently, thrombin-specific inhibitors are in clinical trials as anticoagulant drugs for the treatment of arterial (after coronary artery angioplasty) and deep venous thrombosis. The specificity of these drugs is reflected in the dissociation constants ranging from 2 picomoles/L - 2 nanomoles/L. The thrombin specific inhibitors have demonstrated efficacy in animal models for both treatment and prevention of arterial and venous thrombosis. Recombinant hirudin, derived from the medicinal leech Hirudo medicinalis, is the thrombin-specific inhibitor most studied. Effective doses and their range for therapeutic plasma levels in humans have been determined. The low and high ends of the range are used for venous and arterial thrombosis, respectively.
Several U.S. patents have been issued on anti-thrombin agents for use as medicinals or bound to implantable materials. U.S. Pat. No. 4,944,943 to Eschenfelder, et al. teaches use of hirudin and t-PA for treatment of thrombosis. U.S. Pat. No. 4,952,562 to Klein, et al, discloses anti-thrombotic peptides and pseudopeptides. U.S. Pat. No. 5,019,393 to Ito, et al. teaches use of implantable materials having a thrombogenesis inhibitor immobilized thereon. U.S. Pat. No. 5,053,453 to Ku discloses hirudin or hirudin derivatives covalently linked to support materials to avoid formation of thrombi. U.S. Pat. No. 5,640,814 to Klein, et al. discloses an anti-thrombotic peptide for administration as a medicinal. U.S. Pat. No. 5,087,613 to Courtney, et al. discloses hirudin variants for use as inhibitors of thrombin activity. U.S. Pat. No. 5,095,092 discloses a process for isolation and purification of hirudin. None of the cited patents disclose a quantitative thrombin-time test as taught herein.
Several methods are presently used to assess whether the patient taking the new thrombin-specific inhibitors is therapeutically anticoagulated. It is possible to measure plasma thrombin-specific inhibitors levels by high performance liquid chromatography (HPLC). Though accurate, HPLC is generally available only in large referral hospitals and commercial labs and requires a high level of staff expertise. Even when available in large referral hospitals, the technique is time consuming. Hence, there is delay in obtaining results. This delay is unacceptable when a clinical practitioner is attempting to closely monitor and control the progress of the patient.
Another approach is to monitor the prolongation of the activated partial thromboplastin time (APTT). The APTT is a standard screening test of the coagulation system. This test is commonly used to follow the degree of anticoagulation in patients receiving the anticoagulant heparin. However, in the absence of specific thrombin inhibitors a variety of coagulopathies [e.g. isolated or multiple factor deficiency, abnormal fibrinogen, decreased fibrinogen concentration or antiphospholipid antibodies] tend to prolong the APTT results. Some patients with coagulopathies associated with the nephrotic syndrome or the lupus anticoagulant also develop thrombosis requiring treatment with anticoagulants. In these cases, the APTT is already prolonged and cannot be used to monitor anticoagulation with thrombin specific inhibitors.
Another clotting assay, the standard thrombin time, is routinely used in a qualitative fashion in clinical laboratories to determine the presence of heparin and abnormal or low levels of fibrinogen. The standard thrombin time is not effective and has not been used to quantitate the concentrations of thrombin-specific inhibitors in the blood because of the inherent sensitivity of the standard thrombin time, as presently developed, to factors other than these inhibitors (Walenga J. M. et al. Seminars in Thrombosis Hemostasis 17:103, 1991). In addition, plasmas obtained from patients with liver disease, dysfibrinogenemia, hypofibrinogenemia, or who have received fibrinolytic therapy are likely to produce a prolonged standard thrombin time.
Besides HPLC and the APTT, further methods including amidolytic assays for thrombin and immunologic assays for the hirudinthrombin complex have been suggested. However, the techniques are not easily applied to the clinical laboratory. Because of the potential therapeutic efficacy of the thrombin-specific inhibitors, a new and improved laboratory test for determining plasma levels that can be performed in clinical laboratories with presently available instrumentation is needed. Furthermore, such a new process capable of providing results that are easily interpreted by clinical practitioners would be of great utility.
A novel clinically useful test for monitoring patients on thrombin-specific inhibitor (TSI) therapy, a quantitative thrombin time (QTT) for the management of anticoagulation with thrombin-specific inhibitors, based on the specificity of the TSI for thrombin is disclosed and claimed herein.
The object of the present invention is to provide a method to measure thrombin time that will not be affected by presence of abnormal plasmas which cause a prolongation of standard tests (APTT and standard thrombin time, vide supra), but will be sensitive to the presence of thrombin-specific inhibitors. Another object of the invention is to provide a method from which unknown plasma concentrations of thrombin-specific inhibitors can be determined by generating a standard curve for the therapeutic range of the inhibitor using the QTT. A related object is to provide a method which can be readily performed on laboratory instruments commonly used for measuring the standard thrombin time.
FIG. 1 shows the effect of concentration of the purified fibrinogen on the quantitive thrombin time in abnormal plasmas with prolonged reptilase times and elevated FDP and in normal plasma.
FIG. 2 shows the effect of concentration of purified fibrinogen on the quantitative thrombin time in plasma with low fibrinogen levels.
FIG. 3 shows the effect of heparin concentration on the QTT.
FIG. 4 is the standard curve for recombinant hirudin using the quantitative thrombin time.
FIG. 5 shows the effect of recombinant hirudin concentration on the APTT.
This invention provides a novel method for the quantitative determination of plasma levels of thrombin-specific inhibitors by specifically measuring thrombin time in the presence of exogenous fibrinogen and thrombin. Normal plasmas and plasmas with a coagulopathy have the same baseline for thrombin-specific coagulation time when tested by the QTT. By eliminating the interfering effects of factors in the plasmas from patients with coagulopathy, the prolongation of the QTT will depend only on the presence of thrombin-specific anticoagulant present in the sample and will, therefore, provide a method for evaluation of the anticoagulant independent of other plasma abnormalities that could affect the thrombin time. To eliminate the effect of abnormalities such as antiphospholipid antibodies, and factor deficiencies, the phospholipid based APTT was abandoned in favor of the thrombin-based assay which is independent of phospholipid and coagulation factors other than fibrinogen. Only fibrin degradation products, heparin, and abnormal fibrinogens or a decreased fibrinogen potentially present in plasma could interfere with the assay. The effect of these abnormalities can be removed by diluting plasma 1:10 in buffer and adding excess fibrinogen.
While the test developed has been shown to be effective when using other thrombin-specific inhibitors, the inhibitor exemplified in the testing disclosed herein was recombinant hirudin. Quantification was tested by adding recombinant hirudin to normal plasma at different concentrations within the known therapeutic range. The QTT was then tested with these plasmas and a linear relationship was shown between the log (QTT) and concentration of recombinant hirudin. This allowed the determination of concentrations of recombinant hirudin in plasma. The specificity of the QTT was assessed by adding a known concentration of recombinant hirudin to patient plasma samples demonstrating a coagulopathy (prolonged APTT and/or standard thrombin time). The expected and observed values of recombinant hirudin levels were then compared and shown to be compatible with use of the QTT in the clinical monitoring of patients on recombinant hirudin. It was shown, by comparative evaluation, that the APTT does not provide the specificity provided by the inventive method.
The technique was performed using two different commercially available instruments and equivalent results were obtained.
Plasmas were those submitted to the coagulation laboratory for evaluation of abnormal screening coagulation studies and from healthy volunteers who gave informed consent under a protocol approved by the Human Use Committee of the Walter Reed Army Institute of Research. Human alpha-thrombin (specific activity 2,000-4,000 NIH units/mg protein), recombinant hirudin (specific activity 8,500 units/mg protein) and reptilase (ATROXIN) were purchased from Sigma Chemical Company (St Louis, Mo.). Human fibrinogen was supplied by KabiVitrum (Stockholm, Sweden). APTT reagent was purchased from Organon Teknika Corporation (Durham, N.C.). Sodium heparin (1000 U/mL) was purchased from Elkins-Sinn, Incorporated (Cherry Hill, N.J.).
Blood samples were collected into plastic tubes containing balanced citrate as the coagulant in a ratio of one part anticoagulant to nine parts blood. Each sample was centrifuged at 4� C. and 10,000�g for 20 minutes to obtain platelet-poor plasma which was then stored at -70� C. until ready for use.
The thrombin time was measuring the time to clot formation after adding 100 μL of a 5 U/mL solution of alpha thrombin to 100 μL of plasma and 100 μL VS buffer (vide infra). Either a dataclot-2 fibrometer (Helena Laboratories, Beaumont, Tex.) or an ST4 coagulation instrument (Diagnostica Stago, Parsippany, N.J.) was used.
Human alpha-thrombin was diluted to a concentration of 2.5-20 U/mL in veronal-saline-calcium (VSC) buffer (2.8 mM sodium diethylbarbiturate, 100 mM sodium chloride, 25 mM calcium chloride, pH 7.35) that also contained 1% human serum albumin. Fibrinogen concentrations (0.1-18 μM) were made in VS buffer (as VSC buffer except the buffer contained no calcium, and sodium chloride concentration was 144 mM). Plasma samples were diluted 1:10 in VS buffer. The plasma dilutions, alpha-thrombin and fibrinogen solutions were incubated separately at 37� C. for six minutes before using. Equal volumes of plasma samples and fibrinogen were mixed and incubated at 37� C. for one minute. 200 μL of the plasma-fibrinogen mixture were removed and placed in an assay well. The thrombin time was measured as the time to clot formation after adding 100 μL of the alpha-thrombin solution. Instruments used for the standard thrombin time were also used for the QTT.
The APTT assay was done using a CP-8 photooptic coagulation profiler (BIODATA Corporation, Willow Grove, Pa). 100 μL of plasma was incubated with 100 μL APTT reagent for six minutes. The APTT was measured as the time of clot formation after adding 100 μL 25 mM CaCl2.
The reptilase time was done using a dataclot-2 fibrometer. 100 μL of reconstituted reptilase was added to 200 μL of plasma and the clotting time was recorded.
Recombinant hirudin (lyophilized preparation) was diluted in normal pooled plasma to a concentration of 50 μg/mL. Further dilutions in normal plasma were made to obtain concentrations within the therapeutic range (0.1-3 μg/mL).
The log of the QTTs were calculated and a standard curve was generated after linear regression by plotting the log (QTT) vs concentration of recombinant hirudin (μg/mL). From the standard curve, an unknown plasma level of recombinant hirudin can be determined.
Heparin (initial concentration 1000 u/mL) was diluted in normal pooled plasma to a concentration of 10 U/mL. Further dilutions in plasma were made to obtain heparin concentrations of 0.1-8 U/mL.
The results of the QTT from twenty normal individuals (ten male, ten female) are shown in Table 1; all had a normal standard thrombin time and APTT.
TABLE 1______________________________________Summary statistics based onplasmas from twenty normal individuals, ten maleand ten female. All plasma samples had normal APTT andstandard thrombin time, using standard procedures (see text).______________________________________Mean Quantitative 13.9Thrombin Time, secondss.e.m.a, seconds 0.14s.d.b, seconds 0.66range (mean +/- 2xs.d.), 12.6-15.2secondsr.m.s.c difference, % 4.6______________________________________ a standard error of the mean; b standard deviation; c root mean square difference
The effect of concentration of purified fibrinogen on the quantitative thrombin time in plasma and abnormal plasmas was determined. Different fibrinogen concentrations were used in the quantitative thrombin time to determine at which concentration the effects of the abnormal plasmas were eliminated.
Plasmas were diluted 1:10 in buffer; 100 μL of the diluted plasma was added to 100 μL purified human fibrinogen at different concentrations (0.1-18 μM) and incubated for 30 seconds; 100 μL of 5 U/mL human alpha-thrombin was added to start the reaction; the clotting time was measured. The addition of excess fibrinogen in the assay eliminated the effects of abnormal fibrinogens (FIG. 2) and low fibrinogen (FIG. 2, insert). Plasmas with an abnormal fibrinogen (dysfibrinogenemia: standard thrombin time=45.2 and 18.5 sec [normal 11-14], reptilase time=22.6 and 15.5 sec [normal 8-11]), low fibrinogen (hypofibrinogenemia: fibrinogen=38 mg/dL [normal 200-400]) and plasma with increased fibrin degradation products (FDP=1024 μg/mL [normal<10 μg/mL]) and low fibrinogen (fibrinogen=112 mg/dL) were evaluated. The optimal fibrinogen concentration was determined to be 9-18 μM. This concentration was more than sufficient to overcome the effects of abnormal plasma samples.
Diluting the plasma samples 1:10 in buffer mitigated the effects of fibrin degradation products and therapeutic heparin levels (Table 2). The presence of antiphospholipid antibodies did not interfere with the assay.
Plasmas were diluted 1:10 in buffer; 100 μL of the diluted plasma was added to 100 μL 9 μM purified human fibrinogen and incubated for 30 seconds; 100 μL of 5 U/mL human alpha-thrombin was added to start the reaction; the clotting time was measured. Table 2. Coagulopathies that might otherwise interfere with standard clotting tests, do not interfere with the quantitative thrombin time.a
TABLE 2______________________________________ Quantitative Standard Thrombin ThrombinCoagulopathy Timeb, sec Timec, sec APTTd, sec______________________________________Abnormal fibrinogen 14.2 59.1* 29.4(dysfibrinogenemia)eAbnormal fibrinogen 14.8 45.2* 35.7*(dysfibrinogenemia)fAbnormal fibrinogen 14.4 18.5* 43.3*(dysfibrinogenemia)gAbnormal fibrinogen 13.6 16.0* 37.5*(dysfibrinogenemia)hIncreased fibrin 14.2 23.5* 43.0*degradationproductsiIncreased fibrin 14.2 22.4* 74.8*degradationproductsjMultiple Factor 13.8 13.8 57.6*DeficiencieskHeparin therapyl 14.4 >120* 64.2*Low fibrinogenm 14.2 25.8* >120*Antiphospholipid 14.2 12.3 50.1*AntibodyAntiphospholipid 14.4 13.5 44.4*AntibodyAntiphospholipid 13.2 12.2 57.9*AntibodyAntiphospholipid 13.2 13.7 65.3*AntibodyAntiphospholipid 13.6 12.8 90.0*AntibodyLow factor XIIn 14.2 14.0 45.2*______________________________________ *Abnormal value. b Normal: 12.6-15.2 seconds; c normal: 11-14 seconds; d normal: 22-34 seconds. e reptilase time = 29.4 sec (normal 8-11); f reptilase time = 22.6; g reptilase time = 15.5 h reptilase time = 15.2; i fibrin degradation products = 128 μg/mL (normal < 10), fibrinogen = 225 mg/dL (normal 200-400); j fibrin degradation products = 1024 μg/ml, fibrinogen = 112 mg/dL; k factor II = 37%, factor X = 16% (normal 50-150); l 0.5 U heparin/mL; m fibrinogen = 38 mg/dL; n factor XII < 10% (normal 50-150).
Normal plasma supplemented with heparin at various concentrations were tested in the QTT. Procedure: Plasma was supplemented with porcine heparin at different concentrations (0.1-10 U/mL); these plasmas were diluted 1:10 in buffer; 100 μL of the diluted plasma was added to 100 μL purified human fibrinogen 9 μM) and incubated for 30 seconds; 100 μL of 5 U/mL human alpha-thrombin was added to start the reaction; the clotting time was measured. The QTT became sensitive to heparin at 0.8 U/mL (FIG. 3). The therapeutic range for heparin is 0.3-0.5 U/mL.
The quantitative thrombin time was determined for different known concentrations of recombinant hirudin in plasma. A standard curve was developed by plotting the LOG (quantitative thrombin time) vs concentration of recombinant hirudin. Procedure: Plasma was supplemented with purified recombinant hirudin at different concentrations (0.1-1.75 μg/mL); these plasmas were diluted 1:10 in buffer; 100 μL of the diluted plasma was added to 100 μL purified human fibrinogen (9 μM) and incubated for 30 seconds; 100 μL of human alpha-thrombin (5 U/mL) was added to start the reaction; the clotting time was measured.
Several thrombin concentrations were tested in developing assays for recombinant hirudin. Low thrombin concentrations were too sensitive to high therapeutic plasma levels of recombinant hirudin whereas high thrombin concentrations were insensitive to low therapeutic levels. There was no thrombin concentration that was effective over the entire therapeutic range of recombinant hirudin. Therefore, a thrombin concentration (5 U/mL) sensitive at the low therapeutic range (0.1-1.75 μg/mL) of recombinant hirudin was selected (FIG. 4). The hirudin-free plasma data point was not included in the standard curve. To assay hirudin concentrations greater than 1.75 μg/mL, a 1:1 dilution in pooled plasma was required before the 1:10 dilution in the assay. The optimal conditions for the QTT were: 100 μL 1:10 plasma dilution, 100 μL fibrinogen (9 μM) and 100 μL alpha-thrombin (5 U/mL).
The validity of the QTT in determining plasma recombinant hirudin levels was assessed by adding known concentrations of recombinant hirudin to abnormal and normal pooled plasma. The QTT was measured and the recombinant hirudin concentration determined from the standard curve described above. Table 3 demonstrates that a number of coagulopathies that may prolong the standard thrombin time do not interfere with the measurement of recombinant hirudin levels as determined by the QTT.
Plasma was supplemented with purified recombinant hirudin at different concentrations (0.1-1.75 μg/mL); these plasmas were diluted 1:10 in buffer; 100 μL of the diluted plasma was added to 100 μL purified human fibrinogen (9 μM) and incubated for 30 seconds; 100 μL of 5 U/mL human alpha-thrombin was added to start the reaction; the clotting time was measured.
TABLE 3______________________________________ Measured concentration of QTT, recombinantCoagulopathy seconds hirudin, μg/mL % Errora______________________________________Antithrombin IIIb 22.6 1.11 11deficiencyLow Factor XIIc 23.8 1.21 3.1Abnormal 21.7 1.04 17.0fibrinogendysfibrinogenemiadAbnormal 27.8 1.49 19.5fibrinogendysfibrinogenemiaeAbnormal 23.8 1.21 3.2fibrinogendysfibrinogenemiafElevated fibrin 24.0 1.22 2.2degradationproductsgElevated fibrin 22.4 1.1 12.3degradationproductshElevated fibrin 25.5 1.34 7.0degradationproductsiLow fibrinogenj 24.4 1.25 0Heparin therapyk 26.1 1.38 10.4Multiple factor 24.6 1.27 1.6deficiencieslAntiphospholipid 24.4 1.25 0AntibodyAntiphospholipid 22.4 1.11 12.3AntibodyAntiphospholipid 22.5 1.1 11.7Antibody______________________________________ Recombinant hirudin was added to all plasma samples at a final concentration of 1.25 μg/mL. ##STR1## .sup. b Antithrombin III = 54% (normal 88-140); c factor XII < 10% (normal 50-150); d standard thrombin time = 18.5 sec (normal 11-14) , reptilase time 15.5 sec (normal 8-11); e standard thrombin time = 45.2 sec, reptilase time = 22.6 sec; f standard thrombin time = 18.1 sec, reptilase time = 13.1 sec; g fibrin degradation products = 1024 μg/mL (normal <10), fibrinogen = 183 mg/dL (normal 200-400); h fibrin degradation products = 256 μg/mL, fibrinogen = 360 mg/dL i fibrin degradation products = 1024 μg/mL, fibrinogen = 112 mg/dL; j fibrinogen = 38 mg/dL; k 0.5 U heparin/mL; l factor II = 37%, factor X = 16% (normal 50-150).
The effect of recombinant hirudin concentrations on the APTT was measured. Normal plasma was supplemented with recombinant hirudin at various concentrations and the APTT was measured.
Plasma was supplemented with purified recombinant hirudin at different concentrations (0.1-4 μg/mL); 100 μL of the APTT reagent was added to 100 μL of the plasma supplemented with recombinant hirudin and incubated for 6 minutes; 100 μL of 25 mM CaCl2 was added to start the reaction; the clotting time was measured. FIG. 4 demonstrates that the APTT is not useful at the low end of the therapeutic ranges for recombinant hirudin.
Plasma was supplemented with purified recombinant hirudin at different concentrations (0.1∝4 μg/mL); 100 μL of the APTT reagent was added to 100 μL of the plasma supplemented with recombinant hirudin and incubated for 6 minutes; 100 μL of 25 mM CaCl2 was added to start the reaction; the clotting time was measured.
Table 4 conclusively shows that the presence of a lupus anticoagulant, abnormal fibrinogens, heparin and fibrin degradation products interfere with the APTT assay for the recombinant hirudin.
TABLE 4______________________________________Coagulopathies that interfere with the APTT, interferewith the determination of plasma levels of recombinanthirudin as performed with the APTT.a Measured concentration of APTT, recombinantCoagulopathy seconds hirudin, μg/mL % Errorb______________________________________Low fibrinogenc >300 -- --Abnormal fibrinogen(dysfibrino- 105.8 1.93 54genemia)dAbnormal fibrinogen 132.0 3.66 169(dysfibrino-genemia)eIncreased fibrin 182.1 6.97 458degradationproductsfIncreased fibrin 108.8 2.13 70degradationproductsgHeparin therapyh >300 -- --Low factor XIIi 103.4 1.77 42Antiphospholipid 117.7 2.72 118AntibodyAntiphospholipid 115.6 2.58 106AntibodyAntiphospholipid 162.4 5.67 354Antibody______________________________________ Recombinant hirudin was added to all plasma samples at a final concentration of 1.25 μg/mL. a indicates value could not be determined. ##STR2## c fibrinogen = 38 mg/dL (normal 200-400); d standard thrombin time = 45.2 sec (normal 11-14), reptilase time = 22.6 sec (normal 8-11) ; e standard thrombin time = 18.5 sec, reptilase time = 15.5 sec., f fibrin degradation products = 1024 μg/mL (normal <10), fibrinogen = 112 mg/dL; g fibrin degradation products = 256 μg/mL, fibrinogen = 360 mg/dL h 0.5 U heparin/mL; i factor XII <10% (normal 50-150).
The mean errors for determining the concentration of recombinant hirudin in abnormal plasma using the QTT were less than 10%. In contrast, using the same plasmas, the measured concentration of recombinant hirudin using the standard APTT showed no agreement with the expected value (Table 5).
TABLE 5______________________________________Summary of Data for validation of the QTTin different clinical conditions. QTT APTT______________________________________r.m.s.* difference 0.12 2.82recombinant hirudinconcentration, μg/mLr.m.s. error, % 9.8 225______________________________________ *root mean square
The thrombin-specific inhibitor hirudin and its analogues have demonstrated efficacy in the prevention of thrombosis in preclinical studies. The anticoagulant effects have been monitored by the APTT because the standard thrombin time is too sensitive to the agents to be of clinical utility. The APTT has two major problems that limit its usefulness in monitoring recombinant hirudin: 1) patients with a prolonged baseline APTT cannot be followed (this also holds true for patients receiving heparin therapy); and 2) there is a nonlinear dose-response at low therapeutic levels of recombinant hirudin with regard to the APTT (FIG. 5). The clinical consequence of using the APTT to monitor therapy in the low therapeutic range is excessive anticoagulation for the specific clinical condition. We have developed a quantitative thrombin time that eliminates the inherent difficulties associated with the APTT and the standard thrombin time. The problems which the invention is designed to solve are the sensitivity of standard tests to therapeutic heparin levels, low levels of fibrinogen, abnormal fibrinogens and fibrin degradation products, lupus anticoagulant (antiphospholipid antibodies), and low plasma factor levels.
Some of the novel aspects of the invention include 1) the addition of fibrinogen in excess to compensate for abnormal or low fibrinogen and 2) the plasma samples are diluted 1:10 in buffer which effectively prevents interference of fibrin degradation products, abnormal fibrinogen and heparin. There are several distinct advantages of the invention. The method is simple and can be done in any lab that performs thrombin times routinely (most hospitals). The procedure is specifically adapted for use on instruments presently available to clinical laboratories. The newer automated equipment and methods used in determination of thrombin time may be used for practice of the invention. The method is also quick and requires no additional expertise above that already available in the clinical laboratory. A small amount of patient material is needed (25-50 μL of plasma for 4 tests); where standard tests require 100 μL for a single test. The method allows quantitation of plasma levels of recombinant hirudin and other thrombin-specific inhibitors for use with other thrombin-specific inhibitors (e.g. synthetic or semisynthetic analogues of recombinant hirudin). The mean error is less than 10 %. The standard curve is linear in the therapeutic range, where standard tests are either not linear in the low therapeutic range (APTT) or are too sensitive to recombinant hirudin (standard thrombin time). The method is more sensitive to thrombin-specific inhibitors in comparison to standard tests, as demonstrated by the response of the clotting times to recombinant hirudin concentrations. Finally, the test is inexpensive and fast in comparison to HPLC.
While normal human thrombin was used in the procedure as exemplified, mutant or normal thrombin from other sources may be used in the QTT so long as the alternate thrombin provides a reliable dosage curve when tested against the specific thrombin-specific inhibitor for which concentration is being determined. Similarly, the fibrinogen can be from any source so long as the fibrinogen is functional in normal clot formation. Although a veronal based buffer was used in the development of the QTT, any buffer that provides a reliable dosage curve when tested against a TSI can be used.
The materials for the QTT may be sold as kits. The following components could be provided in the kit:
1) One or more vials of lyophilized plasma containing a known amount of thrombin-specific inhibitor (TSI) which may contain protamine sulfate should be provided. The contents of the vial(s) may be reconstituted with water so that the final concentration of the TSI is such that serial dilutions allow for TSI within the therapeutic range. The addition of the protamine sulfate may be used to effectively remove heparin from the reaction. If protamine sulfate is used, a preferred concentration in the final diluted TSI-containing solution would be about 120 μg/mL. However, in some instances the TSI may be available to laboratories as a stock solution(s).
2) One or more vials of lyophilized alpha-thrombin which may be formulated with a stabilizing agent such as albumin and may contain buffer should be provided. The thrombin could be reconstituted with water to provide the desired concentration.
3) One or more vials of lyophilized fibrinogen to be reconstituted to a final concentration of about 4.75 μM (160 mg/dL). The plasma may contain a neutralizing agent such as protamine sulfate. The kit can, if desired, hold a separate vial of protamine sulfate or another heparin neutralizing agent to be used with TSI and patient samples when a patient is on heparin therapy.
4) Vials of buffer for diluting samples may be included.
Using the kit items, the following procedure may be followed. In a preferred embodiment the standard curve may be developed to identify effective levels of TSI by the following method:
a. Preparing serial dilutions of a thrombin-specific inhibitor in normal plasma in known concentrations to provide samples (though a kit containing varying amounts of TSI within the appropriate range will avoid this procedure).
b. Dilute the samples in buffer to provide 1:10 dilutions.
c. Mixing the dilutions with a 9-18 μM solution of purified human fibrinogen to provide samples.
d. Adding a 5 U/mL solution of purified human alpha-thrombin to the samples of step c to provide solutions.
e. Measuring the clotting times of the solutions in step d.
f. Plotting a standard curve from the results obtained from step e.
The level of TSI concentration in patient plasma is tested in the following manner to determine the TSI level in the patient:
1. Diluting in buffer a plasma sample from patients receiving a thrombin-specific inhibitor to provide a 1:10 plasma to buffer concentration.
2. Mixing the patient samples with a solution of fibrinogen.
3. Adding a thrombin solution to the patient samples prepared in step 2.
4. Measuring the clotting times of the solutions of step 3.
5. Determining the concentration of the inhibitor in the patient samples using the standard curve previously developed.
The invention may also be practiced using whole blood rather than patient plasma.
Vials containing varying amounts of the TSI required for generating the standard curve would be made by diluting the TSI to provide varying dilutions for use in determining the curve. The normal plasma is reconstituted by dilution with buffered water (or the buffer may be added into the diluent) to provide varying concentrations of TSI. A second set of containers containing only plasma is prepared for purposes of comparison. A third container is prepared containing 1 mL of patient plasma with unknown concentration of TSI. To each sample is added 475 μL of reconstituted fibrinogen solution and 50 μL reconstituted thrombin. The clotting time is then measured. Steps a to f may be used as a research tool to evaluate activity of TSI's. However, as more is known about the effect on QTT of various concentrations of a particular TSI, the running of a standard to obtain the curve may be unnecessary. In such instances, the QTT alone without the comparison testing will be sufficient to allow practitioners to evaluate concentration of TSI in the blood.
As an alternative, the kit may contain only fibrinogen and thrombin, whilst one or more stock solutions containing appropriate concentrations of TSI in plasma is maintained for use in the test. (It would also be possible to have one stock concentration at highest level to be tested, which would be diluted to provide lower concentrations for use to provide the appropriate curve.) The extent to which stock solutions are used or multiple vials of TSI containing differing amounts of TSI are provided will depend on the number of tests done in any particular health care center and the quality of staff available to provide testing services. All of the components, plasma, thrombin, and fibrinogen, should be used fresh or be frozen to avoid loss of potency. All of the agents used, including plasma with TSI, normal plasma, thrombin, and fibrinogen may be provided as lyophilized materials in vials for reconstitution. Once reconstituted, the active components are easily inactivated.
Among the uses for which the invention is suitable include quantitative clinical monitoring of patients anticoagulated with the thrombin-specific agents and laboratory research in animal models of thrombosis.
|Cited Patent
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|Burroughs Wellcome Co
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|Haver, et al., "Characterization of Thrombin-Induced Contraction . . . ", ood Vessels 21:53-63 (1984).
|Haver, et al., Characterization of Thrombin Induced Contraction . . . , Blood Vessels 21:53 63 (1984).
|Lefkovitz, et al., "Direct Thrombin Inhibitors . . . ", Circulation 90; No. 3, 1522-1536 (1994).
|Lefkovitz, et al., Direct Thrombin Inhibitors . . . , Circulation 90; No. 3, 1522 1536 (1994).
|Markwardt, "Hiradin and Derivatives as Anticoagulant Agents", Thombosis and Haemostasis, Stuttgart, 1993.
|Markwardt, Hiradin and Derivatives as Anticoagulant Agents , Thombosis and Haemostasis, Stuttgart, 1993.
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|Trapodi, et al., "Effects of Hirudin on Activated Thromboplastin . . . ", Thrombosis and Haemostasis, Stuttgartt, 1993.
|Trapodi, et al., Effects of Hirudin on Activated Thromboplastin . . . , Thrombosis and Haemostasis, Stuttgartt, 1993.
|Zoldhelyi, et al., "Recombinant Hiradinin Patients . . . ", Circulation 88:#5, 2015-2022 (1993).
|Zoldhelyi, et al., Recombinant Hiradinin Patients . . . , Circulation 88: 5, 2015 2022 (1993).
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|Method and kit for testing a multi-channel blood assay cartridge
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|Stb, Ltd.
|Processes For Mixing Fibrinogen and Thrombin Under Conditions That Minimize Fibrin Formation While Preserving Fibrin-forming Ability, Compositions Produced by These Processes, and the Use Thereof
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Kings Inn Hotel
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Historical Traveler Reviews Kings Inn Hotel San Diego
Great Value
We stayed here for 3 nights while visiting Seaworld & the Zoo. The rooms are simple but clean and comfortable. The 2 restaurants serve top class food and the staff are very friendly and attentive.
Our kids loved the large pool and spa area - a definite bonus.
Extremely close to all major attractions - 5/10 mins to Seaworld/Zoo & Old Town.
We would definitely stay here again - great value for the money.
Great Place to Stay on a Budget!
We stayed at the Kings Inn for three nights and would definately consider staying there again in the future. The location was great for the things we wanted to do in San Diego. The staff was very friendly at the hotel and the restaurant. The waffles were very good! I was leary to choose this place because of other reviews but I won't make that mistake again!
Very good choice
I want to tell you just a little great anecdote: i forgot my wallet with money, credit card and passport inside when i left hotel. I then went to L.A. and there i realized that. I called the hotel and they said that they found my wallet. I came back to san diego and they gave me the wallet with everything inside. In Italy this thing wouldn't never happen! Wonderful pool and jacuzzi. Nice and clean rooms. Very quiet and quiet cheap.
Good Budget Motel
Its a reasonable quality and priced hotel. No complaints. The jacuzzi and pool make it worthwhile.
Excellent Budget Vacation Motel
Great motel for vacationers on a budget. We also liked the food at the waffle place. Easy and quick to get to most area attractions.
Nice rooms, location only for those with rental cars
The rooms were attractive and nice. I did not notice the noisy plumbing, but we were rather exhausted. The pool was clean and inviting. The waffles in the breakfast restaurant are EXCEPTIONAL and the service was bright and cheerful. The mexican food in the evening restaurant was good and the staff there especially nice.
This is located near Hotel Circle. If you are attending events downtown, you'll need a rental car. Some conferences have buses that go to hotels in the area, so that might be an option. The nearest trolley station (Old Town) is 2 miles (I'm positive about this distance even though locals will tell you it is about 1/2 mile) down a highway with no sidewalk over 3/4 of the trip and no shoulder. (This is a long story how I know this, but trust me. It is not something you want to walk, especially at night.)
If you are on a budget and without a rental car, try the Padre Trail Inn. If you have a car and can afford an extra $10-15/night, stay here.
It would be worth the extra money just for the nice pool, laundry facilities, great staff, and the great waffles.
It was OK
The rooms were just average, the staff was nice. They let us check in early when we got in sooner than expected. Even though the rooms didn't have a coffee maker they do have coffee in the loby area. We liked the location on hotel circle.
Nice Rooms
The room was nice, had a armour with the T.V in it and a table and a couple of chairs. Staff was very nice. The Waffle resturant was good. But the plumbing is a tad noisy. Great pool area.
King's Inn
Adequate motel style hotel on the outskirts of San Diego. Nice pool area. Hotel has 2 restaurant, althoguh we didn't use them. Bedrooms were ok, plumbing noisy..woken up by someone showering! The hotel is about 10 minutes drive from San Diego centre. A good place to stay if you are on a budget and want to visit San Diego.
Average rating
We booked through Expedia.com which gave it a 3 star rating, but we think it deserved only 2 stars. The rooms were small, no coffee maker, no hair dryer, no iron/ironing board. The room was just average, beds were comfortable though. THe plumbing was noisy, especially when the neighbors were showering.
TripAdvisor Reviews Kings Inn Hotel San Diego
Travel Blogs from San Diego
Day Seven.....Relaxation and Baseball
... in the water while at the ocean. The Pacific Ocean in San Diego is cold. There is no other way to describe it. The sand at the Del is micro fine. I don't know what mineral makes it glisten as if there are diamonds in the sand, but it twinkles in the sunlight at you. The sand is soft and cool to your feet. As you approach the shoreline where the waves are lapping, I love the first few steps in the water logged ...
... the individually designed houses and learning how new everything is in Coronado and San Diego. Houses, buildings nearly all built within last 100-135 years. Leaving Coronado over the bridge gives spectacular views of the whole bay. Wonderful sight.
Lunch was in a pizzeria which came with a delicious pear and ...
Addictive Tendencies of Loneliness
... to hear or read the experience.
There might be someone who needs this:
https://www.lds.org/general-conference/ 2014/10/are-we-not-all-beggars?lang=eng #watch=video
Now, I don't know why the Lord places certain people in our paths, but I do know it is all for our learning. The sooner we can learn from it the faster the recovery. That is the only way I am able to be even slightly patient with all of this. The lessons will come and ...
Day 2 of San Diego
... were very well done, giving them space and plenty of climbing where needed for them, with little homes within rocks etc. All the staff seemed very knowledgable about the types of animals they looked after.
We also decided to ride the Skyfari which takes you from the front of the park to the back near the polar bears - so we rod it up and back!! That was fun to be able to see the park from a birds eye view - although you don't ...
Hey there
... in and cleaned away. I finished with enough time for a quick beer at bootlegger, the bar underneath the hostel. Then back upstairs to Skype with the family back home. Sunday was a bit of a waste thanks to shawn. He was suppose to come get me and surf trestles round lunchtime. He turned up at 315, only 3 hrs late. We drove up to Oceanside for his Mother's Day lunch with his family which was nice, saw all his family again, played some horse shoe, drank and ...

subset: commoncrawl


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-Linebacker Jason Gibson will miss three months after breaking his right foot Monday, Cal head coach Jeff Tedford said.
Gibson also suffered a Lisfranc injury. Doctors must wait for the swelling to subside before determining if he will need surgery.
A redshirt freshman from Gardena (Calif.) Serra, Gibson demonstrated tremendous athleticism in spring practice, but was not expected to compete for a starting role inside with the Bears' tremendous depth.
-Freshman cornerback Jeffrey Coprich will miss two weeks with a fractured jaw.
-Cal's depth at defensive line was wiped out, practicing with just five players for the first hour of practice. Sophomore defensive end Mustafa Jalil and sophomore nose guard Viliami Moala were delayed because of academics, while redshirt junior defensive end Deandre Coleman did not practice at all.
Redshirt junior defensive lineman Keni Kaufusi did not practice for the second consecutive day, but Tedford did not disclose the reason.
-Other players with class conflicts included quarterback Zach Maynard, who missed the first part of practice, and running back Isi Sofele, who did not practice at all.
-Freshman wide receiver Bryce Treggs saw one first-team rep, lining up opposite Keenan Allen. However, Treggs dropped a screen pass and was subbed out immediately for redshirt freshman Maurice Harris.
Treggs did respond with several nice catches later on, including a leaping sideline grab from freshman Zach Kline.
-The first-team offensive line again featured Tyler Rigsbee at left tackle, Jordan Rigsbee at left guard, Brian Schwenke at center, Geoffrey Gibson at right guard, and Matt Summers-Gavin at right tackle.
Gibson was replaced by backup center Chris Adcock for one drive.
-Robert Mullins and David Wilkerson worked as the starting inside linebackers, flanked by Dan Camporeale and Brennan Scarlett on the outside.
-Kameron Jackson worked as the fifth defensive back in nickel personnel.
-The Bears will practice in full pads for the first time this fall Wednesday.
Dan Greenspan is the publisher of Cal Sports Digest and covers the Pac-12 for Fox Sports/Scout.com. Follow him on Twitter @DanGreenspan.


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CBNNews.com - JERUSALEM, Israel - The Shin Bet (General Security Agency) suspects former Arab member of Knesset (MK) Azmi Bishara of actively recruiting for the Lebanese-based Hezbollah terror group.
"According to intelligence gathered by the Shin Bet, Bishara remains in contact with elements that are hostile to the State of Israel," Deputy Attorney General Raz Nizi stated in a letter to the Legal Center for Arab Minority Rights in Israel (ADALAH ).
In the letter, Nizri said the Shin Bet advised members of Balad, the Arab Israeli party that Bishara chaired, not to be in contact with him.
In April 2007, Israel police launched an investigation against Bishara for alleged contact with Hezbollah operatives during the Second Lebanon War in the summer of 2006.
Bishara resigned from the Knesset while in Cairo last year because of allegations that he passed strategic information to Hezbollah during the war.
Bishara has not returned to Israel since, though police detained his wife for questioning when she arrived in the country several days ago.
Source: YNet news
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The Terrorists Funded by the WestBassam Tawil
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La Margarita Restaurant & Oyster Bar
- Today
- 11:00 am - 9:00 pm
- Tomorrow
- 11:00 am - 9:00 pm
- Mon - Thu
- 11:00 am - 9:00 pm
- Fri
- 11:00 am - 11:00 pm
- Sat - Sun
- 8:00 am - 11:00 pm
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REVIEWS(30)Top ▲
Sour beer tasteless food01/20/2015
Don't eat here the food sucks the owner should taste the food he should be ashamed to call this tasteless food Latin Mexican after you eat this slop then you get your overpriced check don't worry about the tip its included they know you won't tip when you are dissatisfied with the food yuck!!!!!!
We enjoyed the food. The carnitas were great. Pricey for Mexican, but it IS in a tourist district in downtown.
Order the campechana(a mexican...06/27/2012
Order the campechana(a mexican seafood cocktail) and a dosarita (dos equis margarita) They have the best of both. This is definitely a people watcher atmosphere and a great place to get fajitas!
Employees are running around like chickens with their heads cut off. Most of them barely speak english! Last time I went my card was over charged an amount of $56.00! Employees and cashiers would not admit to their mistake and did not apologize! The manager didnt even want to credit the money back to my account! It took two weeks and several phone calls before I got my money back. That was just pathetic. The moral of this is employees need to learn english and have no business handling money transactions!
zero stars better describe11/25/2011
Worse high priced food I have ever had. Small servings cold food tasted horrible. I have eaten there many times but it is going down hill fast. $10 a plate you would expect more quality. Fire the cook or close the door!
What a disappointment07/25/2011
What a disappointment. When we were seated the hostess handed us a smaller menu and said that all appetizers and beer etc. were half price for happy hour. We ordered a beer for 4.25 and Quesadillas for about 6.95. We were under the impression that this meant beer would be 2.00 or so and the quesadillas about 3.50. Nope. Supposedly these were the half price prices. Really? Our quesadillas were hardly worth 6.95 meaning they were almost 12.00 for full price. We got 6 chunks of tortilla with some cheese inside. They tried to tell me that this would cost 12.00 regularly. Really? So this meant the Corona at full price would be 8.50. This was very deceptive and should have been explained better when we were seated. My girls got shirley temples for 3.25 each and our chili rellenos were on the soggy side. Surely there is better Mexican food in San Antonio because this is not it.
Great Atmosphere10/11/2010
I like going to this restaurant for the atmosphere. You can sit outside and be a "people watcher", while having good Mexican food. The margaritas are pretty good too! If you go duringi Fiesta, get there early or you will have a long wait!
Very good09/15/2010
I came here for lunch with a large group. The waitstaff was very friendly and the food came out very fast after we ordered. The chips and salsa was excellent. I had the enchilada verde lunch special. The plate was good but average. The price was very reasonable. I will go back.
Great Food and Amazing Service!05/31/2008
For starters, the service was awesome. My girlfriend and I were clueless on what we should order. They were very helpful with suggestions. On top of the great service, the food was delicious. My girlfriend was cold and they brought a table cloth out to use as a blanket to keep her warm. Great atmosphere, I thought i was in Mexico! Will definitely go back any time I am in the area.
Fun, lively, and awesome chips & salsa02/24/2008
A friend and I tried to get into Mi Tierra at 7:00 on a Saturday night but opted for the slightly less busy La Margarita instead. Our table wait--which we were told would be 35 minutes--was about an hour, but the Pomegranite margaritas and people watching made up for it. The chips were hot and the salsa was authentic (soooo good). Our fajitas were yummy, although they only come with pico, guac, rice & charro beans, and some sour cream would have been a welcome addition. The only negative was the slow service, and I can't exactly fault the waitstaff. This is a tourist location and, in our section alone, we saw a softball team, business people in town for a conference, and a ton of vacationers with cameras and fanny packs. If you're in a rush or highly impatient, go somewhere else (out of El Mercado), if you like to people watch and kick back in a festive environment, try La Margarita.
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At the Great Northwoods Sales Warm-Up (GNW) at the University of Wisconsin, students deliver two 20-minute sales presentations to professional buyers. Upon completion of each call, students receive their evaluations sheets, an electronic copy of their presentation and a one-on-one debriefing session with one of their judges.
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text: blackberrystone Play via YouTube OPEN IN APP NSFW Safe Is this playlist safe for work? Home for Christmas '11 December 20, 2011 | 13 tracks | 268 11 | Stats Summer CampOkkervil RiverOtis Redding christmasjoni mitchellholidayssnow Liked Collect Share Edit celebrate Liked! Share this mix with friends: Home for Christmas '11 Wrong video Collect Share Liked! Share this mix with friends:


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Catalogue entry
Inscr. 'S. E. Waller 1882' b.l.
Canvas, 57×79 1/2 (145×202).
Tate Gift 1894.
Coll: Presumably purchased by Henry Tate at the Royal Academy 1882.
Exh: R.A., 1882 (551).
Lit: Walter Armstrong, 'The Henry Tate Collection IV' in Art Journal, 1893, p.197, repr.
Repr: Sir Edward J. Poynter, The National Gallery, III, 1900, p.343.
Exhibited at the R.A. with the following note: 'On the first rumour of impending difficulties between the Scotch and English, the Moss-troopers would sweep swiftly over the country taking every head of cattle within reach - frequently plundering both sides with equal impartiality, and returning to the security of their castles to be welcomed home by their wives and sweethearts, who were nearly as enthusiastic in the matter as themselves.- (Border History).'
Published in:
Mary Chamot, Dennis Farr and Martin Butlin, The Modern British Paintings, Drawings and Sculpture, London 1964, II


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Requests for Reprints: William D. Lawrence, MD, Century Medical Associates, 1616 Kensington Avenue, Buffalo, NY 14215.
Current Author Addresses: Dr. Zielinski: Department of Medicine, Buffalo Veterans Affairs Medical Center, 3495 Bailey Avenue, Buffalo, NY 14215.
Dr. Lawrence: Century Medical Associates, 1616 Kensington Avenue, Buffalo, NY 14215.
The hypereosinophilic syndrome is defined by unexplained eosinophilia (eosinophil count > 1.5 x 109/L) lasting more than 6 months (1). Characteristic clinical features include congestive heart failure with endocardial fibrosis (2), venous and arterial thromboembolism, neuropsychiatric dysfunction (3), anemia, and thrombocytopenia. These disease manifestations have been attributed to tissue infiltration by eosinophils and the direct toxic effects of the products of eosinophilic granules (1, 2, 4).
Therapy has been directed at lowering the absolute eosinophil count to below 1 x 109/L with prednisone and the cytotoxic agent, hydroxyurea (1, 5). This therapeutic approach appears to have dramatically improved median survival
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According to Washington State Patrol Sergeant Scott Davis, at around 1:00 Thursday afternoon, 24-year-old Ryan Tritten was driving southbound on Highway 195 and made an illegal U-turn. His car was hit by a southbound truck that was driving behind it. Tritten and his passenger, 23-year-old James Sullins were air-lifted to Sacred Heart Medical Center in Spokane with possible head injuries.
"There's no criminal charges at this point, nobody's impaired," said Davis. "We just have to nail down why the U-turn was made and then we'll decide what enforcement is necessary."
The driver of the second vehicle, 82-year-old Norman Druffel, wasn't injured. Sergeant Davis said there's a possibility that Tritten was trying to turn around to help a disabled vehicle.
Tritten is currently listed in satisfactory condition, and Sullins is in serious condition. Washington State Patrol is continuing its investigation.


text:
-Label Missing- Unisex black, bended cotton, fleece, having a round neckline and flat hemmed edge, with 2.0in side slits. A small snowman applique details the bodice, with white satin stitched snowflakes, a red satin hat and scarf. This garment measures 25in from shoulder to hem. (made for a woman but would work fine on a guy. If sleeves are too short, just scrunch them up)
Fits 36Bust (Womens Medium) or 36Chest (Mens Small), 30Sleeve
Very Good Condition
1.06 lbs
Price for both: $33.75
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Fact checkJuly 15,2014
Fact check
If defending the morality of our politics, which has seriously been harmed by Rice, Cheney and cohorts, is radical anti-Americanism as Milan Miller sees it, what would be patriotic? Lying a nation into war in which not only thousands of American soldiers are killed and harmed but also a foreign nation gets totally destroyed? (Not to mention the real reason for the war, the big bucks the military-industrial complex, so very connected to that gang, made in that horrible war or should I say: is making?)
Talking about brainwashed: To see the truth and teach the truth has nothing to do with brainwashing. Believe it or not, the Earth is not flat.
Elisabeth Hebert
NorthfieldMORE IN LettersElection-time letters to the papers often sound as though they were written by a machine, but... Full StoryOpposition to the construction of the Dakota Access Pipeline has brought thousands of... Full StoryI am a Berlin School Board member and one of the three members from Berlin on the Act 46, 706B... Full Story
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Style: 609220
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On Tap Today: All things Chesney, and a bit of Bilbo
It's a rare sound indeed: a country music concert at Miller Park. The stadium hasn't played host to any full-scale concert since 2010, but now country stages a comeback when Kenny Chesney's "No Shoes Nation" tour stops there Saturday.
If your memories grow dim about how the park's concert experience works, we're here to help. We give you a look at how Chesney changed country music. If you think you already know all this, we offer a quiz you can take on Chesney, one of country's music's biggest stars. And your score is...? Never mind. We also provide a practical guide to enjoying the Chesney show. »Read Full Blog Post
Foxygen performance a frenetic delight
Indie-rock aficionados have been touting Foxygen this year as a band on the brink of bigger things. There's also been speculation that the band is on the brink of collapse.
Based on the band's wild show Sunday at Turner Hall Ballroom, one could perceive how both perceptions might be true. »Read Full Article
Radio's Brian Kramp resurfaces on WITI-TV's new 'Studio A'
Brian Kramp, the former WLUM-FM morning show host dismissed in May, resurfaces as a "man-about-town" reporter for "Studio A," a new afternoon talk show on WITI-TV (Channel 6).
The hourlong show, hosted by news anchor Ted Perry and "Real Milwaukee" co-host and reporter Katrina Cravy, premieres Sept. 9 at 4 p.m. »Read Full Blog Post(7)


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Trade License.cz / Set up as Self-employed / Freelancer / Sole Proprietor
Registering a Czech Trade License, VAT, Visa / Residence Permit, Tax, Social, Health - all in 1 go!Getting a trade license in Prague / Czech Republic is quick and easy when using our services, because we will guide you through the entire process from initial trade license registration to the first invoice with consultations and email support.
We do not drag you to all kinds of government offices, and we can even register remotely without you being present in Czech Republic!
We not only register your Trade License but ALSO at the Tax Office, Social Office and sort out Health Insurance.
In case you do not have a (Business) Visa or Residence Permit yet, we have several options, ranging from documents-only to full-service.
Since as Alexio.cz Expat Accountant our core business is Accounting, VAT and Taxes, we are very capable of solving complex issues with (foreign) income, invoicing clients abroad / in the EU, Tax optimization, VAT issues and alike.
We have several Trade License Registration Packages, to suit the different needs of different clients.
Included in the price of the Trade License Registration package is 2 hours of consultation:
Consultation before we register your the trade license:
- We'll look at your your visa / residence permit possibilities (obligations),
- whether the trade license is the best option for you (or maybe an sro instead?),
- social / health insurance questions,
- accounting / tax / VAT questions etc. etc.
- we'll teach you how to create an invoice,
- how to invoice clients Europe / abroad,
- how to deal with payments from abroad, cash payments,
- how to pay the monthly social- and health charges,
- how to comply to the czech visa (extension) requirements.
No time for reading all details? Go right to the Orderform
Maybe first a consultatioin? In our office / by email / by phone in Engels, Nederlands nebo v Cestine?
On the next pages we explain what the Trade License requirements are, and give you an introduction of the registration process, including important facts about Czech Visas & Residence Permits, Social Charges, Health Insurance, Income Tax and VAT.
Office closed from Friday 1/7/2016 till Sunday 10/7/2016Our office will be closed for a small summer break between July 1st and July 10th
Emails we will read at least once per day - phones will not be answered during this period.
Thank you for understanding & See you after the break!


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My father, 85, called me the other day, choked up. "Dan, it's Dad. Philly died."
And then he sobbed.
He and Philly Grief were friends for 76 years. There were others: Herbie, Eddie Green, Morenis, Smiley, Wassy — a lifelong bunch of Brighton Beach pals who grew up in the 1930s. My dad, Al, and his friends played "touch" on the dirty lots near the boardwalk, stickball in "the gutter," punchball in the dark "backyards" set between the six-story buildings. As teenagers they went overseas: my father to the Pacific, Philly to Europe. When they came home it was time to reap the benefits of victory. Some went to college to become teachers and accountants. Others married early, worked in the post office, drove a cab, bought a soda route or like my father, sold pots and pans door-to-door in the "colored neighborhoods." These were men who came from immigrant Russian Jewish parents whose mission was to make life "better" for their children, a characteristic they themselves passed down. "The Dream" could work.
Eight of the friends started a ritual that continued for 50 years. Every six weeks at alternating homes or apartments, the "Friends Club" would get together on a Saturday night. The host couple would stock the place with bagels, lox and sturgeon. The men drank J & B; the wives occupied their own corner, blessed for their time away from their kids and routine. I was the first boy born into this circle — and the center of everyone's attention — and well into my teenage years I made every excuse to be home on the given Saturday when Al and Esther hosted "The Friends."
During the summers the group would vacation at bungalow colonies in Monticello and South Fallsburg. The men would come for weekends, then return to the city on Sunday nights for the workweek. When their own boomer children left home, "The Friends," still close, made the move — Florida — first as "snowbirds," all fun and games, the summer camp they never had. Later, their savings accounts and pensions bought them permanency: condos in West Palm, Boynton, Fort Lauderdale. And here too, they would meet, the men playing poker, the women mah jongg. There'd be early-bird specials, group movies at the mall, visits from the grandchildren.
Philly and my father liked to go fishing — a challenge for the latter, but second nature for Philly, who was always different. He attended an agricultural college in Utah, dreaming of becoming a farmer. He loved the land, animals, nature.
Family obligations derailed his hopes. Philly returned to New York where he taught high school science for 30 years.
In their mid-60s to 70s, "The Friends" took trips and cruises to the Mediterranean, the Caribbean and Alaska. Sometimes, the men went to Vegas. And then, it began to change. You'd visit after a year of being a self-absorbed son and they'd look different. Eddie was the first to go. After a jog, Leona found him on the kitchen floor, hoping he was joking. Herbie and Smiley were lost to Alzheimer's. Joe Morenis, cancer. Then, my dad began to erode. A long list of serious medical problems led to a walker, then a wheelchair.
The reversal of roles snuck up on my brother and me; another trip, another emergency room, rehab center, social worker, a helpless mother, another Florida doctor who didn't seem to care. Seen one old timer, seen 'em all — medicine as industry as opposed to age-old "house calls," when the revered doc, carrying his black bag arrived; calm, patient and always right.
My father has no balance left, no strength in his legs or arms. He takes a hard fall at times. His better days are a bit less cruel. But he found relief in the visits from Philly. They would talk about doctors, about their bachelor days, about long deceased brothers — 76 years of talk. And then, as if from nowhere, "Dan," he cried like a baby, "Philly died."
Even I felt helpless, paralyzed — again. I could only listen, fighting back my own tears. Then, after we hung up I came to some temporary comfort in the form of a fantasy. Their next "Friends Club" meeting would take place, not far from now, on a Saturday night. The wives, young and slim on one side of the living room, the men, without any pretentions, relishing the stories of their Sacred Club, the hum of laughter. "Old Friends." The venue doesn't matter.
Dan Klores is a filmmaker and playwright. He is currently working on a new film, "Breslin: The Last Great One."
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Parrish FL Real Estate & Homes for Sale
0 Properties Found
The median home value in Parrish, FL is $256,000.
This is
higher than
the county median home value of $169,700.
The national median home value is $177,414.
The average price of homes sold in Parrish, FL is $256,000.
Approximately 76.7% of Parrish homes are owned,
compared to 6.56% rented, while
16.74% are vacant.
Parrish real estate listings include condos, townhomes, and single family homes for sale.
Commercial properties are also available.
If you see a property you're interested in, contact a Parrish real estate agent to arrange a tour today!
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FTC charges Sage Auto Group with deceptive, unfair sales and financing
The Federal Trade Commission today charged Sage Auto Group with using deceptive and unfair sales and financing tactics, including so-called "yo-yo" financing.
Automakers see high-stakes mobility as an opportunity
The stakes are high for automakers in the future of mass transportation. Andreas Mai, who recently served as the director of smart connected vehicles at Cisco Systems, estimated the size of the mobility industry -- which would include autonomous vehicles, ride-sharing networks and connectivity -- to be worth about $5 trillion over the next decade.
Audi Q5 gains size, power
Audi aims to retain its leadership position in luxury crossovers with a second-generation Q5 that adds size, technology and power to the outgoing
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AutoNews Now is our afternoon video roundup of the top news stories of the day.


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Experts say that surgical robots or robot doctors will be conducting one in three operations in the US in the next five years. The surgical robots are estimated to multiply two folds by 2021, with surgeons guiding mechanical them on comp…
Sales, means the sales volume of Enzymatic Catalyst
Revenue, means the sales value of Enzymatic Catalyst
This report studies sales (consumption) of Enzymatic Catalyst in Global market, especially in USA, China, Europe, Japan, India and Southeast Asia,I50 focuses on top players in these regions/countries, with sales, price, revenue and market share for each player in these regions, covering
- Evonik Industries
- Johnson Matthey
- Exxon Mobil Corporation
- Clariant AG
- Honeywell International Inc.
- LyondellBasell Industries N.V
- Albemarle Corporation
- Chevron Phillips Chemical Company LLC.
- Ineos Group AG
Market Segment by Regions, this report splits Global into several key Regions, with sales (consumption), revenue, market share and growth rate of Enzymatic Catalyst in these regions, from 2011 to 2021 (forecast), like
- China
- Europe
- Japan
- India
- Southeast Asia
Split by product Types, with sales, revenue, price and gross margin, market share and growth rate of each type, can be divided into
- Type I
- Type II
- Type III
Split by applications, this report focuses on sales, market share and growth rate of Enzymatic Catalyst in each application, can be divided into
- Oil & Gas
- Flame Retardants
- Biocide


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Bollywood actress and former Miss World Aishwarya Rai Bachchan looked exquisitely beautiful in the promotional stills for her first big budget Hollywood outing, Pink Panther 2. The stills feature "Ash" giving...
Aishwarya Rai - "Pink Panther 2" Promo Stills
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New Farm
Markets are on every Saturday from 6.30am at New Farm Park, grab a coffee have a wonder around or sit on the grass and watch the world go bye.
Eat Street Markets
The Eat Street Markets is Brisbane's bustling night […]
Brisbane and surrounds are serving up a blockbuster month of entertaining events involving fantastic festivals, fine food, masterful music, fun for kids and sensational stage productions.
Board Games & Burgers
Thursdays 6-10pm @ Grill'd Fortitude Valley
Board Games & Burgers is a tabletop meet-up, run by Vault Games […]
If there is one thing that gets a Brisbane local out of bed on a Saturday morning, it's the promise of a good breakfast.
Fortunately we're spoilt with amazing cafe choices here, but rather than keep the secret to ourselves, we've decided to share our […]Read more
Whether you need the perfect restaurant for a special occasion such as an anniversary, a birthday, or even a proposal, or you're chasing a romantic vibe for a casual night for just the two of you, Brisbane is overflowing with restaurants that fit the bill.
Treat your […]Read more


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Bathroom Warehouse is the largest online retailer of bathroom products in Australia, including a range of bathroom fixtures for the disabled. View Photos, Video and Articles by Bathroomware House or Get a Quote.
Square Landscapes provides a wide range of landscape services, ranging from basic landscape solutions to complete home and business landscaping needs. View Photos by Square Landscapes or Get a Quote.
Leaders in innovative flooring solutions. Expert advice and quality service. Massive savings and great value on a wide range of carpet, rugs, timber flooring, bamboo flooring, laminate flooring, vinyl and luxury vinyl plank flooring. View Photos, Video and Brochures by Harvey Norman The Carpet and Flooring Specialists or Get a Quote.
Strong, durable, and yet flexible enough to create any shape desired. Greenlines Gardenwareâs FormBoss⢠is the steel garden edging solution revolutionising councils, public gardens, parks, and homes right across Australia. View Photos, Video, Brochures and Articles by Formboss Metal Garden Edging or Get a Quote.
Specify CSR Gyprock® high performance walls and ceilings and Fibre Cement external lining products now with your Canberra builder and you can make sure that you have the home youve always wanted. Get a Quote from CSR Gyprock.


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How to Render Transparent Materials in KeyShot
After you saw the quick tip on Glass and Solid Glass Materials you may have become highly addicted to transparent blue objects and creating incredible 3D renderings of transparent blue objects. That's ok. You're among friends. Now, it's time to take that up a level and explore how light and shadows work with the two materials and the settings you can use to fine-tune transparent materials for the look you need.
Rendering Transparent Materials in KeyShot
In this quick, little transparency tip, Brian Townsend explains the difference between the illumination of "Solid glass" and "Glass". He'll also show you the settings to change if the objects under your transparent materials appear too dark.
You can see all of the KeyShot Tutorials, Quick Tips and Webinars in the KeyShot Learning section. Have a look and if you're wondering about how to do something in KeyShot, let us know in the comments!
KeyShot Video Tutorials: Material Creation in KeyShot
Last week we brought you a brief introduction to using textures in That skimmed the surface of all that's possible in Underneath, lies so much So, we've put together a series of video tutorials that takes you through materials, textures and applying them to an example It's three videos with over an hour of solid material that you can watch
All About KeyShot Render Options
You've got your materials set, cameras positioned and an environment that reflects the sheer intensity of the surrounding What do you do next? Render that With KeyShot, you have a lot of options for image or animation output, including three different render But what'st the difference between each mode and what will provide the best results in the


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I recently downloaded the free Killzone 3 multiplayer trial to give it a spin, and to my surprise I've become really addicted! I'm generally not much of an online FPS fan, the last game I really got into was Call Of Duty: Modern Warfare back when the online gameplay that if offered was fresh and exciting. Since then I've dabbled in a few other games like Battlefield: Bad Company 2, and a little bit of COD: World At War, but nothing really holds my interest for very long.
I think that what I like about Killzone 3 is the fact that it's not a "twitch" shooter. Your character has a definite sense of weight and momentum, and jumping takes a second or two to action so bunny hopping about the map isn't a viable option. Other than the demo I haven't actually played the Killzone 3 singleplayer, but I loved Killzone 2 so it hasn't taken me too long to fall back into the rhythm of the game and the weighty controls. I'm actually pretty good at the multiplayer in Killzone 3! This is nice for a change, I'm generally kind of average at online shooters. My K/D ratio is about 0.8, and I'm up to rank 11 so far. Once I hit the rank 10 cap in the trial I was more than happy to pay to buy the full version, especially with the PSN+ discount.
It's early days yet, but I can't see myself getting bored of this game any time soon. Have any of you out there given the trial a go? If so, I'd be interested to hear your thoughts.


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All's fair in love and war.
Along the pristine coastline of Northern California, a battle rages between land developers and environmentalists. Gavin Ransom, a handsome developer, wins the right to build a tract of garish mini-mansions on sensitive coastal real estate over the objections of outraged environmentalists led by his sister, Olive Ransom, an intensely serious woman. When they both meet the beautiful protest singer, Zoe, they fall under her spell and fight for her support and love.
- Length
- Rating PG
- Released:
- Added Dec 30 2005
- Production Year 2004
- Empire SKU 900800
- UPC Code 829567028025
- Studio Virgil Films And Entertainment
- Packaging Keep
- Number of Discs 1
- Disc SS-SL
No Features Specified
- Audio
English Dolby Digital Stereo
- Video
Standard 1.33:1 Color
- Jonathan Parker
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Create a presta shop - oscommerce or zen cart ecommerce website
This project received 34 bids from talented freelancers with an average bid price of $364 CAD.Get free quotes for a project like this
Skills Required
Project Budget$30 - $250 CAD
Total Bids34
Project Description
We need to design a ecommerce platform.
around 100 product
5 main categories
Selling some artistic jewels and other african items.
paypal /card payments.
We will buy a theme on template monster and have you modify it to our needs and then populate it a bit and get it to work perfectly with no bug.
Small work. Tell us your price and what platform you would like to use.
Looking to make some money?
- Set your budget and the timeframe
- Outline your proposal
- Get paid for your work
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mittendrin - Boutique Hotel Berlin, Berlin
- Free full breakfast and WiFi
Main amenities
- 4 smoke-free guestrooms
- Room service
- Daily housekeeping
- ATM/banking services
- Self-serve laundry
- Limited hour front desk
- Tour/ticket assistance
Feel at home
- Sofa bed
- Private bathroom
- Separate sitting area
- Television
- Daily housekeeping
- Laundry facilities
What's around
- In Charlottenburg
- Kaiser Wilhelm Memorial Church (0.2 mi / 0.4 km)
- Berlin Zoo (0.7 mi / 1.2 km)
- Victory Column (1.1 mi / 1.8 km)
- Brandenburg Gate (2.1 mi / 3.5 km)
- Reichstag Building (2.2 mi / 3.5 km)
mittendrin - Boutique Hotel Berlin, Berlin
Hotel Info: 866-539-8117
- Double Room (Romantic)
- Double Room, Shared Bathroom (Klassiko)
- Single Room, Shared Bathroom (Mediterrano)
- Double Room (Modern)
More great choices
At a glance
Key facts
Hotel size
- This hotel has 4 rooms
- This hotel is arranged over 1 floor
- Check-in time 3 PM-7 PM
- Check-out time is 11 AM
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
- Minimum check-in age is 18
Travelling with others
- One child (1 years old and younger) stays free when occupying the parent or guardian's room, using existing bedding
- No cribs (infant beds)
- No pets or service animals allowed
- Free WiFi and wired Internet access in public areas
- Free WiFi in rooms
Other information
- Smoke-free property
- Resort fee included
In the hotel
- Free full breakfast daily
- Room service (during limited hours)
- Bicycle rentals nearby
- Front desk (limited hours)
- Tours/ticket assistance
- Laundry facilities
- Number of buildings/towers - 1
- Elevator/lift
- ATM/banking
In the room
- Ceiling fan
- Minibar
- Coffee/tea maker
- Bathrobes
- Slippers
- Iron/ironing board
- Hypo-allergenic bedding available
- Pillow menu
- Blackout drapes/curtains
- Soundproofed rooms
- Sofa bed
- Premium bedding
- Sleep Number by Select Comfort mattress
- Individually furnished
- Separate sitting area
- Private bathroom
- Shower/tub combination
- Hair dryer
- IPod docking station
- Desk
- Free WiFi
- Free local calls
- Daily housekeeping
mittendrin - Boutique Hotel Berlin, Berlin's small print
Also known as
- mittendrin Boutique
- mittendrin Boutique Berlin
- mittendrin Boutique Hotel
- mittendrin Boutique Hotel Berlin
Only registered guests are allowed in the guestrooms.
Mandatory fees
The charges below are included in the total price displayed:
- Resort fee
Optional extras
Rollaway beds are available for an additional charge
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.
Also popular with travelers from the USA
Recent reviews
Outstanding 5.0 /5 from 23 reviews
A beautifully decorated bed and breakfast on the 4th floor of a restored early 20th century apartment building just a block off the Kurfurstdendamm. Restaurants and shops are plentiful nearby and the U-Bahn station is only a few blocks away. Your host is warmly engaging and tries to make everyone feel at home. Breakfast each morning was an absolute feast with the table spread with about everything anyone could imagine eating for breakfast, served in a beautifully decorated dining room. I recommend it without reservation and will look forward to staying there on my next visit to Berlin.
In all respects the B&B was excellent. In particular it is only a half block off of the Tauentzienstrasse which places it only one block from the Ka De We, t5wo blocks from the U-Bahn, 100 meters from the Europa Center, two blocks from the Kaiser Whilhelm Kirche, etc.The another outstanding characteristic of our stay was the service provided by our hostess. She was a tour director besides a breakfast caterer of of the first rank.It is a great value.
Sabine was a great host. ,he wen't well above and beyond my expectations. As soon as i got to the hotel she was helpful and able to suggest things to do, places to eat, etc. I would highly recommended this BnB!
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Oracle Snatches Sun, Foiling IBM
Oracle Corp. ORCL 0.41 % struck a surprise deal to buy Sun Microsystems Inc. for $7.38 billion, elbowing aside International Business Machines Corp. in the latest sign that a few well-heeled giants are exploiting the recession to snap up weaker players.
Oracle, which has spent more than $30 billion buying software rivals in recent years, swung into action Thursday after talks between IBM and Sun had stalled, people familiar with the matter said.
IBM still held out hope over the weekend that Sun would accept a bid from the big computer maker, these people said, but Sun's board voted in favor of Oracle's offer, which was viewed as less likely to face an antitrust challenge since there was little business overlap.
IBM is highly unlikely to re-enter the bidding for Sun, said people familiar with the matter.
After factoring in Sun's cash and debt, the companies valued Oracle's offer at about $5.6 billion. Sun shareholders will get $9.50 a share in cash. Sun's stock surged 37% on the news Monday to $9.15.
Sun, whose server computers have long been sold along with Oracle's database software, was widely believed to need a bigger partner to compete with giants such as IBM, Hewlett-Packard Co. HPQ 0.91 % and Dell Inc. Sun has suffered from erratic profitability and revenue problems since a boom phase ended with the collapse of the dot-com bubble.
Oracle has emerged as one of the most powerful players on the tech landscape, buying rivals to assemble a long list of programs used by companies.
The deal gives Oracle control over important Sun software -- notably the popular programming technology called Java, which is used to build software for Web sites and cellphones. Oracle Chief Executive Larry Ellison described Java as "the single most important software asset we have ever acquired."
Some analysts said they were stunned by Mr. Ellison's move, predicting that Oracle may eventually choose to divest Sun's hardware business. But Safra Catz, one of two Oracle presidents, said Oracle intends to make Sun's hardware operations a profitable business unit.
Write to Don Clark at email@example.com and Ben Worthen at firstname.lastname@example.com


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web basedAutomobile showroom and service station management software
This project received 8 bids from talented freelancers with an average bid price of ₹34375 INR.Get free quotes for a project like this
Skills Required
Project Budget₹12500 - ₹37500 INR
Total Bids8
Project Description
we are looking for complete fro web absed Automobile showroom and service station management software with complete fetuers. with multi user login means we can create multiple store login or password
means one software for multi store
Looking to make some money?
- Set your budget and the timeframe
- Outline your proposal
- Get paid for your work
Hire Freelancers who also bid on this project
Looking for work?
Work on projects like this and make money from home!Sign Up Now
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Lavon Hooks, the No. 1 juco player in The Clarion-Ledger Juco Top 10, signed a Letter of Intent with Ole Miss on Thursday. "It was a lot of stress, a whole bunch," Hooks told the Gwinnett Daily Post. "I wanted to go somewhere I knew I could make an immediate impact and play on a big stage, and go some place that would make me happy and make my family happy."
Lavon Hooks Signs with Ole Miss
From ClarionLedger.com
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Pentair Minimax Module, (Fenwal), IID Ignition Control, 472447
No.41 Image Shown 472447 Module, Ignition Control Used On Pentair Purex Minimax NT STD w/ DDTC Pool Heaters Part Number Used On Stud w/ DDTC Model Numbers 200, 250, 300, 400. Also Fits NPT Ln w/ DDTC Control Board. Does Not Fit Minimax NT TSI, See 472150. This IID Controller Controls The Ignition of Electronic Heaters, Made By Fenwal, Cat No. 35-662902-113 472447 Can also be found on Minimax NT Pool Heaters parts pages.
Related parts recommended for installation or repair
Pentair Minimax Temperature Controller Assy. Ddtc - 472100
|Now $273.81 was $301.19
|More info >