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Peptides derived from three regions of the lectin binding region of GMP-140 have been found to selectively interact with "selectins", including GMP-140, ELAM-1, and lymphocyte homing receptor. The peptides can be as short as eight to thirteen amino acids in length and are easily prepared and modified by standard techniques. Critical elements of the counter-receptor or ligand on the neutrophils which binds GMP-140 are also identified. The peptides are useful as diagnostics and
The U.S. Government has rights in this invention by virtues of grants from the National Heart, Lung and Blood Institute.
1. An isolated peptide between eight amino acids and 118 amino acids in length comprising an amino acid sequence selected from the group consisting of
and X15 X16 X7 E P N N X17 X18 X19 X20 E D,
X1 =Q or R;
X2 =N, Q, or D;
X3 =R or N;
X4 =D or H;
X5 =N, E or A;
X6 =N, V, or I;
X7 =N or G;
X8 =K, N, or G;
X9 =T, V, or I;
X10 =T or V;
X11 =K, Q, or N;
X12 =A, P, or S;
X13 =N or E;
X14 =E or K;
X15 =A or G;
X16 =D or P;
X17 =K or R;
X18 =R, Q, or K;
X19 =N or K;
X20 =N, D, or K;
and the peptide inhibits binding of neutrophils and monocytes to GMP-140.
2. The peptide of claim 1 wherein the amino acid sequence is selected from the group consisting of CQNRYTDLVAIQNKNE, AENWADNEPNNKRNNED, RKNNKTWTWVGTKKALTNE, and KKALTNEAENWAD.
3. The peptide of claim 1 wherein the amino acid sequence is selected from the group of lectin domain peptides amino acids 19-34, 19-30, 23-34, 21-30, 22-30, 23-30, 54-72, 54-63, 66-78, 73-89, 73-83, 73-85, 77-89.
4. A peptide conjugate comprising the peptide of claim 1 bound to a carrier molecule.
5. A prosthetic comprising the peptide of claim 1 bound to an inert substrate for implantation in the body.
6. A pharmaceutical composition comprising the peptide of claim 1 in a pharmaceutical carrier acceptable for administration to a patient.
7. A diagnostic comprising the peptide of claim 1 further comprising a detectable label selected from the group consisting of fluorescent, radioactive, and enzymatic labels.
The U.S. Government has rights in this invention by virtues of grants from the National Heart, Lung and Blood Institute.
This is a continuation of copending application Ser. No. 07/554,199 filed on Jul. 17, 1990 abandoned, which is a continuation-in-part of U.S. Ser. No. 07/320,408 entitled "Method for Modulation of Inflammatory Responses" filed Mar. 8, 1989 by Rodger P. McEver.
This invention is generally in the field of methods for the treatment and prevention of inflammatory responses involving binding reactions with selections including GMP-140, ELAM-1, and lymphocyte-homing receptor.
The adherence of platelets and leukocytes to vascular surfaces is a critical component of the inflammatory response, and is part of a complex series of reactions involving the simultaneous and interrelated activation of the complement, coagulation, and immune systems.
The complement proteins collectively play a leading role in the immune system, both in the identification and in the removal of foreign substances and immune complexes, as reviewed by Muller-Eberhard, H. J., Ann. Rev. Biochem. 57:321-347 (1988). Central to the complement system are the C3 and C4 proteins, which when activated covalently attach to nearby targets, marking them for clearance. In order to help control this process, a remarkable family of soluble and membrane-bound regulatory proteins has evolved, each of which interacts with activated C3 and/or C4 derivatives. The coagulation and inflammatory pathways are regulated in a coordinate fashion in response to tissue damage. For example, in addition to becoming adhesive for leukocytes, activated endothelial cells express tissue factor on the cell surface and decrease their surface expression of thrombomodulin, leading to a net facilitation of coagulation reactions on the cell surface. In some cases, a single receptor can be involved in both inflammatory and coagulation processes.
Leukocyte adherence to vascular endothelium is a key initial step in migration of leukocytes to tissues in response to microbial invasion. Although a class of inducible leukocyte receptors, the CD11-CD18 molecules, are thought to have some role in adherence to endothelium, mechanisms of equal or even greater importance for leukocyte adherence appear to be due to inducible changes in the endothelium itself.
Activated platelets have also been shown to interact with both neutrophils and monocytes in vitro. The interaction of platelets with monocytes may be mediated in part by the binding of thrombospondin to platelets and monocytes, although other mechanisms have not been excluded. The mechanisms for the binding of neutrophils to activated platelets are not well understood, except that it is known that divalent cations are required. In response to vascular injury, platelets are known to adhere to subendothelial surfaces, become activated, and support coagulation. Platelets and other cells may also play an important role in the recruitment of leukocytes into the wound in order to contain microbial invasion.
Endothelium exposed to "rapid" activators such as thrombin and histamine becomes adhesive for neutrophils within two to ten minutes, while endothelium exposed to cytokines such as tumor necrosis factor and interleukin-1 becomes adhesive after one to six hours. The rapid endothelial-dependent leukocyte adhesion has been associated with expression of the lipid mediator platelet activating factor (PAF) on the cell surface, and presumably, the appearance of other endothelial and leukocyte surface receptors. The slower cytokine-inducible endothelial adhesion for leukocytes is mediated, at least in part, by an endothelial cell receptor, ELAM-1, that is synthesized by endothelial cells after exposure to cytokines and then transported to the cell surface, where it binds neutrophils. The isolation, characterization and cloning of ELAM-1 is reviewed by Bevilacqua, et al., in Science 243, 1160-1165 (1989). A peripheral lymph node homing receptor, also called "the murine Mel 14 antigen", "Leu 8", the "Leu 8 antigen" and "LAM-1", is another structure on neutrophils, monocytes, and lymphocytes that binds lymphocytes to high endothelial venules in peripheral lymph nodes. The characterization and cloning of this protein is reviewed by Lasky, et al., Cell 56, 1045-1055 (1989) (mouse) and Tedder, et al., J. Exp. Med. 170, 123-133 (1989).
GMP-140 (granule membrane protein 140), also known as PADGEM, is a cysteine-rich and heavily glycosylated integral membrane glycoprotein with an apparent molecular weight of 140,000 as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). GMP-140 was first purified from human platelets by McEver and Martin, J. Biol. Chem. 259:9799-9804 (1984). The protein is present in alpha granules of resting platelets but is rapidly redistributed to the plasma membrane following platelet activation, as reported by Stenberg, et al., (1985). The presence of GMP-140 in endothelial cells and its biosynthesis by these cells was reported by McEver, et al., Blood 70(5) Suppl. 1:355a, Abstract No. 1274 (1987). In endothelial cells, GMP-140 is found in storage granules known as the Weibel-Palade bodies. GMP-140 (called PADGEM) has also been reported to mediate the interaction of activated platelets with neutrophils and monocytes by Larsen, et al., in Cell 59, 305-312 (October 1989) and Hamburger and McEver, Blood 75:550-554 (1990).
The cDNA-derived amino acid sequence, reported by Johnston, et al., in Cell 56, 1033-1044 (Mar. 24, 1989), and in U.S. Ser. No. 07/320,408 filed Mar. 8, 1989, indicates that it contains a number of modular domains that are likely to fold independently. Beginning at the N-terminus, these include a "lectin" domain, an "EGF" domain, nine tandem consensus repeats similar to those in complement binding proteins, a transmembrane domain (except in a soluble form that appears to result from differential splicing), and a cytoplasmic tail.
When platelets or endothelial cells are activated by mediators such as thrombin, the membranes of the storage granules fuse with the plasma membrane, the soluble contents of the granules are released to the external environment, and membrane bound GMP-140 is presented within seconds on the cell surface. The rapid redistribution of GMP-140 to the surface of platelets and endothelial cells as a result of activation suggests that this glycoprotein could play an important role at sites of inflammation or vascular disruption.
ELAM-1, the homing receptor, and GMP-140 have been termed "selectins", based on their related structure and function.
The in vivo significance of platelet-leukocyte interactions has not been studied carefully. However, in response to vascular injury, platelets are known to adhere to subendothelial surfaces, become activated, and support coagulation. Platelets and other cells may also play an important role in the recruitment of leukocytes into the wound in order to contain microbial invasion. Conversely, leukocytes may recruit platelets into tissues at sites of inflammation, as reported by Issekutz, et al., Lab. Invest. 49:716 (1983).
The coagulation and inflammatory pathways are regulated in a coordinate fashion in response to tissue damage. For example, in addition to becoming adhesive for leukocytes, activated endothelial cells express tissue factor on the cell surface and decrease their surface expression of thrombomodulin, leading to a net facilitation of coagulation reactions on the cell surface. In some cases, a single receptor can be involved in both inflammatory and coagulation processes.
Proteins involved in the hemostatic and inflammatory pathways are of interest for diagnostic purposes and treatment of human disorders. However, there are many problems using proteins therapeutically. Proteins are usually expensive to produce in quantities sufficient for administration to a patient. Moreover, there can be a reaction against the protein after it has been administered more than once to the patient. It is therefore desirable to develop peptides having the same, or better, activity as the protein, which are inexpensive to synthesize, reproducible and relatively innocuous.
It is therefore an object of the present invention to provide peptides having activity of selectins, including GMP-140, ELAM-1, and lymphocyte homing receptor.
It is another object of the present invention to provide methods for using these peptides to inhibit leukocyte adhesion to endothelium or to platelets.
It is a further object of the present invention to provide methods for using these peptides to modulate the immune response and the hemostatic pathway.
It is yet another object of the present invention to provide peptides for use in diagnostic assays relating to GMP-140, ELAM-1, and lymphocyte homing receptor.
It is yet another object of the present invention to provide carbohydrate-based reagents, based on the structure of the leukocyte ligand for GMP-140, to inhibit GMP-140 - mediated adhesive interactions.
Peptides derived from three regions of the lectin binding region of GMP-140 have been found to function as "selectins", including GMP-140, ELAM-1, and lymphocyte homing receptor. The three regions include amino acids 19-34, 54-72, and 66-89, based on the numbers of the residues contained in the peptide, with residue 1 defined as the N terminus of the mature protein after cleavage of the signal peptide. The peptides can be as short as eight to thrirteen amino acids in length and are easily prepared by standard techniques. They can also be modified to increase in vivo half-life, by chemical modification of the amino acids or by attachment to a carrier molecule or inert substrate. Critical elements of the counter-receptor or ligand on the neutrophils which binds GMP-130 are also identified.
Examples demonstrate that the peptides bind neutrophils and inhibit binding of GMP-140 to neutrophils, with an IC50 (the dose required to inhibit adhesion of neutrophils to immobilized GMP-140 by 50%) ranging from 50 to 300 micromolar. The binding affinity can be modulated using sequences from the EGF domain and divalent cations which bind to both the EGF and lectin domains. An assay is also demonstrated that is useful for screening for variations of these peptides that interfere with binding of all selectins or individual selectins, especially GMP-140.
The peptides are useful as diagnostics and, in combination with a suitable pharmaceutical carrier, for clinical applications in the modulation or inhibition of coagulation processes or inflammatory processes.
FIG. 1 is the nucleotide and deduced amino acid sequence of endothelial cell GMP-140. The translated amino acid sequence of the open reading frame is given in the single-letter code. The stop codon is shown by the asterisk. The thin underlines show the matching positions of amino acid sequences determined from the N-terminus and from 26 peptides of platelet GMP-140. The signal peptide corresponds to positions -41 to -1. The putative transmembrane domain is heavily underlined. The cysteine residues are circled and potential asparagine-linked glycosylation sites (NXS/T) are shown by the dark circles. Two potential polyadenylation signals in the 3' untranslated region are underlined and overlined. This figure is from U.S. Ser. No. 07/320,408.
FIG. 2 is the proposed folding pattern of the domains in GMP-140. The cysteine residues are shown by the dark circles and the potential free sulfhydryl in the cytoplasmic domain is indicated by --SH. This figure is from U.S. Ser. No. 07/320,408.
FIG. 3 is a graph demonstrating inhibition of binding by peptides from the GMP-140 lectin domain, amino acids 66-78 (triangle), amino acids 73-83 (square), amino acids 54-63 (circle), and amino acids 23-30 (dark circle), comparing number of cells bound to mM of peptides.
FIG. 4A and B are comparisons of the specific adhesion of neutrophils to microtiter wells coated with no peptide (1); coated with lectin domain peptide 19-34 conjugated to KHL (2); or coated with a control carboxyterminus peptide (amino acid residues 761-777) conjugated to KLH (3), blocked with Hank's Balanced Salt Solution containing human serum albumin prior to addition of 2�105 neutrophils to each well, in the presence of fluid-phase competitors. Fluid-phase competitors added to the neutrophils prior to transfer to the wells were: Panel A, none (dark bar), purified platelet glycoprotein IIb-IIIa (slashed bar), or purified GMP-140 (stippled bar); Panel B, none (dark bar), 1.5 mM C-terminal peptide 761-777 (slashed bar), 1.5 mM lectin domain peptide 19-34 (stippled bar).
The structure and biosynthesis of GMP-140 has now been analyzed in detail. The entire amino acid sequence of GMP-140 has been determined by a combination of protein sequencing of peptide fragments derived from purified platelet GMP-140 and by cloning of cDNAs encoding GMP-140 from a human endothelial cell cDNA library. Based on the structure and on functional studies, GMP-140 acts as a receptor for neutrophils, and interacts with complement protein C3b and the anticoagulant cofactor protein S.
Cloning of the gene for GMP-140 was first reported by G. I. Johnston, R. G. Cook and R. P. McEver in Abstract 1238 Supplement II Circulation 78(4) (October 1988). Oligonucleotides were prepared based on N-terminal amino acid sequencing of GMP-140 peptides and used to screen a human endothelial cell cDNA library. A 3.0 kb clone was isolated which encoded a protein of 727 amino acids. An N-terminal domain of 118 residues containing many cysteines, lysines, and tyrosines, which is similar to the asialo glycoprotein receptor, is followed by an EGF-type repeating domain structure, and eight tandem repeats of 62 amino acids each, except for the sixth tandem repeat which has 70 amino acids. The repeats are homologous to those found in a family of proteins that include proteins regulating C3b and C4b, but are unique in having six conserved cysteines per repeat instead of the typical four. These are followed by a 24 amino acid transmembrane region and a 35 amino acid cytoplasmic tail. There appear to be at least two forms of the protein derived by alternative splicing of transcripts of the same gene: a soluble form and a membrane or granule bound form.
The predicted amino acid sequence of endothelial cell GMP-140, shown in FIG. 1, suggests the presence of six different structural domains, shown schematically in FIG. 2. One of these regions, a 24-residue hydrophobic segment near the C-terminus, is characteristic of membrane spanning domains. Most of the protein is located on the N-terminal side of this domain and appears to be extracytoplasmic, i.e., facing the lumen of the secretory granule, or, following activation of the cell, exposed to the extracellular environment.
Beginning at the N-terminus, the first domain contains 41 residues (labeled -41 to -1 in FIG. 1) and has the characteristic features of a signal peptide. These include several positively charged amino acids, followed by a hydrophobic domain and then a region rich in polar residues. The small uncharged residues found at positions -3 and -1 are typical of those found at sites of cleavage by signal peptidase. In addition, the amino acid sequence of the N-terminus of platelet GMP-140 matches the deduced endothelial cell sequence at 25 out of 27 positions from residues 1 to 27.
Following the signal peptide, the translated cDNA sequence predicts a mature protein of 789 residues. Comparison of the sequences of platelet GMP-140 peptides to the deduced endothelial cell sequence showed that 337 of 341 assigned amino acids match, suggesting that both cell types synthesize the same protein.
There are 12 potential asparagine-linked glycosylation sites having the consensus sequence NxS/T. All are located on the extracytoplasmic portion of the molecule and all appear to be glycosylated based on the carbohydrate composition of platelet GMP-140. The mature protein contains 65 cysteines accounting for 8% of the total amino acids. Most of these are predicted to be organized into disulfide bridges since only a small amount of carboxymethyl cysteine can be identified in samples of nonreduced GMP-140 treated with iodoacetamide.
The second domain begins at residue 1 and encompasses the first 118 amino acids of the mature protein. This region is rich in lysine (12%), tyrosine (10%), asparagine (13%), and tryptophan (6%) residues. This region of GMP-140 is designated as a "lectin" domain, since many of the proteins containing this motif bind carbohydrate.
The third domain, which begins at residue 119, has a sequence of 40 amino acids that contains six cysteines. Comparison of this region of GMP-140 to sequences in the NBRF database reveals many proteins that contain the same arrangement of cysteines. The first protein described with this motif is the epidermal growth factor (EGF) precursor, which contains ten homologous copies (Gray, et al., Nature 303:236-240 (1983; Scott, et al., Nature 221:236-240 (1983).
The fourth domain, which begins at residue 159, consists of nine tandem consensus repeats, each containing 62 amino acids; in addition, extensions of eight and four residues, respectively, are found at the ends of the seventh and ninth repeats. The boundaries of this domain are set arbitrarily at the first cysteine of the initial repeat and at the last residue before the putative transmembrane domain. A consensus sequence shows that many amino acids occur in at least five out of the nine repeats. All repeats contain six conserved cysteines, three glycines, and one tryptophan, phenylalanine, proline, and leucine. The cysteine residues are arranged in a different motif than the six cysteines found in the "EGF" domain. The repeats are 31% to 56% identical to each other at the amino acid level and 42% to 62% identical at the nucleotide level. No gaps are required to maximize the alignment among the repeats.
The fifth domain, beginning at residue 731, is the 24-residue putative transmembrane domain. Following this is the sixth domain, a presumed cytoplasmic segment of 35 residues that begins with several highly charged residues and ends at the C-terminus of the protein at residue 789. There are possible phosphorylation sites at serine, threonine, and tyrosine residues, as well as a cysteine that might undergo posttranslational modification.
Two different in-frame deletions are identified among the sequences of the four endothelial-cell clones examined in detail. The first deletion is 186 bp. This deletion removes 62 amino acids from the seventh consensus repeat and predicts a protein containing eight instead of nine repeats. The second deletion is 120 bp, which removes 40 amino acids just after the end of the ninth repeat. The region deleted includes the transmembrane domain and the first few residues of the cytoplasmic domain. The remaining 28 residues at the C-terminus are predicted to continue just after the ninth repeat. A hydrophilicity plot (Kyte and Doolittle, J.Mol.Biol. 157:105-132 (1982) predicts that this form of GMP-140 is soluble.
GMP-140 has been demonstrated to be a receptor on platelets and endothelial cells that binds to a surface ligand on neutrophils and monocytes, thereby facilitating the inflammatory process.
The conclusion that GMP-140 serves as receptor for the adherence of leukocytes to activated endothelial cells and platelets was originally based on several observations: the rapid appearance of GMP-140 on the surface of endothelium stimulated with thrombin or histamine which parallels the inducible adherence of neutrophils to endothelium stimulated with these agonists; the interaction of neutrophils or monocytes with platelets only after platelet activation with agonists such as thrombin which cause redistribution of GMP-140 to the cell surface and not with platelet agonists such as ADP; concentration of GMP-140 in postcapillary venules which are the predominant sites for binding of leukocytes to endothelium prior to their migration across the endothelium into the tissues; specific adherence of purified neutrophils to GMP-140 coated on tissue culture microtiter wells; blocking by polyclonal antibodies to GMP-140 of 60-90%, of the adherence of neutrophils to cultured human umbilical vein endothelial cells stimulated with histamine; similarity of the cDNA-derived amino acid sequence of GMP-140 to that of ELAM-1, an endothelial cell protein already known to bind neutrophils, and to lymphocyte homing receptor. Subsequent studies have demonstrated that GMP-140 also mediates adhesion of neutrophils to stimulated platelets, but not unstimulated platelets, in the presence of Ca2+. The binding of platelets to neutrophils was inhibited by a monoclonal antibody to GMP-140 and by purified GMP-140.
Other studies have demonstrated that GMP-140 binds to C3b, a complement system protein, and protein S, an anticoagulant cofactor protein. GMP-140 shares sequence homology with the plasma protein C4b-binding protein (C4bp), which not only interacts with the plasma protein C4b but also with protein S.
Peptides derived from GMP-140 have now been discovered that are useful in diagnostics and in modulating the hemostatic and inflammatory responses in a patient wherein a therapeutically effective amount of a peptide capable of blocking leukocyte recognition of GMP-140 is administered to the patient.
It has now been discovered that peptide sequences within the lectin domain of GMP-140, having homology with the lectin domains of other proteins, especially ELAM-1 and the homing receptor, selectively inhibit neutrophil adhesion to purified GMP-140, can therefore be used in diagnostic assays of patients and diseases characterized by altered binding by these molecules, and in screening assays for compounds altering this binding, and should be useful clinically to inhibit or modulate interactions of leukocytes with platelets or endothelial cells involving coagulation and/or inflammatory processes.
The cDNA-derived primary structure of GMP-140 provides several insights into functions for GMP-140 in the vascular system. The most remarkable observation is the striking structural similarity of GMP-140 to two other receptors found on vascular cells which have been recently cloned.
The first of these similar receptors is ELAM-1. ELAM-1 is an endothelial cell protein that is not present in unstimulated endothelium. However, when endothelium is exposed to cytokines such as tumor necrosis factor or interleukin-1, the gene for ELAM-1 is transcribed, producing RNA which in turn is translated into protein. The result is that ELAM-1 is expressed on the surface of endothelial cells 1-4 hours after exposure to cytokines, as reported by Bevilacqua et al., Proc.Natl.Acad.Sci.USA 84:9238-9242 (1987) (in contrast to GMP-140, which is stored in granules and presented on the cell surface within seconds after activation). ELAM-1 has been shown to mediate the adherence of neutrophils to cytokine-treated endothelium and thus appears to be important in allowing leukocytes to migrate across cytokine-stimulated endothelium into tissues. The cDNA-derived primary structure of ELAM-1 indicates that it contains a "lectin" domain, an EGF domain, and six (instead of the nine in GMP-140) repeats similar to those of complement-regulatory proteins, a transmembrane domain, and a short cytoplasmic tail. There is extensive sequence homology between GMP-140 and ELAM-1 throughout the both proteins, but the similarity is particularly striking in the lectin and EGF domains.
The second molecule with overall structural similarity to GMP-140 is a homing receptor found on lymphocytes. Homing receptors are lymphocyte surface structures that allow lymphocytes to bind to specialized endothelial cells in lymphatic tissues, termed high endothelial cells or high endothelial venules (reviewed by Yednock and Rose, Advances in Immunology. vol. 44, F. I. Dixon,ed., 313-378 (Academic Press, New York 1989). This binding allows lymphocytes to migrate across the endothelium into the lymphatic tissues where they are exposed to processed antigens. The lymphocytes then re-enter the blood through the lymphatic system. The homing receptor contains a lectin domain, an EGF domain, two complement-binding repeats, a transmembrane domain, and a short cytoplasmic tail. The homing receptor also shares extensive sequence homology with GMP-140, particularly in the lectin and EGF domains.
A comparison of the lectin domains between GMP-140, ELAM-1, and the homing receptor (LEU-8) is shown in Table I. Based on these sequence similarities it should be possible to select those peptides inhibiting binding of neutrophils to GMP-140 which will inhibit binding of ELAM-1, the homing receptor, and other homologous selectins, to components of the inflammatory process, or, conversely, which will inhibit only GMP-140 binding.
The role of the GMP-140 lectin domain was tested by synthesizing a series of peptides spanning almost all of the 118 residues of the lectin domain, except for two hydrophobic stretches predicted to be sequestered in the interior of the molecule. Peptides were also synthesized encompassing the EGF-like domain (36 residues) which follows the lectin domain. Peptides were also made from one of the consensus repeats, the transmembrane region, and the C-terminus (cytoplasmic tail) of the molecule as controls. Active peptides derived from the lectin domain are shown in Table I which also aligns the related sequences of the lectin domains of ELAM-1 and the homing receptor, LEU-8. Peptides were prepared either on an Applied Biosystems Model 430A automated peptide synthesizer using t-Boc chemistry or on a Dupont RAMPS manual peptide synthesizer using Fmoc chemistry. After cleavage from the resin on which they were synthesized, all peptides were purified by reverse phase high performance liquid chromatography.
Peptides were screened for their ability to inhibit neutrophil adhesion to purified GMP-140 immobilized on plastic wells, using the assay described by Geng, et al., Nature 343, 757-760 (1990).
Human neutrophils are isolated from heparinized whole blood by density gradient centrifugation on Mono-Poly resolving media, Flow Laboratories. Neutrophil suspensions are greater than 98% pure and greater than 95% viable by trypan blue exclusion. For adhesion assays, neutrophils are suspended at a concentration of 2�106 cells/ml in Hanks' balanced salt solution containing 1.26 mM Ca2+ and 0.81 mM Mg2+ (HBSS, Gibco) with 5 mg/ml human serum albumin (HBSS/HSA). Adhesion assays are conducted in triplicate in 96-well microtiter plates, Corning, incubated at 4� C. overnight with 50 microliters of various protein solutions.
GMP-140 is isolated from human platelet lysates by immunoaffinity chromatography on antibody S12-Sepharose� and ion-exchange chromatography on a Mono-Q� column (FLPC, Pharmacia Fine Chemicals), as follows.
Outdated human platelet packs (100 units) obtained from a blood bank and stored at 4� C. are pooled, adjusted to 5 mM EDTA at pH 7.5, centrifuged at 4,000 rpm for 30 min in 1 liter bottles, then washed three times with 1 liter of 0.1 M NaCl, 20 mM Tris pH 7.5 (TBS), 5 mM EDTA, 5 mM benzamidine.
The pellets are then resuspended in a minimum amount of wash buffer and made 1 mM in DIFP, then frozen in 50 ml screwtop tubes at -80� C.
The frozen platelets are thawed and resuspended in 50 ml TBS, 5 mM benzamidine, 5 mM EDTA pH 7.5, 100 M leupeptin. The suspension is frozen and thawed two times in a dry ice-acetone bath using a 600 ml lyophilizing flask, then homogenized in a glass/teflon mortar and pestle and made 1 mM in DIFP. The NaCl concentration is adjusted to 0.5 M with a stock solution of 4 M NaCl. After stirring the suspension at 4� C., it is centrifuged in polycarbonate tubes at 33,000 rpm for 60 min at 4� C. The supernatant (0.5 M NaCl wash) is removed and saved; this supernatant contains the soluble form of GMP-140. Care is taken not to remove the top part of the pellet with the supernatant. The pellets are then homogenized in extraction buffer (TBS, 5 mM benzamidine, 5 mM EDTA, pH 7.5, 100 M leupeptin, 2% Triton X-100). After centrifugation at 19,500 rpm for 25 min at 4� C., the supernatant is removed. The extraction procedure is repeated with the pellet and the supernatant is combined with the first supernatant. The combined extracts, which contain the membrane form of GMP-140, are adjusted to 0.5 M NaCl.
The soluble fraction (0.5 M NaCl wash) and the membrane extract (also adjusted to 0.5 M NaCl) are absorbed with separate pools of the monoclonal antibody S12 (directed to human GMP-140) previously coupled to Affigel (Biorad) at 5 mg/ml for 2 h at 4� C. After letting the resins settle, the supernatants are removed. The S12 Affigel containing bound GMP-140 is then loaded into a column and washed overnight at 4� C. with 400 ml of 0.5 M NaCl, 20 mM Tris pH 7.5, 0.01% Lubrol PX.
Bound GMp-140 is eluted from the S12 Affigel with 100 ml of 80% ethylene glycol, 1 mM MES pH 6.0, 0.01% Lubrol PX. Peak fractions with absorbance at 280 nm are pooled. Eluates are dialyzed against TBS with 0.05% Lubrol, then applied to a Mono Q column (FPLC from Pharmacia). The concentrated protein is step eluted with 2 M NaCl, 20 mM Tris pH 7.5 (plus 0.05% Lubrol PX for the membrane fraction). Peak fractions are dialyzed into TBS pH 7.5 (plus 0.05% Lubrol PX for the membrane fraction).
GMP-140 is plated at 5 micrograms/ml and the control proteins: human serum albumin (Alb), platelet glycoprotein IIb/IIIa (IIb), von Willebrand factor (vWF), fibrinogen (FIB), thrombomodulin (TM), gelatin (GEL) or human serum (HS), are added at 50 micrograms/ml. All wells are blocked for 2 h at 22� C. with 300 microliters HBSS containing 10 mg/ml HSA, then washed three times with HBSS containing 0.1% Tween-20 and once with HBSS. Cells (2�105 per well were added to the wells and incubated at 22� C. for 20 min. The wells were then filled with HBSS/HSA, sealed with acetate tape (Dynatech), and centrifuged inverted at 150 g for 5 min. After discarding nonadherent cells and supernates, the contents of each well are solubilized with 200 microliters 0.5% hexadecyltrimethylammonium bromide, Sigma, in 50 mM potassium phosphate, pH 6.0, and assayed for myeloperoxidase activity, Ley, et al., Blood 73, 1324-1330 (1989). The number of cells bound was derived from a standard curve of myeloperoxidase activity versus numbers of cells. Under all assay conditions, the cells released less than 5% of total myeloperoxidase and lactate dehydrogenase. Results are presented in Table I. 100% adhesion is that seen in the presence of the C-terminal peptide (amino acid residues 761-777 of GMP-140) as a negative control; this value is the same as controls in which neither peptide nor antibody is added to the cells. Inhibition is read as a lower percent adhesion, so that a value of 5% mean that 95% of the specific adhesion was inhibited.
None of the peptides from the EGF domain of GMP-140 inhibited adhesion. However, peptides from three noncontiguous regions of the lectin domain inhibited adhesion. The three regions from the lectin domain are from amino acid 19 to 34, amino acid 54 to 72, amino acid 73 to 89, and an overlapping peptide of amino acids 66-78. Amino acids are numbered based on the number of the residues contained in the peptide, with residue 1 defined as the N terminus of the mature protein after cleavage of the signal peptide.
Currently, the shortest peptide sequences derived from these sequences that are known to have activity range from eight to thirteen amino acids, varying somewhat depending on the area of the lectin domain from it was derived. Some of the shorter peptides have more activity than the longer peptide sequences. The shortest active peptide characterized at this time is lectin domain amino acids 23 to 30, derived from lectin domain amino acids 19 to 34. This peptide is identical among GMP-140, ELAM-1, and the homing receptor with the exception of a single amino acid difference in ELAM-1, and is therefore expected to inhibit cell-cell contacts mediated by all three selectins. The shortest active peptide derived from lectin domain amino acids 54 to 72 known at this time is from amino acid 54 to 63. The shortest active peptide derived from lectin domain amino acids 78-89 known at this time is from amino acids 73 to 83. In addition, an overlapping peptide, spanning amino acids 66 to 78, is very active. It may be possible to design two active shorter, non-overlapping peptides from the region spanning amino acids 66 to 83.
As shown in Table I, some of these regions are more highly conserved among the selectins than others. As a result, it is possible to use peptides derived from the highly conserved regions to modulate interactions involving all of the selectins and peptides from the regions that are less conserved among the selectins to modulate interactions only involving GMP-140. For example, the central core of lectin region 19-34 (residues 23-30) is extraordinarily conserved among the three molecules. In contrast, the amino acid sequence 54 to 60, derived from lectin region 54 to 72, has a number of differences among the selectins.
TABLE I__________________________________________________________________________Comparison of Lectin Domains of GMP-140,ELAM-1, and LEU-8 and Percentage ofNeutrophils Bound to GMP-140 in thePresence of GMP-140-derived peptides.Protein/Peptide Amino Acids % Bounda__________________________________________________________________________ 19 34GMP-140 CQ NRYTDLVAIQ NKNEELAM-1 CQ QRYTHLVAIQ NKEELEU-8 CR DNYTDLVAIQ NKAECONSENSUS Cq .rYTdLVAIQ NK.EGMP-140 peptides19-34 5% 19-26 92% 27-34 91% 19-30 12% 23-34 30% 21-30 20% 22-30 6% 23-30 7% 21-29 71% 21-28 85% 21-27 65% 54 72 GMP-140 RKNNKTW TWVGTKKALT NE ELAM-1 RKVNNVW VWVGTQKPLT EE LEU-8 RKIGGIW TWVGTNKSLT EE CONSENSUS RK.n..w tWVGT.K.LT eEGMP-140 peptides54-72 15% 54-63 13% 64-72 92% 54-58 108% 59-63 102% 54-60 63% 57-63 69% 55-60 70% 56-60 96% 54-59 86% 66 73 89 GMP-140 KKALT ENAENWADNE PNNKRNNED ELAM-1 QKPLT EEAKNWAPGE PNNRQKDED LEU-8 NKSLT EEAENWGDGE PNNKKNKED CONSENSUS .K.LT eEAeNWadgE PNNk.n.EDGMP-140 peptides73-89 22% 73-80 100% 81-89 104% 73-85 41% 77-89 68% 73-83 17% 66-78 6%__________________________________________________________________________ a THe number of cells bound to immobilized GMP140 was determined as described in Geng, et al., Nature 343:757-760 (1990). The number of cells bound in the presence of the control Cterminal peptide (residues 761777) was normalized to 100%. This value was identical to that observed in the absence of peptide. Peptide inhibition of adhesion is indicated by an adhesion value significantly less than 100%; for example, a value of 5% indicates 95% inhibition of adhesion seen in cont rols. All peptides were added to a final concentration of 1.5 mM.
Example 1 demonstrates that peptides from three regions of the lectin domain of GMP-140 inhibit binding of neutrophils to GMP-140 immobilized on a surface. A study was also done to determine whether the peptides would also inhibit binding of monoclonal antibodies to the immobilized GMP-140.
Three monoclonal antibodies (mAb) that block adhesion of neutrophils to GMP-140 were developed, designated G1, G2, and G3. Based on competitive ELISAs with the purified protein, G1, G2, and G3 each recognize distinct or only partially overlapping epitopes. 1.5 mM peptide was added to biotinylated mAb at a concentration of 2.5 micrograms/ml, which were then added to the wells containing GMP-140, as described in example 1. Binding was measured by an ELISA with an avidin detection system.
The monoclonal antibodies were biotinylated as follows: to 0.5 ml of purified IgG antibody (1 mg/ml in PBS, pH 7.4) was added 50 μl of 3.2 mM N-hydroxysuccinimide biotin in dimethyl sulfoxide and 50 μl of 1 M NaHCO3. After a 2 hour incubation at room temperature in the dark, the reaction was stopped with 50 μl of 1 M NH4 Cl. The biotinylated antibody was then separated from other components by gel filtration on a PD-10 column equilibrated in PBS. The ELISA was performed as follows; all steps were performed at room temperature.
Biotinylated antibody (2.5 μg/ml) with or without 1.5 mM peptide was incubated with wells coated with GMP-140 as described in example 1. After a 2 hour incubation, the antibody was removed, the wells were washed, and 0.1 ml of horseradish peroxidase-conjugated stepavidin (Pierce), diluted 1:1000 in HBSS/HSA, was added for 30 min. The wells were then washed and 0.1 ml of peroxidase substrate (Pierce) was added for 15 min. The color reaction was read at 405 nm.
The results are shown in Table 2.
TABLE 2______________________________________Effect of soluble peptides on binding ofbiotinylated monoclonal antibodies to immobilized GMP-140. % Binding of Biotinylated MAbsbPEPTIDESa G1 G2 G3c______________________________________LECTIN 19-34 23 26 24LECTIN 19-30 54LECTIN 54-72 106 125 104LECTIN 73-89 95 99 94LECTIN 91-108 97 93 96LECTIN 110-116 98 100 108LECTIN/EGF 118-126 92 92 104EGF 128-132 107 92 104EGF 134-141 97 104 109EGF 145-158 94 92 108REPEATS 544-556 107 96 111TRANSMEMBRANE 755-761 100 119 104SECRETORY 720-721/762-765 106 99 104C-TERMINUS 761-777 100 100 100______________________________________ a The peptide (1.5 mM) was added to the mAb (2.5 micrograms/ml) prio to addition of the solution to the microtiter well containing immobilized GMP140. b Binding of the biotinylated mAb G1, G2, or G3 to immobilized GMP14 was measured by ELISA with an avidin detection system. c G1, G2, and G3 are antibodies binding to GMP140 that prevent neutrophil adhesion to immobilized GMP140.
The ability of the lectin 19-34 peptide to prevent binding to GMP-140 of all three monoclonal antibodies that block interactions of GMP-140 with leukocytes provides additional proof of the importance of the lectin domain in leukocyte recognition. It is postulated from this data that the conformation of the lectin domain is modulated by interactions with the EGF domain; these interactions in turn are modulated by divalent cations, which may bind to both the lectin and EGF domains. The result is a three-dimensional conformation of the lectin domain that confers affinity and specificity of binding to its receptor(s) on neutrophils and monocytes.
This receptor is believed to be a glycoprotein containing sialic acid and other oligosaccharides that are recognized by the lectin domain of GMP-140. Using the assay described in example 1, and an assay in which 125 I[GMP-140] binds specifically, saturably and reversibly to human neutrophils, as described by Moore and McEver, Clin.Res. 38, 364A (1990), certain critical structural features of the ligand (or, perhaps more accurately, "counter-receptor") for GMP-140 on human neutrophils have been defined.
Neutrophils were treated with proteases, either trypsin or elastase, to determine if the receptor contains a protease-sensitive protein component. Greater than 70% of the adhesion of neutrophils to immobilized GMP-140 was abolished by the proteases, but not by elastase or trypsin inactivated with diisopropylfluorophosphate, indicating that at least a substantial fraction of the leukocyte counter-receptors for GMP-140 contain a protease-sensitive protein component.
Treatment of neutrophils with neuraminidase purified from Vibrio cholera abolished both binding of 125 [GMP-140] to human neutrophils and adhesion of neutrophils to immobilized GMP-140, indicating that sialic acid residues constitute an essential component of the leukocyte counter-receptor(s) for GMP-140.
A monoclonal antibody to the Lewis X antigen, Lacto-N-fucopentaose III, "LNF-III", blocked binding of 125 [GMP-140] to human neutrophils in a dose-dependent fashion, indicating that LNF-III, a repeating structure found in polylactosaminoglycans of neutrophils and monocytes, is a component of the leukocyte counter-receptor(s) for GMP-140.
These results indicate that the counter-receptor, or ligand, on leukocytes for GMP-140 is a glycoprotein wherein a combination of sialic acid and LNF-III are required for receptor function. The predominant polyfucosylated lactosaminoglycan on human granulocytes is a tretraantennary structure. As described by Spooncer, et al., J. Biol. Chem. 259, 4792-4801 (1984), this structure contains four linear polylactosaminoglycan chains attached to core mannose residues of complex oligosaccharides, which are attached to asparagine residues of core proteins. Only one of these four chains is modified by both fucose and sialic acid, as reported by Fukuda, et al., J. Biol. Chem. 259, 10925-10935 (1984). This chain is linked by a β→6 linkage to the core mannose. This chain is terminated by a sialic acid linked α2→3 to a galactose which is part of an LNF-III unit. As shown by Fukuda, et al., (1984), the structure is: ##STR1##
It has not been determined whether the sialyated form of LNF-III operates within the context of the larger tetraantennary oligosaccharide, which can have minor variations, or the combination within the context of a core protein. Since fucosylated polylactosaminoglycans are also found on glycolipids of neutrophils and monocytes, as reported by Spooncer, et al., (1984), and proteases do not block all GMP-140 binding, it is possible that some of the GMP-140 may be interacting with oligosaccharides that form part of glycolipids. Conversely, a role for direct protein-protein interactions between GMP-140 and protein components of the glycoprotein receptor cannot be excluded.
The three peptide regions of the lectin domain interact with the leukocyte counter-receptor. When presented in the three-dimensional context of the lectin domain of the native protein, the three regions bind cooperatively with precise specificity and high affinity to the appropriate receptor. However, each region is sufficient to competitively inhibit leukocyte binding to GMP-140 alone. As a result, it is possible to use peptides containing one or more of these regions, alone or in combination with additional or substituted amino acids, to modulate binding of leukocytes, platelets and endothelial cells via GMP-140.
Peptides from the lectin-like domain of GMP-140 were assayed for their ability to inhibit adhesion of neutrophils to immobilized GMP-140 in the assay described in example 1. Concentrations tested ranged from 0.1 mM to 1.5 mM.
The results for four peptides are shown in FIG. 3. It is apparent that the peptides from the GMP-140 lectin domain, amino acids 66-78, amino acids 73-83, amino acids 54-63, and amino acids 23-30, inhibited binding in a dose-dependent manner. The IC50, the dose of peptide required to inhibit adhesion by 50%, ranges from about 50 μM to about 300 μM, depending on the peptides. These ranges are well within the effective concentrations for the in vivo administration of peptides, based on comparison with the RGD-containing peptides, described, for example, in U.S. Pat. No. 4,792,525 to Ruoslaghti, et al., used in vivo to alter cell attachment and phagocytosis.
In some cases, modification of the peptides by alteration of the amino acids themselves or by attachment to a carrier molecule is required to increase half-life of the molecule in vivo.
Lectin peptide 19-34 was conjugated to the carrier protein keyhole limpet hemocyanin by its N-terminal cysteine by standard procedures such as the commercial Imject kit from Pierce Chemicals. This peptide-KLH conjugate was then tested in the assay described in example 1 and the numbers of cells bound determined.
FIG. 4A and B are comparisons of the specific adhesion of neutrophils to microtiter wells coated with no peptide (1); coated with lectin domain peptide 19-34 conjugated to KHL (2); or coated with a control carboxyterminus peptide (amino acid residues 761-777) conjugated to KLH (3), blocked with Hank's Balanced Salt Solution containing human serum albumin prior to addition of 2�105 neutrophils to each well, in the presence of fluid-phase competitors. Fluid-phase competitors added to the neutrophils prior to transfer to the wells were none (control), purified platelet glycoprotein IIb-IIIa (control), or purified GMP-140 (Panel A); or none (control), 1.5 mM C-terminal peptide 761-777 (control), 1.5 mM lectin domain peptide 19-34 (Panel B).
It is apparent from the graphs that the lectin peptide-KHL conjugate, when immobilized on plastic, directly supports neutrophil adhesion, although not as efficiently as native GMP-140. For purposes of comparison, when 200,000 neutrophils are added to a
well, the lectin peptide 19-34 binds 40,000 and GMP-140 binds 120,000. The peptide is coupled to KLH simply to facilitate its coating to plastic. The ability to compete for adhesion with fluid-phase purified GMP-140 and the lectin peptide 19-134, but not with other proteins or peptides, shows that the adhesion is specific. In other studies, a large number of peptides from other regions of the lectin domain, the EGF domain, and scattered regions of GMP-140 do not inhibit neutrophil adhesion to immobilized GMP-140.
The peptides described above have a variety of applications as diagnostic reagents and, potentially, in the treatment of numerous inflammatory disorders.
The GMP-140 binding peptides can also be used for the detection of human disorders in which GMP-140 ligands might be defective. Such disorders would most likely be seen in patients with increased susceptibility to infections in which leukocytes might not be able to bind to activated platelets or endothelium. Cells to be tested, usually leukocytes, are collected by standard medically approved techniques and screened. Detection systems include ELISA procedures, binding of radiolabeled antibody to immobilized activated cells, flow cytometry, or other methods known to those skilled in the arts. Inhibition of binding in the presence and absence of the lectin domain peptides can be used to detect defects or alterations in selectin binding. Such disorders would most likely be seen in patients with increased susceptibility to infections in which leukocytes would have defective binding to platelets and endothelium because of deficient leukocyte ligands for GMP-140. The GMP-140 peptide is labeled radioactively, with a fluorescent tag, enzymatically, or with electron dense material such as gold for electron microscopy. The cells to be examined, usually leukocytes, are incubated with the labeled GMP-140 peptides and binding assessed by methods described above with antibodies to GMP-140, or by other methods known to those skilled in the art. If ligands for GMP-140 are also found in the plasma, they can also be measured with standard ELISA or radioimmunoassay procedures, using labeled GMP-140 peptide instead of antibody as the detecting reagent.
A similar approach can be used to determine qualitative or quantitative disorders of GMP-140 itself. In this case, sialyated LNF-III oligosaccharides, or appropriate derivatives thereof, are labeled and tested for their ability to bind to GMP-140 on activated platelets from patients with disorders in which GMP-140 might be defective.
Since GMP-140 has several functions related to leukocyte adherence, inflammation, and coagulation, clinically, compounds which interfere with binding of GMP-140 and/or the other selectins, including ELAM-1 and LEU-8, such as the GMP-140 peptides, can be used to modulate these responses.
For example, GMP-140 peptides can be used to competitively inhibit leukocyte adherence by competitively binding to GMP-140 receptors on the surface of leukocytes. This kind of therapy would be particularly useful in acute situations where effective, but transient, inhibition of leukocyte-mediated inflammation is desirable. Chronic therapy by infusion of GMP-140 peptides may also be feasible in some circumstances.
Since GMP-140 is believed to stabilize irreversible platelet aggregates after initial cell-cell contact is initiated by binding of adhesive proteins to GPIIb-IIIa, inhibition of this interaction could enhance the ability of competitive antagonists of the GPIIb-IIIa-adhesive protein interaction to block platelet aggregation. An example of a clinical situation where this might be useful would be in the respiratory distress syndrome, tumor metastasis, rheumatoid arthritis and atherosclerosis.
Reperfusion injury is a major problem in clinical cardiology. Therapeutic agents that reduce leukocyte adherence in ischemic myocardium can significantly enhance the therapeutic efficacy of thrombolytic agents. Thrombolytic therapy with agents such as tissue plasminogen activator or streptokinase can relieve coronary artery obstruction in many patients with severe myocardial ischemia prior to irreversible myocardial cell death. However, many such patients still suffer myocardial neurosis despite restoration of blood flow. This "reperfusion injury" is known to be associated with adherence of leukocytes to vascular endothelium in the ischemic zone, presumably in part because of activation of platelets and endothelium by thrombin and cytokines that makes them adhesive for leukocytes (Romson et al., Circulation 67: 1016-1023, 1983). These adherent leukocytes can migrate through the endothelium and destroy ischemic myocardium just as it is being rescued by restoration of blood flow.
There are a number of other common clinical disorders in which ischemia and reperfusion results in organ injury mediated by adherence of leukocytes to vascular surfaces, including strokes; mesenteric and peripheral vascular disease; organ transplantation; and circulatory shock (in this case many organs might be damaged following restoration of blood flow).
Bacterial sepsis and disseminated intravascular coagulation often exist concurrently in critically ill patients. They are associated with generation of thrombin, cytokines, and other inflammatory mediators, activation of platelets and endothelium, and adherence of leukocytes and aggregation of platelets throughout the vascular system. Leukocyte-dependent organ damage is an important feature of these conditions.
Adult respiratory distress syndrome is a devastating pulmonary disorder occurring in patients with sepsis or following trauma, which is associated with widespread adherence and aggregation of leukocytes in the pulmonary circulation. This leads to extravasation of large amounts of plasma into the lungs and destruction of lung tissue, both mediated in large part by leukocyte products.
Two related pulmonary disorders that are often fatal are in immunosuppressed patients undergoing allogeneic bone marrow transplantation and in cancer patients suffering from complications that arise from generalized vascular leakage resulting from treatment with interleukin-2 treated LAK cells (lymphokine-activated lymphocytes). LAK cells are known to adhere to vascular walls and release products that are presumably toxic to endothelium. Although the mechanism by which LAK cells adhere to endothelium is not known, such cells could potentially release molecules that activate endothelium and then bind to endothelium by mechanisms similar to those operative in neutrophils.
Tumor cells from many malignancies (including carcinomas, lymphomas, and sarcomas) can metastasize to distant sites through the vasculature. The mechanisms for adhesion of tumor cells to endothelium and their subsequent migration are not well understood, but may be similar to those of leukocytes in at least some cases. The association of platelets with metastasizing tumor cells has been well described, suggesting a role for platelets in the spread of some cancers.
Platelet-leukocyte interactions are believed to be important in atherosclerosis. Platelets might have a role in recruitment of monocytes into atherosclerotic plaques; the accumulation of monocytes is known to be one of the earliest detectable events during atherogenesis. Rupture of a fully developed plaque may not only lead to platelet deposition and activation and the promotion of thrombus formation, but also the early recruitment of neutrophils to an area of ischemia.
Another area of potential application is in the treatment of rheumatoid arthritis.
In these clinical applications, the peptide, or mixture of peptides, in an appropriate pharmaceutical carrier, is preferably administered intravenously where immediate relief is required. The peptide(s) can also be administered intramuscularly, intraperitoneally, subcutaneously, orally, as the peptide, conjugated to a carrier molecule, or in a drug delivery device. The peptides can additionally be modified chemically to increase their in vivo half-life.
The peptides can be prepared by proteolytic cleavage of GMP-140, or, preferably, by synthetic means such as those used to prepare the peptides in example 1. These methods are known to those skilled in the art. An example is the solid phase synthesis described by J. Merrifield, J. Am. Chem. Soc. 85, 2149 (1964), used in U.S. Pat. No. 4,792,525, and described in U.S. Pat. No. 4,244,946, wherein a protected alpha-amino acid is coupled to a suitable resin, to initiate synthesis of a peptide starting from the C-terminus of the peptide. Other methods of synthesis are described in U.S. Pat. Nos. 4,305,872 and 4,316,891. These methods can be used to synthesize peptides having identical sequence to GMP-140, or substitutions or additions of amino acids, which can be screened for activity as described in examples 1 to 3.
The peptide can also be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
Peptides containing cyclopropyl amino acids, or amino acids derivatized in a similar fashion, can also be used. These peptides retain their original activity but have increased half-lives in vivo. Methods known for modifying amino acids, and their use, are known to those skilled in the art, for example, as described in U.S. Pat. No. 4,629,784 to Stammer.
Peptides that are biologically active are those which inhibit binding of neutrophils and monocytes to GMP-140, or which inhibit leukocyte adhesion to endothelium that is mediated by ELAM-1 and/or the homing receptor. Sialyated LNF-III oligosaccharides that are active can be screened in a similar manner.
Suitable pharmaceutical vehicles are also known to those skilled in the art. For parenteral administration, the peptide will usually be dissolved or suspended in sterile water or saline. For enteral administration, the peptide will be incorporated into an inert carrier in tablet, liquid, or capsular form. Suitable carriers may be starches or sugars and include lubricants, flavorings, binders, and other materials of the same nature. The GMP-140 peptide can also be administered locally at a wound or inflammatory site by topical application of a solution or cream.
Alternatively, the peptide may be administered in liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to a patient are known to those skilled in the art. U.S. Pat. No. 4,789,734 describe methods for encapsulating biological materials in liposomes. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary. A good review of known methods is by G. Gregoriadis, Chapter 14. "Liposomes", Drug Carriers in Biology and Medicine pp. 287-341 (Academic Press, 1979). Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the bloodstream. Alternatively, the peptide can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time, ranging from days to months. See, for example, U.S. Pat. Nos. 4,906,474, 4,925,673, and 3,625,214.
The subject peptides are generally active when administered parenterally in amounts above about 1 μg/kg of body weight. For treatment of most inflammatory disorders, the dosage range will be between 0.1 to 30 mg/kg of body weight. A dosage of 70 mg/kg may be required for some of the peptides characterized in the examples. This dosage will be dependent, in part, on whether one or more peptides are administered. As discussed with respect to binding of the three regions of the lectin domain, a synergistic effect may be seen with combinations of peptides from different, or overlapping, regions of the lectin domain, or in combination with peptides derived from the EGF domain of GMP-140.
The peptides can also be coated onto substrates for use as prosthetics that are implanted into the body to prevent leukocyte adhesion to platelets or endothelium.
The criteria for assessing response to therapeutic modalities employing antibodies to GMP-140 is dictated by the specific condition and will generally follow standard medical practices. For example, the criteria for the effective dosage to prevent extension of myocardial infarction would be determined by one skilled in the art by looking at marker enzymes of myocardial necrosis in the plasma, by monitoring the electrocardiogram, vital signs, and clinical response. For treatment of acute respiratory distress syndrome, one would examine improvements in arterial oxygen, resolution of pulmonary infiltrates, and clinical improvement as measured by lessened dyspnea and tachypnea. For treatment of patients in shock (low blood pressure), the effective dosage would be based on the clinical response and specific measurements of function of vital organs such as the liver and kidney following restoration of blood pressure. Neurologic function would be monitored in patients with stroke. Specific tests are used to monitor the functioning of transplanted organs; for example, serum creatinine, urine flow, and serum electrolytes in patients undergoing kidney transplantation.
Modifications and variations of the present invention, methods for modulating binding reactions involving GMP-140, will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the appended claims.
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Johnson -- Sutherland
Evangeline Johnson and Brandon Sutherland, both of Spicer, are engaged to be married July 29 at Carlos Creek Winery in Alexandria.
Johnson is the daughter of Amy and Steve Combs of New London and Joel and Julie Johnson of New London.
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text: Bing teams with Klout to enhance social search Published on October 01, 2012. 0 Reprints Reprints Ad Age LookBook Weekly Feature Get Help Organizing Your Mobile Data and Start Tracking What's Working Learn more Redmond, Wash.—Microsoft Corp.'s Bing search engine has formed a partnership with social analytics company Klout Inc. to highlight the social influence of contacts found through search queries on both platforms. According to the Bing blog, Klout social influencer data, scores and topics these influencers specialize in will appear in Bing's People Who Know sidebar. In return, Bing will provide Klout with additional data to augment its own search function, including how often a person shows up in Bing's sidebar. In this article: BtoB Advertisement Read These Next Coming Soon: Jim Beam Apple E.J. Schultz on 03.31.2015 16 0 Newell's Design Chief Leaves in 'Mutual Decision,' Company Says Jack Neff on 03.31.2015 5 0 Tumblr's First CMO Stephanie Dolgins Will Also Oversee Its Media Deals Tim Peterson on 03.30.2015 121 0 Comments (0)


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CHARLESTON, W.Va. (AP) — The Associated Press decided not to cover the West Virginia gubernatorial debate Tuesday because of restrictions placed on media coverage by its organizers.
The debate's sponsors — the West Virginia Broadcasters Association and AARP — barred still photographers and reporters from covering the debate in the same room as the candidates, allowing only video coverage of the event.
The organization said in an advisory it sent to journalists that it would allow only one still photographer before the debate began, but not during the hourlong debate. No reporters were allowed into the theater before or during the debate at the Clay Center for the Arts and Sciences. The debate was held in a theater that seats up to 200 people invited by the sponsors.
The broadcasters association did not respond to several calls and emails from the AP.
"There is no reason a small pool of reporters and photographers could not be accommodated in the hall during this important event," said AP Senior Managing Editor Michael Oreskes. "This is the one opportunity the people of West Virginia have to watch the two candidates engage. We need to offer as many perspectives as possible to capture the event and the candidates' positions accurately and with fairness and balance. If organizers of presidential debates can figure out how to do it, the organizers of this debate could, too."
Some other West Virginia newspapers, including The Dominion Post of Morgantown, joined the AP in refusing to cover the debate.
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Repairing Knothole Crack In Floor Joist
While opening the ceiling in our garage to install Uponor Joist Trak under the subfloor of a bathroom above I noticed that a crack had developed in a 2x12 joist knothole. The unsupported joist span of the 2x12 is 19' and the ends rest on 2x6 plates. The crack is located 60" (5') from the wall. The crack extends up 3-1/2" on one side of the joist and 2" on the other side. There is no sagging of the joist and the crack does not extend past the knothole.
I was thinking of sistering two 2x12 boards, one on either side of the joist, that extend 300% of the depth of the 2x12, extending out from both sides of the crack. That would be 3 x 11-1/4 = 33.75" x 2 = 67.5". So the repair 2x12's would be 67.5" long and be centered on the crack. I would apply construction adhesive to the mating surfaces and secure the sandwich together with 1/4" Simpson SDS screws or 5/16" GRK structural screws. How does this plan sound? What schedule (pattern) is recommended for the screws?
Thanks for taking the time to read my post and consider my questions.
Use 3/8" bolts instead of screws. And one board should be sufficient. Two is a bit much, but nothing wrong with doing two.
Also, use a lally column(the kind used in basements that have a screw adjustment on the top) to push up that joist just a bit before adding the repair.
Bolting pattern would be one on either side of the crack, about 6" away and about 4" up from the bottom. Two more about 4" each side of the crack and about 3&1/2" down from the top.
Then a bolt every 18" to each end, being careful to not place them on the same level as the neighboring bolts.
Don't remove the lally column for a couple days to let the construction adhesive to set well.
Your only problem is that a very small area of the bathroom floor might deflect slightly more than the rest of the floor and cause the tile to crack even though it has apparently not done so yet. I would put a long 2x4 against the lower side of the joist and put as many 10d nails in it as I could find.
I would also reinforce the other ones with low knots since they have little more capacity than the cracked one.
Sister with structural grade fir (no knots in vulnerable areas) with as much length as possible; best if one end had bearing, then jack the free end into place while you bolt them together. Use a glue if you wish, one that has minimal "creep" factor.
This floor is over designed for bending by a large factor so the crack doesn't reduce the load carrying capacity below what the code requires but it does allow that one joist to deflect more than the others when subjected to a very heavy load which is highly unlikely to occur in a bathroom.
It should be reinforced if you plan to add a whirlpool spa or use very large ceramic tiles.
I decided to contact a structural engineer to make sure my plan would meet code. He did the calculations and said my repair would meet code with his schedule for the 1/4" x 3" Simpson SDS screws. I installed the repair pieces yesterday and now I have piece of mind knowing I don't have to worry about further deterioration of the joists.
Thanks everyone for your comments.
"He did the calculations and said my repair would meet code..."
"meeting code" os often not adequate for supporting tile floors.
The code allow a deflection of 1/360 of span.
Adequate for plaster and drywall (at solid wood spans) but not for anything larger than mosaic tile.
For 4-6 inch tiles, 1/480 is needed, and for stone or tiles above about 6 inches 1/720 is needed to prevent grout and tile cracking.
The I-joist manufacturers ran into trouble years ago by fabricating I-joists with spans of 14 feet and higher (rare for solid wood).
The allowed deflection of almost 1/2 inch resulted in 'bouncy' floors and unhappy customers (while still being structurally adequate to code).
Hi Brickeyee's,
His calculations are based on adding a 1" mortar bed with 3/8" tile. Our neighbors in the adjoining townhouse with the same floor plan did the same addition to their bathroom 16 years ago and it has held up perfectly. BTW, our floors are solid and not bouncy.
The building code should never be a standard or a goal for remedial work; it is a minimum standard for new construction and in the matter of acceptable deflection for large sized ceramic tile it should always be exceeded as has been pointed out already. I'm surprised an engineer would even mention the building code.


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Dilettos, the sprawling Italian restaurant formerly known as Papa Razzi, has a new chef, but shows little change on the menu. Located in Westbury, this bustling restaurant has plenty of delicious choices -- certain dishes will make you want to come back for more.
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UI Design: Applying CSS Based on Screen Orientation
By Thoriq Firdaus. Filed in Coding, UI/UX, Web Design
With the introduction of CSS3 Media Queries, we are able to shift and apply CSS between different viewport or device screen sizes.
We can detect the viewport width threshold, which we want the style rules to be applied to, with the
min-width or
max-width declaration within the Media Queries, as we have shown in our Responsive Design Series.
Device Orientation
Mobile devices can be operated with two orientations, Portrait and Landscape. In particular cases, we might also need to apply these styles based on the devices orientation apart from the device viewport width. To serve different stylesheets based upon screen and orientation declaration in the stylesheet link:
<link rel="stylesheet" media="all and (orientation:portrait)" href="portrait.css"> <link rel="stylesheet" media="all and (orientation:landscape)" href="landscape.css">
This will apply the styles for iPad, iPhone, Android, Safari, and Firefox. Alternatively, we can also add the orientation declaration within the Media Queries, like so.
@media all and (orientation:portrait) { /* Styles for Portrait screen */ } @media all and (orientation:landscape) { /* Styles for Landscape screen */ }
As W3C described, regardless of the device type (mobile or desktop), when the viewport height is larger than the width, the orientation is described as
portrait. Conversely, it is described as
landscape when the width viewport size is larger than the height.
Below are some of the test preview in iPhone, iPad and Desktop with Landscape and Protrait orientation.
iPhone (Portrait)
iPhone (Landscape)
iPad (Portrait)
iPad (Landscape)
Desktop (Portrait)
Desktop (Landscape)
You can see for yourselves the demo in action and download the source from the following links. Resize the browser window or turn your device screen to see different results as shown in the screenshots above.
Further Resource
- iPad Orientation CSS — Cloud Four
- CSS3 Media Queries — W3C
Author: Thoriq Firdaus
Thoriq is a writer for Hongkiat.com with a passion for web design and development. He is the author of Responsive Web Design by Examples, where he covered his best approaches in developing responsive websites quickly with a framework.


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Sprinkle gelatin over cold water in mixing bowl; set aside for 10 minutes to soften.
Add boiling water and stir until gelatin is completely dissolved, and add sugar replacement, lemon juice and rind.
Stir to thoroughly blend.
Cool until mixture is consistency of unbeaten egg whites.
With an electric beater, whip lemon mixture until frothy.
Fold beaten egg whites into lemon mixture.
Spoon into 6 serving dishes; refrigerate until set.


text:
Accessibility tools
Service tools
Navigation path
In the coming decades, all EU countries will experience steep increases in the share of elderly persons in the total population and a significant decline in the share of young people and those of working age.
While longer lives are a major achievement of European societies, the ageing of the population also poses significant challenges for their economies and welfare systems. The demographic transition is considered to be one of the most important challenges facing the EU.
Evaluation of demographic change at micro and macro level
DG ECFIN's responsiblity is to carry out economic analysis and evaluation at both the micro and macro levels to contribute to an understanding of the real and potential impacts of demographic change; it also provides policy advice on possible responses to this change. As part of this, DG ECFIN monitors and analyses the performance of European welfare states, with a special focus on analysing and projecting the economic and budgetary implications of ageing populations.
The age-related expenditure projections cover pensions, long-term health care, education and unemployment transfers, and feed into a variety of policy debates at EU level. In particular, they are used in the annual assessment of the sustainability of public finances carried out as part of the Stability and Growth Pact; in the context of the Open-Method of Co-ordination on pensions, health care and social inclusion; and the analysis on the impact of ageing populations on the labour market and potential growth which is relevant for the Lisbon Strategy and Broad Economic Policy Guidelines.
Outputs include notes to the Economic and Financial Committee and the Economic Policy Committee and its working groups, analytical reports and publications, briefings, speeches and inputs to the Commission's work in general.
Related publications
>> Ageing Report 2012 Economic and budgetary projections for the 27 EU MS
>> Ageing Report 2012 Underlying Assumptions and Projections Methodologies
>> Sustainability Report 2009. European Economy 9/2009
>> EC-EPC Report on the economic and budgetary impact of ageing, statistical annexes and information on demographic and macroeconomic assumptions
>> The 2009 Ageing Report: Economic and budgetary projections for the EU-27 Member States (2008-2060). European Economy 2/2009
>> The 2009 Ageing Report: Underlying Assumptions and Projection Methodologies for the EU-27 Member States (2007-2060). European Economy 7/2008.
>> EPC Report - The impact of ageing on public expenditure: projections for the EU25 Member States on pensions, health care, longterm care, education and unemployment transfers (2004-2050). European Economy. Special Report 1/2006
>> EPC Report - The 2005 projections of age-related expenditure (2004–50) for the EU-25 Member States: underlying assumptions and projection methodologies. European Economy. Special Report 4/2005
>> Setting targets for government budgets in the pursuit of intergenerational equity. Economic Paper 358/2009
>> Public pension expenditure in the EPC and the European Commission projections: an analysis of the projection results. Economic Paper 268/2007
>> The economic impact of ageing populations in the EU-25 Member States. Economic Paper 236/2005
>> Long-term sustainability of public finances in the European Union. European Economy 4/2006
>> Actuarial neutrality across generations applied to public pensions under population ageing: effects on government finances and national saving. Economic Paper 230/2005
>> Population ageing and public finance targets. Economic Paper 196/2003
>> Pension reform: key issues illustrated with an actuarial model. Economic Paper 174/2002
>> A case for partial funding of pensions with an application to the EU Candidate Countries. Economic Paper 149/2001
>> The economic consequences of ageing populations. Economic Paper 138/1999
>> Generational accounting in Europa. European Economy. Reports and Studies. 6/1999


text:
Fedora 9 Released
The Fedora project, which is supported financially by Red Hat, has just released Fedora 9. The free distribution is based on kernel 2.6.25 and includes the KDE 4.0.3 and Gnome 2.22 desktops.
Xfce 4.4.2 completes the selection of desktops. Users will immediately notice the new look at the installation stage. The Anaconda installer can now resize partitions with Ext2/3 and NTFS filesystems, and is also capable of creating encrypted filesystems. The developers offer the Ext3 successor Ext4 as an optional filesystem.
NetworkManager 0.7 is enabled by default to handle connections to the outside world. Software management is handled by PackageKit which acts as a front-end for the underlying Yum tool.
Other packages on board with Fedora 9 include the free Java environment, OpenJDK 6, gcc 4.3.0, FreeIPA, Perl 5.10.0, MySQL 5.0.51a, PostgreSQL 8.3.0, and Xserver 1.5.
Popular applications such as Firefox 3 Beta 5 and Open Office 2.4 are also included. The Release Notes have a full list of new features.
Users who keep to the pre-upgrade approach should be able to update their existing Fedora systems to version 9 without installation media, after downloading the required files. Otherwise, Fedora 9 is available for various systems and with various download options from the following website.
Issue 174/2015
Buy this issue as a PDF
Microsoft Frees Code for .NET Build Engine
MSBuild is now just another GitHub project as Redmond continues its path to the light.
Researchers Demonstrate Hack by Heat
Malware could pass data and commands between disconnected computers without leaving a trace on the network.
Kernel Developers Adopt Code of Conflict
New rules emphasize collegiality in coding.
Ubuntu Switches to systemd
Upstart lands in the dust bin as a new era begins for Linux.
Old Vulnerabilities Are Kept Alive Through Bad Configuration
HP's annual Cyber Risk report offers a bleak look at the state of IT.
Linus Torvalds Announces Linux 4.0
But what do the big numbers really mean?
Microsoft Frees CoreCLR
.NET Core execution engine is the basis for cross-platform .NET implementations.
New Trojan Attacks Linux Servers
The Xnote trojan hides itself on the target system and will launch a variety of attacks on command.
Cisco Releases Annual Security Report
Spammers go low-volume, and 90% of IE browsers are unpatched.
Zero Day Exploits Target Flash
Adobe scrambles to release patches for vulnerable Flash Player.


text:
I'm a perl programmer doing a bit of C#. Facing an odd issue with Regex.Replace in regard to the zero-or-more assertion, *.
Say I wanted to replace zero or more letters with a single letter. In perl, I could do this:
my $s = "A";
$s =~ s/\w*/B/;
print $s;
$s now = "B"
But if I try and do the same in C#, like this:
string s = Regex.Replace("A", @"\w*", "B");
s now = "BB"
The docs do say "The * character is not recognized as a metacharacter within a replacement pattern"
Why? And is there any work around if you want a bit of your regex to slurp up some left over string which may not be there (like ".*?" on the end)
(this is a silly example, but you get the point)


text:
Kukla's Korner Hockey
Theodore Starts Tonight
by Paul on 10/12/07 at 11:27 AM ET
Comments (0)
from the Rocky Mountain News,
Six weeks after undergoing arthroscopic knee surgery, Avalanche goalie Jose Theodore will make his regular- season debut tonight.
He will start against the St. Louis Blues at the Scottrade Center in a game that could set the tone for the kind of season it will be for him and for the Avalanche.
"It's good to be back in," he said Thursday. "It was a long rehab, but it's fun to get the start. I'm anxious to get out there and help the team."
Filed in: NHL Teams, Colorado Avalanche, | KK Hockey | Permalink
Tags: colorado+avalanche, goalies, jose+theodore
Be the first to comment.
Add a Comment
Please limit embedded image or media size to 575 pixels wide.
Add your own avatar by joining Kukla's Korner, or logging in and uploading one in your member control panel.
Captchas bug you? Join KK or log in and you won't have to bother.
Most Recent Blog Posts
KK- How To Help Kukla's Korner
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Using Advanced Stats In The Scouting Process
Auston Matthews Another Example Of Hockey's Growth
Tom Wilson Fined For Diving/Embellishment
Make Or Break Road Trip Starts For The Calgary Flames
About Kukla's Korner Hockey
Paul Kukla founded Kukla's Korner in 2005 and the site has since become the must-read site on the 'net for all the latest happenings around the NHL.
From breaking news to in-depth stories around the league, KK Hockey is updated with fresh stories all day long and will bring you the latest news as quickly as possible.
Email Paul anytime at firstname.lastname@example.com


text:
Hang tight!
Recipe by Judy
"Mild, slightly sweet with taste of sesame seeds."
Hmm. None of these ingredients are on sale today.
Show ingredients on sale
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Save money at local stores when ingredients are on sale!
Watch video tips and tricks
1/2 cup
1/4 cup
white sugar
1/4 cup
packed brown sugar
1 teaspoon
vanilla extract
2 cups
all-purpose flour
2 teaspoons
baking powder
1/4 teaspoon
1/4 cup
3/4 cup
sesame seeds
Simple flavor - just a hint of sweetness. Not bad, but the Texture is more like a scone or buiscut - not a "typical" cookie. They also get a lot bigger once cooked - i would have made them half the size.
1 Rating
* Percent Daily Values are based on a 2,000 calorie diet.
Sesame Seed Cookies II
Serving Size: 1/18 of a recipe
Servings Per Recipe: 18
Amount Per Serving
Calories: 163
Calories from Fat: 80
Bunny cakes, lamb cakes, chocolate eggs, and carrot cakes to nibble on.
Breakfast casseroles, drinks, fancy eggs, and brunch ideas for a sweet Easter event.
Delicious recipes, party ideas, and cooking tips! Get a year of Allrecipes magazine for $7.99!
A quick and easy way to bake cookies using a package of cake mix.
Become a cookie decorating artist!
Follow this simple recipe for perfect homemade cookies.


text:
| Share
Pronunciation: (di-spōz'), Strict Standards: Non-static method FenSites::linkTo() should not be called statically in /site/html/dictionary/dispose.html on line 73 [key]
—v., -posed, -pos•ing,
1. to give a tendency or inclination to; incline: His temperament disposed him to argue readily with people.
2. to put in a particular or the proper order or arrangement; adjust by arranging the parts.
3. to put in a particular or suitable place: The lamp was disposed on a table nearby.
4. to make fit or ready; prepare: Your words of cheer dispose me for the task.
1. to arrange or decide matters: to do as God disposes.
2. Obs.to make terms.
3. dispose of,
a. to deal with conclusively; settle.
b. to get rid of; discard.
c. to transfer or give away, as by gift or sale.
d. to do away with; destroy.
1. Archaic.disposition; habit.
2. Obs.arrangement; regulation; disposal.
Random House Unabridged Dictionary, Copyright © 1997, by Random House, Inc., on Infoplease.
See also:


text:
More Videos...
Matching #'s, 2.0 liter aluminum head injected 4cyl @ 135hp, 4-speed close
ratio trans, new mirror finish Taiga green paint, black vinyl bucket seat
interior, full factory gauges, new front suspension, new Mini lites w/
fresh hi speed radials, great sounding stainless Exhaust, detailed engine + trunk compartments,
freshly rebuilt fuel injection. A stunning and very rare car that can be
driven every day and vintage raced on Sunday.
1970 Chevrolet Shortbed Pickup FOR SALE
Freshly restored classic truck, fresh crate 350/300hp V-8, automatic trans,
12 bolt rear, great looking medium blue metallic paint, new grey cloth
interior, factory gauges, 15 Chevrolet wheels w/ BFG radial T/As, great
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stainless, show or drive.
1969 Chevrolet Camaro Z/28 FOR SALE flemings ultimate garage
Original X33 coded real Z28, double hump heads "010" bolt main Z28 engine,
rare matching #'s M21 close ratio 4-speed, original 12 bolt 4.10
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stripes, correct coded white bucket seat interior, factory console w/
console gauges, cowl induction hood with correct air cleaner, disc brakes,
Delco AM radio, front & rear spoilers, date coded glass, new chrome,
stainless + emblems, rally wheels w/ Goodyear Polyglas tires, very nice.
1969 Chevrolet Camaro Z/28 Cross Ram FOR SALE flemings ultimate garage
Ultra rare & genuine Z28, less than 500 miles on a complete nut & bolt
rotisserie restoration (look underneath this car), factory correct DZ coded
302 Chevrolet V-8 w/ Cross Ram intake and correct 2X4bbl Holley carbs,
correct 4-speed manual trans, original BU1009 coded 12 bolt posi rear
differential w/ 3.73 gears, factory correct chambered Exhaust, mirror finish Cortez silver paint w/
black stripes, brand new black bucket seat interior w/ factory console,
correct Hurst shifter and factory console gauges, 120mph speedo & 7Krpm
tach, power steering, power disc brakes, tilt wheel, correct rally wheels
w/ new BFG radial TA's, show detailed engine compartment & trunk, well
documented, original owners manual, recent $25K in update receipts on a
restored American Icon - Spectacular and Real Z28!
1969 Chevrolet Camaro Z/28 for sale with test drive, driving sounds, and walk through video
Real X33 coded factory original Z-28, nicely done restoration, enjoyed and
show driven, Chevrolet legendary small block, deep groove pulleys, correct
cowl induction air cleaner, rare Heavy Duty M21 4-speed trans, original GM
12 bolt posi rear diff, brand new modern air conditioning, brand new mirror
finish correct coded "65" Garnet red paint with cowl hood, white stripes
and callouts, Black houndstooth bucket seat interior, factory center
console w/ gauges plus factory 8,000 RPM tach, fast ratio Z/28 steering
box, power disc brakes, 15" rally wheels with correct Goodyear Polyglas
GT's, all new chrome and stainless, great sounding bumper exit dual Exhaust, detailed engine and trunk
compartments, true Z/28 with correct colors and great sound.
1969 Camaro Z 28 Silver
Nice Restored Aztec Silver Z/28 4 Speed. This car has been Sold. Go to
kumberamotors.com to look at others like it For Sale.
1969 Chevrolet Camaro Z28 RS DZ302 Crossram Dual Carb Muscle Car
Be sure to check us out on Facebook at:
We just got back from taking this baby for a spin and I thought it deserved
another look. This is a beautiful 1969 Camaro Z28 that's equipped with a
302c.i. 2x4BBL (Cross Ram) 290 H.P. (#s Matching) V8, 4 Speed Manual
M21-Muncie (#s Matching) Transmission, 12 Bolt Rear w/ 4.10 Posi-Trac,
Quick Ratio P. Steering (N44), P. 4 Wheel Disc Brakes (JL8), Tilt Wheel,
Factory AM Radio, Buckets/Console w/ Gauge Package, Correct GM Numbered
Winters Cross Ram Intake, GM Off-Road Cam Shaft, Original GM Cowl Induction
Hood, Front & Rear Spoilers, Hide-Away Headlights, Ralley Wheels, Rally
Sport Trim Package, Correct LeMans Blue Exterior (Paint Code 71-71),
Correct Blue Vinyl Interior (Trim Code-715), Very Correctly Detailed from
Top to Bottom, Very Fresh Nut & Bolt Rotisserie Restoration. X33 D80 Cowl
Coded Car. Fine Example of Bowtie Muscle! Very Correct & Desirable Classic
Z28 Rally Sport!! Thanks for watching and commenting, I appreciate it.
muscle car
collector vehicle
hot rod
rat rod
lead sled
show car
69 Camaro Girl - Beautiful car and Hot Girl!- FOR SALE!
This is a beautiful 1969 Camaro...Cortez Silver and Black
with Ghost stripes. One of a kind paint job and boasts a Chevrolet DZ-302
engine. The video is not the best of quality so you can not appreciate the
way this car sounds or looks but WOW! And the girl is pretty slick
too.....:-) I'm trying to sell it but you have never seen a car turn so
many heads! email: email@example.com
1969 Z/28 Camaro, Cortez Silver
No aluminum wheels or fuel injection on this one. It's all Old School.
SOLD!! 1969 Camaro Z28 X77 DZ 302 Car For Sale Test Drive!
Jeff Whitaker 301-816-1000
horsepower A
favorite in Norway, Sweden, Germany, Netherlands, Denmark, Canada,
Australia, New Zealand and the U.S.A.
1969 Camaro RS Z28
camaro 302 idle
another clip taken with a different camera, im way too close and the sound
is horrible but it shows the fast throttle response
Dyno ARAO 32v 302 cu in Bonneville record holder SB Chevy www.araoengineering.com
www.araoengineering.com ARAO 32v 302 cu in 208 MPH Bonneville record holder
SB Chevy engine, normally aspirated. Arao Engineering, Racing, Street
Racing, Racing Engines, Heads, Cylinder heads, 32v, 32valve, Russell Arao,
Russ Arao, Aluminum heads, Arao Racing Engines, 4v, 4valve, Performance
heads, 2valve heads, Chevy, Ford, Chevy small block, Chevy big block, Ford
small block, Ford Windsor, Ford Cleveland, LS, LSX, Warhawk, Records,
Speed, horsepower,
1969 Camaro Z28 JL8 Cross Ram
Lot S125 1969 Chevrolet Camaro Z/28 Coupe
JL8 Power 4-Wheel Discs, Crossram 302 4-Speed
Mecum Kissimmee High Performance Auction
January 24 - 26, 2008
This is a 302 cu in SBC Z28 engine in action...
I've borrowed this from 'pepsikatt'
It gives an idea of what the car should sound like.
Dodge Ram Crushes GT-R in 1/4-Mile Pass
Drag Racing Down Under — 1500HP VW
Wheelstand Wednesday — Compilation Video
All Show No Go Blown/Turbocharged 1969 Camaro
Which car is faster?
Similar 1/4 mile timeslips to browse:
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1968 Chevrolet Camaro rs: 6.150 @ 224.880
Shannon Jenkins, Engine: 748 Cheverolet, Tires: goodyear
1999 Chevrolet Camaro pro modified: 6.321 @ 232.780
Jordan S. Comilang, Engine: Kieth Black 527, Supercharger: Weiand 1471 Tires: Goodyear Eagles
1991 Chevrolet Camaro nitro coupe: 6.321 @ 225.670
tommy howes, Engine: blown 526, Supercharger: 1471
1996 Chevrolet Camaro : 6.440 @ 221.080
TJ Stewart,
1969 Chevrolet Camaro SS 641 Blown Pro: 6.740 @ 220.700
Nick D'Amico, Engine: 641 Blown Pro Kill, Supercharger: a bigger one Turbos: n/a
1997 Chevrolet Camaro Z28: 6.900 @ 196.000
jeff fleming, Engine: 819 CI, Supercharger: no Turbos: no
1990 Chevrolet Camaro IROC-Z28: 6.959 @ 195.620
Marvin Blaney, Engine: Blaney's Competition,
1979 Chevrolet Camaro Z 28: 7.025 @ 210.000
Zoltan Kohari, Engine: Pat Musi Dart Big M Block, Supercharger: 14-71 Little field Tires: 34.5/17.0-16
1968 Chevrolet Camaro SS: 7.050 @ 198.000
GREG CASON, Engine: 565,
1967 Chevrolet Camaro Pro-Street: 7.470 @ 192.000
Dale L Collins Jr, Engine: 540, Turbos: 106 Precision Tires: Mickey Thompson
1968 Chevrolet Camaro RS: 7.470 @ 179.000
Ken Staib, Engine: 532 in. big block, Tires: M.T. 33 x10.5W
2002 Chevrolet Camaro bickel: 7.520 @ 175.300
Steve Tiona, Engine: 588 cid, Tires: 17x15x33
1989 Chevrolet Camaro Z28: 7.561 @ 189.000
Steve Toppe, Engine: 615, Tires: 33.5x17
1967 Chevrolet Camaro ss632: 7.860 @ 165.800
Thomas Coffman, Engine: 632, Supercharger: none Turbos: none Tires: 33x17x15 goodyear
1992 Chevrolet Camaro : 7.936 @ 172.210
Todd, Engine: 548 BBC,
1978 Chevrolet Camaro Z28: 7.970 @ 176.000
Aldo Aka Junior, Engine: 572ci, Tires: Goodyear
1990 Chevrolet Camaro : 8.220 @ 165.000
JCE, Engine: 468 small block chevy, Tires: 10.5 W
1978 Chevrolet Camaro z-28: 8.280 @ 160.000
john begglry, Engine: 565, Tires: goodyear
1987 Chevrolet Camaro : 8.300 @ 165.000
Jamie Oxendine, Engine: 540cu chevy, Supercharger: none Turbos: none Tires: 33.5/15/15
©2015 DragTimes - Disclaimer - Contact Us


text:
Oceanside Palms
No availability found through our partners. Please contact the business directly or check some of our recommended alternatives.
How has this motel rated in the past?
- Meeting rooms/conference facilities
- Continental Breakfast
- Drycleaning onsite
- Wireless internet connection in public areas
- Refrigerator in room
- Microwave in room
Photos of Oceanside Palms
Travelers also recommend:
- More recommendations
TripAdvisor Reviews Oceanside Palms
Travel Blogs from Oceanside
We are now finishing up our first week at the beach house.
Weather is sunny and about mid-teens. There is a cool breeze off the ocean otherwise it would be a lot warmer. During the day shirt sleeves are fine but in the evening a fleece is definitely in order.
As mentioned earlier the house is small but functional. It is behind one row ...
Keeping Your Online Audience Serious
... gains in business productivity. He furthermore reduced company journey and travel expenses by introducing plus implementing various cooperation technologies.
His expertise includes company analysis; project management; management of global cross-matrix groups; systems anatomist and evaluation, architecture, prototyping and integration; technologies evaluation and evaluation; systems development; performance assessment; and administration of off-shore advancement.
Shamu, sun and surprisingly soaking rapids
... No! So we both fell asleep for an hour and woke up to scorching sun, and now a few red bits. But perfect. We ended the day with a 'posh meal' at the resort, it was delicious and the setting is breathtaking, the sun setting over the bay as we ate. A perfect end to a pretty amazing day. If today is anything to go by, we really are in for the holiday of a lifetime. Callum xx Ps. We will add photos with every entry so don't forget to ...
Cruisin' down the West Coast.....on a ship!
... good (we got 2 adjoining rooms to save our sanity) and the food decent, albeit repetitive. Most of the programs and shows were at night which was nice for the adults but not great for the kids. Stellar dining room staff (we loved Elena!) and friendly cabin stewards. The ports of call did not interest us so we hung out on the ship enjoying the peace and quiet. Seasickness was kept to a minimum (thank you Lord!), although ...
Livin' the dream
... br> Megan and Matt joined us after work at the beach for some frescoball, surfing, and catching up. The sunset was absolutely stunning, and the boys surfed until they couldn't see.
Life in Leucadia consisted of surfing, playing beach volleyball, reading on the beach, napping, ping pong tournaments, and cooking up some seriously good meals with our friends. We were having so much fun, we stayed for 10 days. This was definitely ...


text:
Flamenco Spin on Lewis Carroll's Alice: Wonderland
* Additional fees apply. No coupon or promo codes necessary to enjoy the displayed discount price.
The last date listed for Wonderland was Sunday May 18, 2014 / 7:30pm.
Venue Details
When Alice is beckoned by the Cheshire Cat to leave her boring day to day life and walk through a mirror, she enters a whole new world, and her life definitely gets "curious and curiouser." She grows and shrinks in size and meets all manner of crazy characters —talking mice and rabbits, a Mad Hatter who drinks tea to the rhythm of flamenco Bulerías, Royal flower-painting Playing Cards, a wise Caterpillar, and a King who fears nothing . . . except his wife . . . the Red Queen! But things get even crazier when the Knave of Hearts tries to steal some of the Red Queen's treasured tarts, leading to a climactic trial that could end with both the Knave and Alice losing their heads! Will it be "Off with their heads!" or will Alice find a way to dance her way through this curious land of flamenco and find her way home? The only way to find out is by going down the rabbit hole with us and joining us in WONDERLAND!
About the Ticket Supplier: Calo Gitano Productions
Caló Gitano is a Venezuelan Flamenco Dance Company and Academy located in Atlanta, spreading the Duende throughout the South East!


text:
Illustrating the Socioeconomic Divide With iOS and Android 161
from the your-phone-is-your-class-marker dept.
We've improved Slashdot's video section; now you can view our video interviews, product close-ups and site visits with all the usual Slashdot options to comment, share, etc. No more walled garden! It's a work in progress -- we hope you'll check it out (Learn more about the recent updates).
... "Manhattan, a generally affluent area."
"the average value of an order from an iPad is $155, compared to $110 from Android tablets."
The funny thing is, that often it's for the exact same thing both of them bought.
Sites check the user-agent and rich guys (IOS) are shown a higher price for the same objects, as it has been noticed quite a few times.
So if you want a bargain, you need a user-agent-changer for your iPad to mimic a poor people's OS.
Real Users find the one combination of bizarre input values that shuts down the system for days.


text:
Nicotinate + 5-phospho-alpha-D-ribose 1-diphosphate + ATP + H2O = beta-nicotinate D-ribonucleotide + diphosphate + ADP + phosphate.
The Protein Feature View requires a
browser that supports
SVG (Scalable Vector
Graphics). Mouse
over tracks and
labels for more
The RCSB PDB (citation) is managed by two members of the Research Collaboratory for Structural Bioinformatics:
RCSB PDB is a member of the
The RCSB PDB is funded by a grant from the
National Science Foundation, the
National Institutes of Health, and the
US Department of Energy.


text:
Tenofovir Renal Toxicity and Glomerular Filtration Rate (GFR) Validation
The recruitment status of this study is unknown because the information has not been verified recently.
Verified February 2011 by The HIV Netherlands Australia Thailand Research Collaboration.
Recruitment status was Recruiting
Recruitment status was Recruiting
The HIV Netherlands Australia Thailand Research Collaboration
Chulalongkorn University
Kirby Institute
Information provided by:
The HIV Netherlands Australia Thailand Research Collaboration
ClinicalTrials.gov Identifier:
First received: June 4, 2010
Last updated: February 3, 2011
Last verified: February 2011
- Full Text View
- Tabular View
- No Study Results Posted
- Disclaimer
- How to Read a Study Record
No Study Results Posted on ClinicalTrials.gov for this Study
|Study Status:
|This study is currently recruiting participants.
|Estimated Study Completion Date:
|December 2011
|Estimated Primary Completion Date:
|June 2011 (Final data collection date for primary outcome measure)
No publications provided by The HIV Netherlands Australia Thailand Research Collaboration
Publications automatically indexed to this study by ClinicalTrials.gov Identifier (NCT Number):
Publications automatically indexed to this study by ClinicalTrials.gov Identifier (NCT Number):
Avihingsanon A, Jitmitraparp S, Tangkijvanich P, Ramautarsing RA, Apornpong T, Jirajariyavej S, Putcharoen O, Treeprasertsuk S, Akkarathamrongsin S, Poovorawan Y, Matthews GV, Lange JM, Ruxrungtham K; HIV-NAT125 study team. Advanced liver fibrosis by transient elastography, fibrosis 4, and alanine aminotransferase/platelet ratio index among Asian hepatitis C with and without human immunodeficiency virus infection: role of vitamin D levels. J Gastroenterol Hepatol. 2014 Sep;29(9):1706-14. doi: 10.1111/jgh.12613.
Avihingsanon A, Apornpong T, Ramautarsing RA, Ubolyam S, Tangkijvanich P, Ananworanich J, Lange JM, Matthews G, Lewin SR, Ruxrungtham K; HIV-NAT 105 study team. Decline in serum 25 hydroxyvitamin D levels in HIV-HBV-coinfected patients after long-term antiretroviral therapy. Antivir Ther. 2014;19(1):41-9. doi: 10.3851/IMP2673. Epub 2013 Aug 23.


text: Alison Interact My Recent Posts Lead Jun 13 02 1:28 AM Tags : : Go to .....www.wireimage.com. & you will see 160 pic's of kd. there are some i have not seen....have a look. carn't remember if i typed in "kd lang or "k d lang".. Quote Reply Remove this ad Remove this ad