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5 bedroom detached house to rent Heddon House, Heddon On The WallEasiliving are proud to present this beautiful detached house in the fantastic countryside location of Heddon on the Wall. The property briefly comprises of five bedrooms, two with en suites, kitchen, living room, games room, study, utility & cloak room. More details ›
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1 bedroom semi-detached house to rent Turpins Hill, Heddon on the Wall, ne15We are pleased to offer for rent a delightfully located one bedroom semi detached cottage in a pleasant country setting with open views! The accomodation benefits from double glazing and Lpg central heating. This property comprises of: lounge, fitted kitchen, one bedroom, fitted bathroom, enlcose More details ›
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Clay Buchholz's Hamstring, Daniel Bard's Rebound Attempt Among Top 10 Red Sox Spring Training Storylines (Photos)
Originally written on NESN.com | Last updated 2/12/13
Spring training is officially under way, and the Red Sox are one of the more interesting teams to watch. There's no telling whether this season will yield better results for the Sox. There are still a lot of questions, and after a 69-93 campaign, there are plenty of reasons to be skeptical. One thing that's certain, however, is that this season's Red Sox team looks significantly different than last year's version. And with a number of high character guys joining the mix, the 2013 Red Sox should also be a much more likeable team than a year ago. The most notable new face is one that really isn't all that new. Manager John Farrell, who was once the team's pitching coach under Terry Francona, will be the man tasked with making sure that with new pieces comes news results. There's really nowhere to go but up after last year's debacle under manager Bobby Valentine, but given the depth of the American League East, climbing out of the cellar isn't going to be easy. Spring training often times isn't that revealing. But in a season with so many question marks, it will go a long way toward showing us what to expect in a number of areas. Will Clay Buchholz be healthy? Can Daniel Bard rebound? Is John Lackey really capable of turning his Red Sox career around? These are just a few of the things to watch this spring. Check out what else you should be keeping an eye on before Opening Day by clicking the link below. Daniel Bard's Uncertain Future, John Lackey's High Expectations Among Spring Training Storylines to Watch » Have a question for Ricky Doyle? Send it to him via Twitter at @TheRickyDoyle or send it here.
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A method for substantially inhibiting initiation or spread of infection by rhinovirus or Coxsackie A virus of host cells expressing the major human rhinovirus receptor (ICAM-1), comprising the step of contacting the virus with a soluble polypeptide comprising the HRV binding site in domains I and II of ICAM-1; which polypeptide is capable of binding to the virus and reducing infectivity thereof; the contact being under conditions which permit the virus to bind to the polypeptide.
1. A method for substantially inhibiting initiation or spread of infection by human rhinovirus of host cells in tissue susceptible to such infection, comprising the step of contacting the virus with a soluble polypeptide comprising domains I and II of ICAM-1; which polypeptide is capable of binding to rhinovirus of the major receptor class and reducing infectivity thereof, said contact being under conditions which permit said rhinovirus to bind to said polypeptide.
2. The method of
3. The method of
I and II
I, II, and III
I, II, III, and IV
I, II, III, IV, and V.
4. A method for substantially reducing initiation or spread of infection by Coxsackie A virus of host cells expressing the major human rhinovirus receptor, comprising the step of contacting the virus with a soluble polypeptide comprising the HRV binding site in domains I and II of ICAM-1; which polypeptide is capable of binding to Coxsackie A virus and reducing infectivity thereof, said contact being under conditions which permit said Coxsackie A virus to bind to said polypeptide.
This application is a continuation of application U.S. Ser. No. 08/103,303 filed Aug. 6, 1993, now abandoned, which is a continuation of U.S. Ser. No. 07/631,313 filed Dec. 20, 1990, abandoned, which is a continuation-in-part of U.S. Ser. No. 07/556,238 filed Jul. 20, 1990, abandoned, and a continuation-in-part of U.S. Ser. No. 07/390,662 filed Aug. 10, 1989, abandoned; U.S. Ser. No. 07/390,662 is a continuation-in-part of U.S. Ser. No. 07/262,428 filed Oct. 25, 1988, abandoned, and a continuation-in-part of U.S. Ser. No. 07/239,571 filed Sep. 1, 1988, abandoned; U.S. Ser. No. 07/262,428 is a continuation-in-part of U.S. Ser. No. 07/239,571.
The present invention relates to novel truncated forms of intercellular adhesion molecule (ICAM), designated "tICAMs", which effectively bind to human rhinovirus (HRV) and to lymphocyte-function associated antigen-1 (LFA-1). The present invention also pertains to DNA sequences coding for various tICAMs and to methods for preventing or amelio-rating infection and inflammation using said tICAMs.
Human rhinoviruses, the major causative agent of the common cold, belong to the picornavirus family. The three-dimensional structure of several rhinovirus serotypes have now been determined to atomic resolution by Rossmann, M. G., E. Arnold, J. W. Erickson, E. W. Frankenberger, P. J. Griffith, H. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend, Nature (1985) 317:145-153; Kim, S., T. J. Smith, M. M. Chapman, M. G. Rossmann, D. C. Pevear, F. J. Dutko, P. J. Felock, G. D. Diana, and M. A. McKinlay, J. Mol. Biol. (1990) 210:91-111. The virion is composed of a protein capsid of 60 protomeric units, consisting of the four protein subunits VP1-4, surrounding an RNA genome. Each of the 60 protomeric units possesses a recessed "canyon" that is believed to contain the site that binds to the receptor on the target cell surface [reviewed in Rossmann, M. G., J. Biol. Chem. (1989) 264:14587-14590]. The dimensions of the canyon are such that it is too small to admit the combining site of an antibody but is apparently large enough to admit the virus-binding site of the receptor.
In order to infect host cells, viruses must bind to and then enter cells to initiate an infection. Since 1959, evidence has accumulated in the literature indicating that the presence of specific binding sites (receptors) on host cells could be a major determinant of tissue tropism of certain viruses. [Holland, J. J., and L. C. McLaren, "The mammalian cell-virus relationship. II. Absorption, reception, and eclipse of poliovirus by HeLa cells," J. Exp. Med. 109:487-504 (1959); Holland, J. J., "Receptor affinities as major determinants of enterovirus tissue tropisms in humans," Virology 15:312-326 (1961)]. Among picornaviruses such as poliovirus, Coxsackie virus, and rhinoviruses, specific binding to host cells has been demonstrated. By competition experiments, it has been demonstrated that some of these receptors are distinct from one another in that the saturation of the receptor of one virus had no effect on the binding of a second virus. [Lonberg-Holm, K., R. L. Crowell, and L. Philipson. "Unrelated animal viruses share receptors," Nature 259:679-681 (1976)].
Rhinoviruses can be classified according to the host cell receptor to which they bind. Tomassini, J. E. and R. J. Colonno, "Isolation of a receptor protein involved in attachment of human rhinoviruses," J. Virol. 58:290 (1986) reported the isolation of a receptor protein involved in the cell attachment of HRV. Approximately 90% of the more than 115 serotypes of rhinoviruses, as well as several types of Coxsackie A virus, bind to a single common receptor termed the "major" human rhinovirus receptor (HRR) [Abraham, G., and R. J. Colonno, "Many rhinovirus serotypes share the same cellular receptor," J. Virol. 51:340-345 (1984)]; the remaining 10% bind to one or more other cell receptors.
The major human rhinovirus receptor has been transfected, identified, purified, and reconstituted as described in co-pending U.S. patent applications Ser. No. 07/262,428 and 07/262,570, both filed Oct. 25, 1988. Greve, J. M., G. Davis, A. M. Meyer, C. P. Forte, S. C. Yost, C. W. Marlor, M. E. Kamarck, and A. McClelland, "The major human rhinovirus receptor is ICAM-1," Cell 56:839 (1989), identified the major HRR as a glycoprotein with an apparent molecular mass of 95 kD and having an amino acid sequence essentially identical to that deduced from the nucleotide sequence of a previously described cell surface protein named intercellular adhesion molecule (ICAM-1). ICAM-1 had first been identified based on its role in adhesion of leukocytes to endothelial cells [Rothlein, R., et al., J. Immunol. 137:1270-1274 (1986); see also Simmons, D., M. W. Makgoba, and B. Seed, Nature (1988) 331:624; Staunton, D. E., S. D. Marlin, C. Stratowa, M. L. Dustin, and T. A. Springer, "Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families," Cell (1988) 52:925-933]. Induction of ICAM-1 expression by cytokines during the inflammatory response may regulate leukocyte localization to inflammatory sites. Subsequently, Staunton, D. E., et al., Cell 56:849 (1989) confirmed that ICAM-1 is the major cell surface receptor for HRV. See also Staunton, D. E., M. L. Dustin, H. P. Erickson, and T. A. Springer, "The arrangement of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and rhinovirus," Cell (1990) 61:243-254. The precise extent of the virus-binding site on ICAM-1 remains to be determined, although results from mouse-human chimeras and site-directed mutagenesis indicate that the two N-terminal domains play a major role in virus binding [Staunton, et al., Cell (1990) 61:243-254], and a model has been developed for the interaction of the N-terminal domain of ICAM-1 with HRV14 [Giranda, V. L., M. S. Chapman, and M. G. Rossmann, Proteins (1990) 7:227-233].
European Patent Application 0 289 949 describes membrane-associated ICAM-1, which mediates attachment of many cell types, including endothelial cells, to leukocytes expressing lymphocyte function associated molecule-1 (LFA-1; CD18/CD11a, a member of the beta-2 integrin family). This patent application provides a discussion of the prior research in the field of intercellular adhesion molecules.
Heterotypic binding of LFA-1 to ICAM-1 mediates cellular adhesion of diverse cell types and is important in a broad range of immune interactions [Marlin, et al., Cell (1987) 51:813-819]. ICAM-1 also binds to MAC-1 (CD18/CD11b), another beta-2 integrin, but not to p150/95 (CD18/CD11c) [Staunton, D. E., S. D. Marlin, C. Stratowa, M. L. Dustin, and T. A. Springer, Cell (1988) 52:925-933]. MAC-1 and p150/95 differ from LFA-1 by their alpha subunit. Although minimal peptide recognition sites have been identified for many other integrins, the recognition site for LFA-1 on ICAM-1 remains obscure. Staunton, et al., Cell (1990) 61:243-254 have reported that a transmembrane form of the first two domains of ICAM-1 retains some LFA-1-binding activity and that a number of mutations in the first two domains of the full-length molecule cause reductions in LFA-1-binding activity.
The primary structure of ICAM-1 is homologous to two other cellular adhesion molecules: neural cell adhesion molecule (NCAM) and myelin-associated glycoprotein (MAG). This suggests that ICAM-1 is a member of the immunoglobulin supergene family [Simmons, et al., Nature (1988) 331:624-627; Staunton et al., Cell (1988) 52:925-933]. The cDNA sequences are described in the above-referenced papers by Simmons et al. and Staunton et al., from which the amino acid sequence of ICAM-1 has been deduced.
ICAM-1 is an integral membrane protein 505 amino acids long and has: i) five immunoglobulin-like extra-cellular domains at the amino-terminal (extracellular) end (designated domain 1 [amino acid residues 1-88], domain 2 [89-185], domain 3 [186-284], domain 4 [285-385], and domain 5 [386-453] [Staunton, et al., Cell (1988) 52:925-933]); ii) a hydrophobic transmembrane domain (454-477); and iii) a short cytoplasmic domain at the carboxy-terminal end (478-505). Electron microscopy has indicated that ICAM-1 is a highly elongated molecule [Staunton, et al., Cell (1990) 61:243-2541].
Several approaches to decreasing infectivity of viruses in general, and of HRV in particular, have been pursued including: i) developing antibody to the cell surface receptor for use in blocking viral binding to the cell; ii) using interferon to promote an anti-viral state in host cells; iii) developing various agents to inhibit viral replication; iv) developing antibodies to viral capsid proteins/peptides; and v) blocking viral infection with isolated cell surface receptor protein that specifically blocks the viral binding domain of the cell surface receptor.
In 1985, the isolation of a monoclonal antibody that appeared to be directed against the major rhinovirus receptor was described. [Colonno, R. J., P. L. Callahan, and W. J. Long, "Isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses," J. Virol. 57:7-12 (1986)]. This monoclonal inhibited infection of cells with the appropriate serotypes of rhinovirus and it inhibited binding of radiolabeled rhinovirus to cells. Colonno et al. subsequently reported that the monoclonal antibody bound to a protein with an apparent molecular weight of 90 kD [Tomassini, et al., J. Virol. (1986) 58:290-295]. This monoclonal antibody has been utilized in clinical trials with primates and humans and is understood to provide some protection against rhinovirus infection.
There are several other reports of attempts at therapeutic intervention in rhinovirus infections. Intranasal application of interferon in humans has been attempted. [Douglas, R. M. et al., "Prophylactic efficacy of intranasal alpha2-interferon against rhinovirus infections in the family setting," N. Eng. J. Med. 314:65-75 (1986)]. In this case, significant reduction in the severity of the infection was found, although nosebleeds were observed as a side-effect. Also, several analogs of disoxaril ("WIN" compounds) that reduce the infectivity of a number of picornaviruses (with widely varying effectiveness, depending on the serotype) have been tested in tissue culture and in some animal models [Fox, M. P., M. J. Otto, and M. A. McKinlay, Antimicrob. Ag. and Chemotherapy (1986) 30:110-116]. These compounds appear to inhibit replication at a step subsequent to receptor binding, probably at some step of virus uncoating. The atomic coordinates of the binding sites of these compounds within the viral capsid of the serotype HRV14 have been determined by x-ray crystallography, and are located in a hydrophobic pocket present in each protomeric unit of the capsid [Smith, T. J., et al., "The site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating," Science (1986) 233:1286-1293]. The specific function of the binding pocket, if any, is unknown, but drug-resistant mutants with a single amino acid interchange in this region arise at high frequency and are viable [Badger, J., et al., "Structural analysis of a series of antiviral agents complexed with human rhinovirus 14," PNAS 85:3304-3308 (1988); see also Dearden et al., Arch. Virol. (1989) 109:71]. This result calls into question the efficacy of such compounds as drugs. The production of anti-peptide antibodies in rabbits has been reported using peptides derived from amino acid sequences of the viral capsid proteins that line the "receptor canyon" of HRV14 [McCray, J., and G. Werner, "Different rhinovirus serotypes neutralized by antipeptide antibodies," Nature (1987) 329:736-738]. While the titers of these sera are quite low, cross-serotype protection of cells in tissue culture from rhinovirus infection was demonstrated, raising the possibility of a vaccine.
It is an object of the present invention to provide an HRV receptor protein having the property of blocking HRV infection. Given the high affinity the virus has for its receptor, a therapeutic agent effective against HRV infection is the receptor itself, or more specifically, the virus-binding domain of the receptor. A protein, protein fragment, or peptide that comprises the virus-binding domain can block the ability of virus to bind to host cells by occupying (blocking) the receptor binding cleft on the virus. Furthermore, since such a molecule makes some or all of the molecular contacts with the virus capsid that the receptor does, virus mutations that adversely affect binding of the molecule also adversely affect binding of the receptor, and thus are deleterious or lethal for the virus; therefore, the likelihood of drug-resistant mutants is very low.
Using this approach, Greve, et al., Cell (1989) 56:879, reported that purified transmembrane ICAM-1 (tmICAM-1) could bind to rhinovirus HRV3 in vitro. Other results with HRV2, HRV3, and HRV14 demonstrate a positive correlation between the ability to bind to rhinovirus and the ability to neutralize rhinovirus. Results using HRV14 and HRV2 demonstrate a positive correlation between the receptor class of the virus and the ability to bind to tmICAM-1 in vitro. That is, ICAM-1, being the major receptor, can bind to HRV3, HRV14, and other major receptor serotypes and neutralize them, while it does not bind or neutralize HRV2, a minor receptor serotype. Further studies, using purified tmICAM-1, demonstrate that it effectively inhibits rhinovirus infectivity in a plaque-reduction assay when the rhinovirus is pretreated with tmICAM-1 (50% reduction of titer at 10 nM receptor and one log reduction of titer at 100 nM receptor protein). These data were consistent with the affinity of rhinovirus for ICAM-1 of HeLa cells, which has an apparent dissociation constant of 10 nM, and indicates a direct relationship between the ability of the receptor to bind to the virus and to neutralize the virus.
The ICAM of the prior art is an insoluble molecule which is solubilized from cell membranes by lysing the cells in a non-ionic detergent. Because large-scale production of tmICAM-1 is not presently economically feasible, and because maintenance of tmICAM-1 in an active form requires the use of detergents, alternate soluble forms of receptor protein suitable for use as a rhinovirus inhibitor are desirable. See generally copending applications U.S. Ser. No. 07/130,378; U.S. Ser. No. 07/239,570; U.S. Ser. No. 07/239,571; U.S. Ser. No. 07/262,428; U.S. Ser. No. 262,570; U.S. Ser. No. 07/390,662; and U.S. Ser. No. 07/556,238, all incorporated herein by reference.
U.S. Ser. No. 07/130,378 (filed Dec. 8, 1987) and its CIP application U.S. Ser. No. 07/262,570 are directed to transfected non-human cell lines which express the major human rhinovirus receptor (HRR), and to the identification of HRR as intercellular adhesion molecule (ICAM).
U.S. Ser. No. 07/301,192 (filed Jan. 24, 1989) and its CIP applications U.S. Ser. No. 07/445,951 (abandoned) and U.S. Ser. No. 07/449,356 are directed to a naturally-occurring soluble ICAM (sICAM) related to but distinct from tmICAM in that said sICAM lacks the amino acids spanning the hydrophobic transmembrane region and the carboxy-terminal cytoplasmic region; in addition this sICAM has a novel sequence of 11 amino acids at its C-terminus.
Parent application U.S. Ser. No. 07/239,571 (filed Sep. 1, 1988) and its CIP applications U.S. Ser. No. 07/262,428 and U.S. Ser. No. 07/390,662 are directed to the use of detergent-complexed tmICAM as an inhibitor of HRV infectivity, using the non-ionic detergent complex to maintain the transmembrane protein in solution. These cases are also directed to truncated forms of ICAM comprising one or more of the extracellular domains I, II, and III, IV and V of tmICAM, which truncated forms do not require the presence of non-ionic detergent for solubilization.
Parent application U.S. Ser. No. 07/556,238 (filed Jul. 20, 1990) is directed to multimeric configurations and forms of tmICAM-1 and tICAM-1 with improved ability to prevent HRV infection. When tmICAM-1, tICAM(453), or tICAM(185) is first adsorbed on any of a number of insoluble supports, such as nitrocellulose, PVDF (polyvinylidene difluoride), or DEAE (diethylaminoethyl) membranes, and then incubated with radioactive HRV, the virus-binding activity of tICAM(453) becomes comparable to that of tmICAM-1. This binding of multimeric tICAM(453) or of multimeric tICAM(185) to HRV has the same properties as the binding of HRV to ICAM-1 on HeLa cells: it is inhibited by anti-ICAM-1 monoclonal antibodies, it is specific for HRV of the major receptor group, and it has the same temperature-dependence pattern as the binding of HRV to cells (i.e., binds well at 37� C. and undetectably at 4� C.).
The present application contains further data demonstrating the properties and efficacy of various forms of tICAM.
The present invention provides truncated forms of ICAM-1 designated tICAM-1 which are soluble without the addition of a detergent.
The present invention also provides purified and isolated tICAM-1, or functional derivatives thereof, substantially free of natural contaminants.
The present invention further provides a purified and isolated DNA sequence encoding tICAMs as well as host cells encoding said sequences.
The present invention provides a method of recovering tICAMs in substantially pure form comprising the steps of cloning a genome coding for the desired tICAM form in a suitable expression vector in a suitable host.
Also provided by the invention are novel pharmaceutical compositions comprising a pharmaceutically acceptable solvent, diluent, adjuvant or carrier, and as the active ingredient, an effective amount of a polypeptide characterized by having HRV binding activity and reduction of virus infectivity.
The present invention includes monoclonal antibodies against ICAM-1 and tICAMs, and hybridoma cell lines capable of producing such monoclonal antibodies.
This invention further includes the therapeutic use of antibodies specifically directed to tICAMs to decrease cell adhesion mediated by ICAM-1 and LFA-1.
The invention further includes a method of inhibiting LFA-1 and ICAM-1 interaction comprising the step of contacting LFA-1-containing cells with a tICAM or a functional derivative thereof.
A further aspect of the invention is use of fragments, functional domains or analogs of LFA-1 to disrupt interactions between HRR and HRV and thereby treat HRV infections.
This invention further includes a method for substantially reducing infection by HRV of the major receptor group, comprising the step of contacting the virus with tICAM-1 or a functional derivative thereof.
This invention further includes a method of diagnosis of the presence and location of an LFA-1-expressing tumor cell.
Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description thereof which includes numerous illustrative examples of the practice of the invention.
FIG. 1 is a schematic diagram illustrating the domains included in several representative tICAM constructs of the present invention.
FIG. 2 shows secretion of soluble ICAM-1 proteins. A. Diagram of progressively truncated forms of ICAM-1 used in transfection experiments. Crosshatched indicates signal sequence and filled region indicates the transmembrane region. B. Fluorograph of 35S-cysteine-labelled products secreted by COS cells analyzed by SDS-PAGE; loading of lanes containing tICAM(283) and tICAM(88) is 10-fold higher than other lanes. C. Silver-stained gel of purified tICAM(453) and tICAM(185) produced by CHO cell transfectants.
FIG. 3 shows circular dichroism spectra of IgG and ICAM-1 proteins. A: c92.5 IgG; B: ICAM-1; C: tICAM(453); D: tICAM(185). The spectra for c92.5 IgG, tICAM(453), and tICAM(185) were collected in 20 mM sodium phosphate buffer (pH 7.5) and the spectrum for ICAM-1 was collected in 0.1% octylglucoside/150 mM NaCl/10 mM sodium phosphate (pH 7.5). The data were averaged from 5 repetitive scans, buffer-subtracted and smoothed.
FIG. 4 shows inhibition of ICAM-1/LFA-1-mediated cell adhesion by soluble ICAM-1. JY cells were preincubated with various concentrations of tICAM(453) (filled circles) or tICAM(185) (open squares) for 30 min and then allowed to bind to ICAM-1-coated plates for 60 min at 37 C. The binding of JY cells is >90% inhibited by monoclonal antibody c78.4 to ICAM-1 and IOT 16 (AMAC Inc.) to LFA-1.
As used herein, the following abbreviations and terms include, but are not necessarily limited to, the following definitions:
The ICAM-1 terminology has been used although it is now recognized that the terms HRR and ICAM-1 are interchangeable.
"Multimerization" and "multimeric" include, but are not limited to dimerization and dimeric, and include any multimeric configuration of the ICAM-1 molecule, or fragment thereof, that is effective in reducing viral binding and infectivity.
"Transmembrane" generally means forms of the ICAM-1 protein molecule which possess a hydrophobic membrane-spanning sequence and which are membrane-bound.
"Non-transmembrane" generally means soluble forms of the ICAM-1 protein including truncated forms of the protein that, rather than being membrane bound, are secreted into the cell culture medium as soluble proteins, as well as transmembrane forms that have been solubilized from cell membranes by lysing cells in non-ionic detergent.
"Truncated" generally includes any protein form that is less than the full-length transmembrane form of ICAM.
"Form" is generally used herein to distinguish among full-length and partial-length ICAM forms; whereas "configuration" is generally used to distinguish among monomeric, dimeric, and multimeric configurations of possible ICAM forms.
All forms and configurations of the ICAM-1 molecule, whether full length or a fragment thereof, including muteins, whether monomeric or multimeric, may be fully or partially glycosylated, or completely unglycosylated, as long as the molecule remains effective in reducing viral binding and infectivity.
"Ligand" is generally used herein to include anything capable of binding to at least one of any of the forms and configurations of ICAM and includes, but is not limited to, HRV, other viruses that bind to the "major" group human rhinovirus receptor, LFA-1, and Plasmodium falciparum (the causative agent of malaria).
"Human rhinovirus" or "HRV" generally includes all human serotypes of human rhinovirus as catalogued in Hamparian, V., et al., Virol. (1987) 159:191-192.
The sequence of amino acid residues in a peptide is designated in accordance with standard nomenclature such as that given in Lehninger's Biochemistry (Worth Publishers, New York, 1970).
Full-length ICAM-1, also known as human rhinovirus receptor (HRR), is termed transmembrane ICAM (tmICAM-1). The present invention provides non-transmembrane truncated forms of ICAM-1 designated tICAM-1, i.e., ICAM substantially without the carboxyl-terminal cytoplasmic domain and without the hydrophobic transmembrane domain, which are soluble without the addition of a detergent. Non-transmembrane forms of ICAM can include functional derivatives of ICAM and mutein forms of tICAM. Preferred embodiments are tICAM(453), tICAM(283), tICAM(185), and tICAM(88).
The present invention further provides purified and isolated tICAMs, or functional derivatives thereof, substantially free of natural contaminants. tICAMs can be obtained from transfected cells, e.g., CHO cells, and are characterized by being soluble in the absence of nonionic detergents and being the translation product defined by novel DNA oligomers. These products have the advantage of being secreted from cells in a soluble form and of not being immunogenic. These truncated products differ from the natural insoluble product in that the truncated soluble products do not contain the membrane-spanning and cytoplasmic domains present in the insoluble form.
The present invention provides purified and isolated DNA sequences encoding tICAMs as well as host cells encoding said sequences. The nucleotide sequences coding for tICAMs can be contained in vectors, such as plasmids, and the vectors can be introduced into host cells, for example eukaryotic or prokaryotic cells. Examples of suitable eukaryotic cells are mammalian cells, e.g. Chinese hamster ovary cells, and yeast cells. Examples of suitable prokaryotic cells are, e.g, E. coli. Eukaryotic cells are preferred; mammalian cells are particularly preferred.
Also provided by the invention are novel pharmaceutical compositions comprising a pharmaceutically acceptable solvent, diluent, adjuvant or carrier, and as the active ingredient, an effective amount of a polypeptide characterized by having HRV-binding activity and reduction of virus infectivity.
The invention further provides novel pharmaceutical compositions comprising a pharmaceutically acceptable solvent, diluent, adjuvant or carrier, and as the active ingredient, an effective amount of a polypeptide characterized by having LFA-1-binding activity and reduction of LFA-1 activity.
The present invention further provides methods of recovering a tICAM in substantially pure form comprising the steps of cloning a gene coding for the desired tICAM form in a suitable expression vector in a suitable host. The secretion of a soluble protein eliminates the problems associated with production and purification of an insoluble, cell-membrane-bound protein, since cell lysis is not required and thus continuous culture can be employed along with simplified procedures for purification and isolation of the tICAMs. The protein may be purified using an immunoaffinity column, lectin or wheat germ agglutinin column. Other purification steps may include sizing chromatography, ion chromatography, and gel electrophoresis. The antibody capable of binding to tICAM is selected from antibodies against ICAM-1 (tmICAM or HRR) or fragments thereof.
The present invention includes polyclonal antibodies against tICAM. For a method for producing peptide antisera see Green et al., Cell 28:477-487 (1982).
The invention also includes hybridoma cell lines capable of producing monoclonal antibodies to tICAMs, and the monoclonal antibodies produced by said cell lines.
This invention further includes the therapeutic use of antibodies specifically directed to tICAMs to decrease cell adhesion mediated by ICAM and LFA-1.
Pharmaceutical preparations of proteins, protein fragments, functional domains, and analogs have an application in a plurality of diseases. Since the various forms of ICAM are ligands for both LFA-1 and HRV, they may also be used to block tmICAM interaction with LFA-1, which is critical to many cell adhesion processes involved in the immunological response. This method of inhibition of ICAM-1-mediated adhesion has application in such disease states as inflammation and graft rejection, and for suppression of LFA-1-expressing tumor cells and other processes involving cell adhesion.
A further aspect of the invention is use of fragments, functional domains or analogs of LFA-1 to disrupt interactions between HRR and rhinovirus and thereby treat rhinovirus infections.
This invention further includes a method of diagnosis of the presence and location of an LFA-1-expressing tumor cell.
This invention further includes a method for substantially reducing infection by other pathogens of the major HRR group, e.g., Coxsackie virus and Plasmodium falciparum, comprising the step of contacting the pathogen with one or more tICAMs or functional derivatives thereof.
The following examples describe preparation and characterization of representative compounds of the present invention and representative compositions containing said compounds. As used hereinabove and below unless expressly stated to the contrary, all temperatures and temperature ranges refer to the centrigrade system; the term percent or (%) refers to weight percent; and the term mole and moles refer to gram moles.
Rhinoviruses were grown, purified, and assayed essentially as described in Abraham, G., et al., J. Virol. (1984) 51:340 and Greve, et al., Cell, (1989) 56:839. The serotypes chosen for these studies include HRV14, the standard in the field, and HRV3, which has an approximately 10-fold higher affinity for ICAM than does HRV14. HRV2, which binds to the "minor" receptor rather than the "major" receptor, was used as a negative control.
Rhinoviruses HRV2, HRV3, and HRV14 were obtained from the American Type Culture Collection, plaque purified, and isolated from lysates of infected HeLa-S3 cells. Purified rhinovirus was prepared by polyethylene glycol precipitation and sucrose density gradient sedimentation. Viral purity was assessed by SDS-PAGE analysis of capsid proteins and by electron microscopy. Infectivity was quantified by a limiting-dilution infectivity assay scoring for cytopathic effect, essentially as described by Minor, P. D., "Growth, assay and purification of picornaviruses," in Viroloqy: A Practical Approach, B. W. J. May, ed. (Oxford, IRL Press, 1985), pp. 25-41.
BALB/cByJ female mice were immunized by intraperitoneal injection of 107 intact HeLa cells in 0.5 ml of phosphate-buffered saline (PBS) three times at 3-week intervals. Two weeks later the mice were bled and aliquots of serum were tested for protective effects against HRV14 infection of HeLa cells. Positive mice were boosted by a final injection of 107 HeLa cells, and 3 days later spleen cells were fused to P3X63-Ag8.653 myeloma cells [Galfre, et al., Nature (1977) 266:550-552] to produce a total of approximately 700 hybridoma-containing wells. Each well was tested by incubating 3�104 HeLa cells in 96-well plates with 100 μl of supernatant for 1 hour at 37� C.; the cells were then washed with PBS, and a sufficient amount of HRV14 was added to give complete cytopathic effect in 24-36 hours. Wells that were positive (protected from infection) were scored at 36 hours.
Cells were removed from wells which scored positive in the first screen and cloned by limiting dilution in 96-well microtiter plates. Supernatants from these wells were tested in the cell protection assay and positive wells were again identified. Further clonings were performed until all of the hybridoma-containing wells were positive indicating a clonal population had been obtained. Four cloned cell lines, and their corresponding antibodies, were obtained and were designated c78.1A, c78.2A, c78.4A, c78.5A. An additional two cell lines were obtained by hyperimmunizing mice with L-cells transfected with human genomic DNA comprising HRR and were designated c92.1A and c92.5A.
C92.1A was deposited on Nov. 19, 1987 with the American Type Culture Collection, and was designated HB 9594.
ICAM-1 cDNAs encoding soluble forms were constructed by using polymerase chain reaction (PCR) [Saiki, et al., Science (1985) 230:1350-54] to insert stop codons within the reading frame of the ICAM-1 cDNA pHRR-2, [Greve, et al., Cell (1989) 56:839-847]. The plasmid DNA was digested with EcoR1 to excise the ICAM-1 insert and treated with alkaline phosphatase to prevent recircularization of the vector in subsequent ligation steps. 10 ng of template DNA was subjected to 10 cycles of PCR amplification using the 5′ oligonucleotide primer GGAATTCAAGCTTCTCAGCCTCGCTATGGCTCCCAGCAGCCCCCGGCCC (SEQ ID NO:1) and the 3′ oligonucleotide primers GGAATTCCTGCAGTCACTCATACCGGGGGGAGAGDACATT (SEQ ID NO:2) [for tICAM (453)], TTCTAGAGGATCCTCAAAAGCTGTAGATGGTCACTGTCTG (SEQ ID NO:3) [for tICAM (283)], TTCTAGAGGATCCTCAAAAGGTCTGGAGCTGGTAGGGGG (SEQ ID NO:4) [for tICAM (185)], and TTCTAGAGGATCCTCACCGTTCTGGAGTCCAGTACACGG (SEQ ID NO:5) [for tICAM (88)]. The PCR reaction products were digested with EcoR1 [tICAM(453)] or EcoR1 and BAMH1 [tICAM(283), tICAM(185), and tICAM(88)] and cloned into the polylinker site of Bluescript SK+ (Stratagene). In this manner, stop codons were inserted immediately before the first residue of the transmembrane domain, at the predicted ends of domains I+II+III, domains I+II, and domain I to produce a series of progressively truncated proteins (FIG. 2A). Clones containing the desired inserts were verified by restriction analysis and DNA sequencing.
The inserts were excised by digestion with HindIII and XbaI, inserted into the expression vector CDM8 [Seed, B., Nature (1987) 239:840-42], transfected into COS cells for transient expression, and co-transfected with pSV2-DHFR into CHO cells for establishment of stable cell lines. For transient expression, COS cells were transfected by the DEAE-dextran method [Kingston, R. E., in Current Protocols in Molecular Biology, F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds., pp. 9.0.1-6] and pulse-labeled 72 hours after transfection with [35S]-cysteine for 18 hours; culture supernatants were then immunoprecipitated with c78.4 IgG-Sepharose and analyzed by SDS-PAGE [Greve, et al., Cell (1989) 56:839-847]. Stable CHO transfectants were obtained by cotransfection of ICAM-1 cDNAs with pSV2-DHFR into dihydrofolate reductase-deficient CHO cells by the calcium phosphate method or by electroporation [Bebbington and Hentschel, in DNA Cloning—A Practical Approach, Vol. 3, D. M. Glover, ed. (IRL Press, Oxford, 1987), pp. 163-188].
This experiment indicated that the entire extracellular domain, tICAM(453), and the two N-terminal domains, tICAM(185), were efficiently secreted from transfected COS cells as species of 80 kD and 43 kD, respectively (FIG. 2B). The expression of domains I+II+III [tICAM(283)] was approximately 10-fold lower than the above-mentioned fragments and the secreted protein was more heterogeneous in mobility on SDS-PAGE. Expression of domain I [tICAM(88)] could not be detected in COS cells, and alternative constructs in which the stop codon was shifted to several sites N- and C-terminal to residue 88 also failed to produce detectable amounts of protein.
Transfected cells were cloned and individual clones secreting ICAM-1 protein were identified by radioimmunoassay of culture supernatants. The expression of tICAMs was monitored by immunoprecipitation of [35S]-cysteine-labeled culture supernatants with monoclonal or polyclonal antisera followed by SDS-PAGE. Two monoclonal antibodies, c92.5 (which recognizes the same epitope as c78.4) and c78.5, define two distinct conformational epitopes on ICAM-1. These two antibodies were utilized in an RIA for soluble ICAM-1. c92.5 IgG was absorbed onto Immulon-4 microtiter plates, the plates blocked with 10 mg/ml BSA, and the plates incubated with tICAM-containing samples. The plates were then washed, incubated with 125I-c78.5 IgG, and the bound radioactivity determined. The concentration of tICAM was determined by comparison to a standard curve of purified ICAM-1.
Cell lines secreting tICAM(453) (CT.2A) and tICAM(185) (CD12.1A) were selected for further study and were subjected to gene amplification in methotrexate-containing media [Bebbington et al., supra]. In order to obtain sufficient quantities of protein for functional and structural studies, CHO cell transfectants were cloned and subjected to stepwise gene amplification in increasing concentrations of methotrexate. A clone derived from CT.2A (resistant to 100 nM methotrexate) and a clone derived from CD12.1A (resistant to 1 μM methotrexate) were used for purification of soluble truncated proteins.
This resulted in the derivation of cell lines secreting 1.5 μg/ml of tICAM(453) and 1.0 μg/ml of tICAM(185). A stable cell line expressing tICAM(283) was not obtained, perhaps because the low level of secretion was at the limit of sensitivity of the immunoassay. The cells were adapted to serum-free media and the secreted ICAM-1 proteins purified to homogeneity from culture supernatants (FIG. 2C).
tICAM(453) and tICAM(185) were purified from culture supernatants of their respective CHO transfectant cell lines by monoclonal affinity chromatography as described in Greve et al., Cell (1989) 56:839-847, followed by ion exchange chromatography on Mono-Q [for tICAM(453); absorption in 10 mM Tris (pH 6.0), elution in a 0-0.5 M NaCl gradient] or gel filtration on Superose-12 [for tICAM(185)] columns (Pharmacia). Protein was quantitated by amino acid analysis and by RIA. Amino acid analysis was performed on an Applied Biosystems Model 420A amino acid analyzer.
Blocking studies with the panel of six monoclonal antibodies to ICAM-1 (all of which inhibit virus binding to ICAM-1) indicated that there are two distinct epitopes defined by these antibodies, one defined by c78.4 (containing c78.1, c78.2, c92.1 and c92.5), and the other by c78.5. Immunoprecipitation studies with proteolytic fragments of ICAM-1 and with in vitro translations of truncated ICAM-1 cDNAs indicate that both of these epitopes are contained within the first Ig-like domain.
In vitro virus-binding studies utilizing radiolabeled tICAM(453) and purified HRV have indicated that it can bind to rhinovirus in solution.
ICAM has been predicted, based on homology to NCAM, to be a member of the immunoglobulin gene superfamily. One would expect that the immunoglobulin-like domains in ICAM would have the basic "immunoglobulin fold", as has been shown for two other members of this family, beta-2-microglobulin and the HLA-A2 alpha-3 domain. This fold consists of a "beta-barrel" conformation consisting of two antiparallel beta-pleated sheets, one composed of three and one composed of four beta strands; a disulfide bond between two cysteine residues (separated by approximately 60 amino acids along the chain) connects the two sheets [Williams, A. F., Immun. Today (1987) 8, 298-303]. Two of the disulfide bonds, those corresponding to domains II (C110-C161) and III (C212-C265), have been experimentally determined by us, providing support for the model. This model for the structure provides a basis for designing unique analogs that could mimic the virus-binding site and be useful as receptor blockers. Each pair of antiparallel beta strands in the beta-barrel is linked by a hairpin turn of variable size; such turns or loops that protrude from secondary structures are often found to play roles in recognition of ligands [Lezczynski and Rose, Science (1986) 224:849-855]. Such protruding structures may be of particular interest in the rhinovirus receptor, since the receptor-binding site on the virus capsid is proposed to be in a recessed cavity. Using the sequence of the HRR, such turns and loops can be predicted based on a beta-barrel structure and produced as synthetic peptides with addition of novel cysteine residues at the N- and C-terminus of the peptides; a disulfide bond can then be formed between such residues on the same peptide to close the loop covalently (in contrast to the native protein, wherein the loop would be closed by noncovalent interactions between the adjacent beta-strands). Such peptides would have a conformation more analogous to the conformation in the native protein than a simple linear peptide, and can be tested for virus-binding activity.
The value fexp/fo of truncated ICAM-1 proteins were determined from the apparent Stokes radii (Rs) determined by gel filtration on a Superose-12 column calibrated with protein standards (ferritin, 61.0 A; catalase, 52.2 A; bovine serum albumin, 35.5 A; ovalbumin, 30.5 A; and RNase A, 16.4 A) and the calculated molecular weights of the proteins, using the value of M calculated from the core-glycosylated form of the proteins, determined by synthesis in the presence of swainsonin.
fo (frictional coefficient of solvated sphere) was determined from:
The following values are assumed: v2=0.73 cm3/g (partial specific volume of protein), v1 o=1.0 cm3/g (partial specific volume of solvent), δ1=0.35 g H2O/g protein (solvation of protein), η−0.01 g/(cm.s) (viscosity of solvent), and v1 o=1.0 cm3/g (specific volume of the solvent).
We have examined several of the physical properties of tICAM(453) and tICAM(185). Both proteins were quantitatively immunoprecipitated by two monoclonal antibodies directed against two distinct conformation-dependent epitopes on ICAM-1, indicating that these epitopes were contained within the first two domains and providing evidence that the purified proteins were correctly folded. The frictional ration, f/fo, the ratio between the observed and calculated frictional coefficients shown below for both tICAM (453) and tICAM (185) indicates that both fragments of ICAM-1 are highly asymmetric and elongated molecules.
The ratio of experimental and calculated frictional coefficients, f/fo, of both tICAMs indicate highly asymmetric and elonged shapes, and are consistent with the data reported for a species essentially identical to tICAM(453) [Staunton, et al., Cell (1990) 61:243-254]. Proteins belonging to the immunoglobulin supergene family would be expected to have domains with the "immunoglobulin fold" motif, which is basically two apposed sheets of antiparallel beta-strands with connecting loops. In support of the immunoglobulin homology, disulfide mapping studies of ICAM-1 have revealed the existence of intradomain disulfide bonds predicted by the immunoglobulin fold in domains 1-4. This is consistent with the data of Staunton, et al., Cell (1988) 52:925-933 with regard to tICAM(453).
CD spectra were recorded on an AVIV model 62DS spectrometer. Protein solutions at approximately 0.5 mg/ml (determined by amino acid analysis in the indicated buffers) were scanned at 20 C in a cell with a 0.1 cm pathlength. Five respective scans (1 nm interval, 1.5 nm bandwidth) were averaged and buffer-subtracted for each spectrum.
Circular dichroism (CD) spectra were obtained for the soluble forms of ICAM. A single minima at 210-220 nm is indicative of the presence of beta-structure and should be seen in proteins containing immunoglobulin-like domains because of the extensive amount of beta-structure in the "immunoglobulin fold" [Williams, A. F., and A. N. Barclay, Ann. Rev. Immunol. (1988) 6:381-405]. As internal standards, spectra were collected for beta-2-microglobulin and a monoclonal IgG, immunoglobulin-like proteins of known three-dimensional structure [Becker, J. W. and G. N. Reeke, Proc. Natl. Acad. Sci. USA (1985) 82:4225-4229; Bjorkman, P. J., M. A. Saper, B. Samraoui, W. S. Bennett, J. L. Strominger, and D. C. Wiley, Nature (1987) 329:506-512; Amzel, L. M. and R. J. Poljak, Ann. Rev. Biochem. (1979) 48:961-967]. As expected, the spectra of beta-2-microglobulin (not shown) and IgG (FIG. 3A) possessed single major minima at 215 nm and 217 nm, respectively, indicative of extensive amounts of beta-structure. The spectra of the two truncated proteins tICAM(453) and tICAM(185) were similar to each other and to that of ICAM-1 (FIGS. 3B, C, and D), with minima at 216-217 nm and a broad shoulder at 225-230 nm, although the shoulder was more prominent with tICAM(185) than tICAM(453). Considered qualitatively, these CD spectra provide evidence that the soluble ICAM-1 proteins are folded and possess considerable amounts of beta-structure. The shoulder at 225-230 nM present in the three tICAMs is not found in the spectra of beta-2-microglobulin and IgG, suggesting the presence of secondary structural features not present in proteins with "classical" immunoglobulin-like domains. The fact that the shoulder at 225-230 is more prominent in tICAM(185) suggests that the secondary structural features detected by this method are localized in the first two domains. These CD data are similar to those reported by Sayre, P. H., R. E. Hussey, H-C Chang, T. L. Ciardelli, and E. L. Rheinherz, "Structural and binding analysis of a two domain extracellular CD2 molecule," J. Exp. Med. (1989) 169:995-1009] for a soluble form of CD2 (which is also thought to be a member of the immunoglobulin supergene family) in that significant amounts of alpha-helix (which is essentialy absent from immunoglobulin molecules) was predicted from the CD spectra. When individual domains of ICAM-1 are compared to domains from other members of the immunoglobulin supergene family by the ALIGN program [Dayoff, M. O., W. C. Barker, and L. T. Hunt, Methods in Enzymology (1983) 91:524-545], the similarity is quite limited, with only domains II and III of ICAM-1 having significant scores (above 3 SD; for discussion see Williams, A. F., and A. N. Barclay, Ann. Rev. Immunol. (1988) 6:381-405]. Domain I has a significant score only when compared with domain I from ICAM-2 and VCAM-1, proteins that are closely related in function. In addition, domain I has a number of unusual features, such as a short distance (44 residues) between intradomain disulfide bonds and the presence of four cysteines instead of the usual two in the B and F beta-strands.
The epitopes reactive with monoclonal antibodies c78.4A and c78.5A are conformationally-dependent epitopes and thus can be used an analytical probes for confirming retention of the native ICAM structure. Known amount of purified ICAM were incubated with c78.4A or c78.5A IgG-Sepharose™ and the fraction of the radioactivity bound determined. These experiments showed that the purified tmICAM-1, tICAM(185), and tICAM(453) completely retained the ability to bind to these monoclonal antibodies.
Transfectants were metabolically labeled with [35S]-cysteine, and cell lysates (for transmembrane ICAM) or culture supernatants (for truncated ICAM) were prepared and incubated with c78.4A IgG-Sepharose™ beads. The beads were washed and absorbed proteins were eluted with SDS and analyzed by SDS-PAGE [see Greve, et al., Cell (1989) 56:839-847]. It was found that the isolated proteins were quantitatively bound to the c78.4A and c78.5A Mabs.
Accordingly, the tICAM(185) and tICAM(453) both have retained native ICAM structure.
Described below are three binding assays used to assess binding activity of the various forms of ICAM.
A. Pelleting Assay
[35S]-Cysteine-labeled tmICAM-1 or tICAM was mixed with HRV3 in 100 ul of 10 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% Triton X-100. The mixture was incubated for 30 minutes at 37� C., cooled on ice, layered on top of a cushion of 200 μl of 10% glycerol, 0.2 M triethanolamine (pH 7.5), and centrifuged in a Beckman air-driven centrifuge at 134,000�g for 30 minutes at 4� C. The top 275 μl was removed, and the pellet was analyzed by SDS-PAGE and scintillation counting. Silver-staining of SDS gels of control experiments indicated that essentially all of the HRV3 is pelleted under these conditions and essentially all of the ICAM remains in the supernatant. The results are shown below:
These data show that both truncated forms of ICAM bind to rhinovirus.
B. Solution Binding Assay
Previous work has demonstrated that detergent-solubilized tmICAM-1 can bind to rhinovirus [Greve, et al., Cell (1989) 56:839-847] and that the extracellular domain of ICAM-1 competes for rhinovirus binding to cells [Marlin, et al., Nature (1990) 344:70-72]. To quantitatively compare the virus-binding properties of the soluble ICAM-1 proteins, a solid-phase competition assay was employed. In these experiments, varying concentrations of soluble competitor ICAM-1 proteins were assayed for the ability to compete for the binding of [35S]-HRV3 to immobilized ICAM-1; nonionic detergent (Triton X-100) was included in the buffers so that the different proteins could be compared under identical conditions. First, tmICAM-1 (isolated in the presence of 0.1% octylglucoside instead of Triton X-100) was diluted 10-fold into a Tris/NaCl buffer and allowed to adsorb to the walls of a microtiter plate (Immunlon-4, Dynatech) overnight. Nonspecific binding sites on the plate were then blocked with 10 mg/ml BSA and binding experiments performed in 0.1% Triton X-100/1 mg/ml BSA/Tris/NaCl. Approximately 20,000 cpm of [35S]-HRV3 were mixed with varying amounts of tmICAM, tICAM(453) or tICAM(185); incubated for 1 hour at 37� C., and then added to wells of the microtiter plates and incubated for 3 hours at 37� C. The plates were washed and the bound radioactivity determined.
As shown below, tmICAM-1 inhibits virus binding half-maximally at low concentrations (0.008 μM) while tICAM(453) and tICAM(185) inhibit at much higher concentrations (3.1 μM and 9.7 μM, respectively; or 350- to almost 1,000-fold higher than tmICAM).
These data confirm and extend the earlier observations that tICAM(453) and tICAM(185) do bind to rhinovirus but with lower affinities than does tmICAM-1 and provide evidence that the virus-binding site is encompassed within the two N-terminal domains (first 185 residues) of ICAM-1. The IC50 value of tICAM(453) is comparable to that of tICAM(185). The small (three-fold) lower IC50 of tICAM(185) is reproducible but of unknown significance at present. It has been reported that a mouse-human chimera of transmembrane ICAM-1 in which the first two domains are of human origin is capable of binding rhinovirus as well as human ICAM-1, while a shortened transmembrane form containing only the two N-terminal domains binds rhinovirus at approximately one-tenth the level of full-length ICAM-1 [Staunton, et al., Cell (1990) 61:243-254]. These workers interpret these results as demonstrating that domains I and II are important for rhinovirus binding while the distance from the plane of the membrane determines the efficiency with which the rhinovirus can approach the binding site. The interpretation of these results is limited by the requirement for species-specificity of all possible interactions with the rhinovirus and by the steric limitations of shortened transmembrane molecules. Our results with the soluble ICAM-1 clearly and quantitatively demonstrate that the virus-binding site is contained within the first two N-terminal domains.
C. Dot-Blot Assay
An alternative method of measuring binding activity was utilized in which tmICAM-1, tICAM(453), or tICAM(185) were adsorbed to nitrocellulose filters, the non-specific binding sites on the filters blocked with 10 mg/ml bovine serum albumin (BSA), and radioactive virus or 125I-monoclonal antibody to ICAM-1 incubated with the filter for 60 minutes at 37� C. The filters were washed with buffer and the filters exposed to X-ray film.
The amount of radioactivity bound to the filters was determined by densitometry of the autoradiograms, and the data is expressed as HRV3 binding (in arbitrary units) normalized to the amount of ICAM bound to the blot by a parallel determination of the amount of 125I-monoclonal antibody c78.4A or c78.5A bound to the ICAM (bound to the blot). The results are shown below:
The results from this experiment indicate that under these assay conditions tICAM(453) is capable of binding rhinovirus at levels comparable to those of tmICAM-1 when the amount of virus bound was normalized to the amount of [125I]-Mab bound. Further, these results indicate that the tICAM forms are capable of binding to rhinovirus, but that the binding avidity is dependent to some degree upon the configuration of the tICAM. tmICAM-1 may be a small multimer (probably a dimer) and presentation of tICAM in a multimeric form mimics this multimeric configuration.
Evidence supporting this hypothesis comes from quantitative binding studies, in which the ratio of the maximum number of rhinovirus particles and the maximum number of antibody molecules that can be bound to cells is approximately 1.5. This is in contrast to the earlier work of Tomassini, J., et al., (1986) J. Virol. 58:290, which suggested a complex of five molecules needed for binding. Their conclusion was based on an erroneous interpretation of gel filtration data that failed to take into account bound detergent molecules.
HRVs and [35S]-HRV3 was propagated and purified as described in Example 1). All infectivity assays were performed with HeLa-S3 in Dulbecco's Modified Essential Medium/2% fetal calf serum. Assay I: 0.1 ml of dilutions of HRV3 (with 104−10−1 pfu/ml) in the presence or the absence of tICAM(185) or tICAM(453) were added to 10 wells of a 96-well microtiter plate containing 2�104 HeLa cells/well in 0.1 ml. The cultures were incubated for five days at 34� C. and the titer determined from the number of infected wells at the limiting dilution. Assay II: 107 PFU of HRV3 and various concentrations of ICAM-1 in a final volume of 25 μL of 10 mM HEPES (pH 7.5)/150 mM NaCl were incubated for 30 minutes at 37� C., and then serially diluted into culture medium. The virus was then incubated with HeLa cells at 106 cells/ml for 30 minutes at room temperature, plated into 10 wells of a 96-well microtiter plate at 105 cells/well, and then scored for infectivity as described above. Assay III: 0.1 ml of HRV3 (0.1 μg/ml, 107 PFU/ml) was preincubated for 30 minutes at 37� C. with ICAM-1 and then added to wells of a 96-well microliter dish containing 105 HeLa cells and incubated for 24 hours. The cultures were then scored by staining with crystal violet [Minor, P. D., in Virology: A practical approach, B. W. J. Mahy, ed. (IRL Press, Oxford, 1985), pp. 25-40] and determining the optical density at 550 nm. All experiments were performed in triplicate and the results expressed at the concentration of ICAM-1 needed to reduce the OD550 by 50%.
The effect of soluble ICAM-1 on HRV infectivity was examined in three different infectivity assays, the first two being limiting dilution determinations and the third being a high multiplicity of infection (MOI) experiment using the same virus concentrations as the virus binding assay in Example 16. In assay I, cells were infected with serial dilutions of HRV3 in the continuous presence of various concentrations of ICAM-1 and the reduction in virus titer determined. As shown below, the IC50 of tICAM(185) for neutralization is 8.2 μM, similar to the concentration needed to inhibit virus binding. In contrast, the IC50 for neutralization of tICAM(453) is 0.38 μM, 10-fold lower than the concentration needed to block virus-receptor binding:
Thus, the neutralizing activity of tICAM(185) is directly correlated with its effect on virus receptor binding, while tICAM(453) is neutralizing HRV at a considerably lower concentration and is presumably acting by a mechanism other than direct competition for receptor-binding sites on the virus. Assay II consists of preincubating HRV3 with ICAM-1, serially diluting the mixture into culture medium, and then adding the virus to the cells; under these conditions the ICAM-1 is diluted out to negligible concentrations for the actual infection. tICAM(185) and tICAM(453) had essentially no neutralizing activity in this assay at concentrations as high as 20 uM protein, indicating that the effects of the soluble ICBMs on rhinovirus must be reversible. Assay III consists of a single cycle infection at an MOI of 10 in the continuous presence of various concentrations of soluble ICAM-1. Under these conditions tICAM(453) neutralized HRV with an IC50 of 1.2 μM, an 11-fold lower concentration than that of tICAM(185). The relative difference in the concentrations of the two proteins needed to neutralize rhinovirus in Assay III is essentially the same as in Assay I, although the absolute concentrations are approximately 3-fold higher, perhaps because of a nonlinear relationship between infectious particles and infected cells under conditions of high MOI.
The neutralization activities of the two soluble ICAM-1 species examined here are intriguing in that they indicate multiple mechanisms of virus neutralization by receptor. The first mechanism, exemplified by tICAM(185), appears to be a simple competitive inhibition of virus-receptor binding by soluble receptor. The second, exemplified by tICAM(453), involves neutralization of virus at a concentration below that necessary to inhibit virus-receptor binding. This disparity between the virus-neutralizing and virus-binding activities of tICAM(453) indicates that steps in virus entry and/or uncoating are affected by this protein. These apparently different mechanisms of virus neutralization may be reflecting different steps in the entry and uncoating process in which the membrane-bound receptor participates in a normal infection. The modes of action of the capsid-binding WIN compounds as revealed by their effects on different rhinovirus serotypes [Pevear, D. C., M. J. Fancher, P. J. Felock, M. G. Rossman, M. S. Miller, G. Diana, A. M. Treasurywala, M. A. McKinlay, and F. J. Dutko, J. Virol. (1989) 63:2002-2007], where some serotypes are blocked from binding to the receptor while other serotypes are blocked at an intracellular uncoating step, suggest a similar "hierarchy" of sites at which the infection can be interrupted. Both of the neutralization mechanisms mediated by soluble ICAM-1 are reversible, since neutralization is seen when cells are infected in the continual presence of soluble ICAM but not when the virus is preincubated at comparable concentrations of ICAM-1 followed by dilution of the ICAM-1 to negligible concentrations. During many picornavirus infections, noninfectious "altered" virions can be recovered from cells, presumably as a direct or indirect result of interaction with the receptor [Roesing, T. G., P. A. Toselli, and R. L. Crowell, J. Virol. (1975) 15:654-667; Guttman, N. and D. Baltimore, Virol. (1977) 82:25-36]. The relationship of the receptor-neutralized rhinoviruses to such "altered" virions found in other picornaviruses is unclear at present.
Marlin, et al., Nature (1990) 344:70-72 have reported that the extracellular domain of ICAM-1-neutralized virus at a concentration of ICAM considerably lower than that needed to inhibit virus-receptor binding, although this discrepancy was attributed to differences in virus concentrations between the binding and infectivity two assays. Our results, which show a differential effect of the two soluble ICAM-1 proteins even at virus concentrations identical to that in the virus-binding assay indicate that there is a real difference between binding and neutralizing properties of tICAM(453). Possible explanations include: (1) cooperative interactions between ICAM-1 molecules at the surface of the virus leading to more effective virus inactivation, (2) effects of the size of the ICAM-1/rhinovirus complex on virus internalization or uncoating, or (3) interactions of portions of the ICAM-1 molecule other than the two N-terminal domains with the rhinovirus. In an interesting parallel in another system, the soluble CD-4 mediated neutralization of HIV, a nonlinear relationship between soluble CD4 concentration and neutralization at high fractional occupancy of gp 120 molecules was observed, providing evidence for positive cooperativity between virus-bound CD4 molecules [Layne, S. P., M. J. Merges, M, Dembo, J. L. Spouge, and P. L. Nara, Nature (1990) 346:277-279]. While the molecular basis of tICAM(453)-mediated neutralization or its significance to interaction of the rhinovirus with ICAM-1 at the cell surface remains to be determined, it clearly indicates a role for the three domains C-terminal to residue 185.
JY cells at 107 cells/ml (labeled with 10 μCi/ml [35S]-cysteine for 18 hours) were preincubated with soluble ICAM-1 or indicated monoclonal antibodies for 30 minutes at 37� C. in Dulbecco's Modified Essential Medium/2% fetal calf serum and then added to microtiter plates coated with ICAM-1. The plates were then incubated for 60 minutes at 37� C., the plates washed three times with media, and then the bound cells quantified by scintillation counting.
Soluble ICAM molecules were then examined for their ability to inhibit the adhesion of JY lymphoblastoid cells (which express LFA-1) to ICAM-1-coated tissue culture plates. As shown in FIG. 4, tICAM(185) and tICAM(453) both inhibited JY cell binding at identical concentrations of 10 μM, indicating that the LFA-1 binding site is entirely encompassed within the first two domains of ICAM-1.
The identical inhibitory activities of tICAM(453) and tICAM(185) toward ICAM-1/LFA-1-mediated cell adhesion clearly demonstrate that the LFA-1 binding site is completely encompassed within the first 185 residues of ICAM-1. Previous work has indicated that the two N-terminal domains of ICAM-1 were important in LFA-1 binding, in that site-directed mutagenesis at a number of positions in domains I and II reduce the ability to bind to LFA-1 and a shortened transmembrane molecule containing domains I and II retains one-third of the LFA-1 binding activity of full-length ICAM-1 [Staunton, et al., Cell (1990) 61:243-254]. The relatively high concentration of soluble ICAM-1 needed to inhibit cell adhesion illustrates the fact that ICAM-1/LFA-1-mediated cell adhesion is a cooperative result of multiple weak interactions, as might have beer predicted from the strong dependence of ICAM-1/LFA-1 mediated cell adhesion on the density of ICAM-1 [Dustin, M. L. and T. A. Springer, J. Cell Biol. (1989) 107:321-331]. A micromolar dissociation constant has been reported for the CD2/LFA-3 interaction [Sayre, P. H., R. E. Hussey, H-C. Chang, T. L. Ciardelli, and E. L. Rheinherz, "Structural and binding analysis of a two domain extracellular CD2 Molecule," J. Exp. Med. (1989) 169:995-1009], suggesting that low adhesion molecule affinity is a general phenomenon, perhaps to allow for tight regulation of the adhesion process by control of copy number. The identical inhibitory activities of both soluble ICAM-1 proteins toward cell adhesion provide additional evidence that both proteins are correctly folded and underscores the fact that the enhanced neutralizing activity of tICAM(453) relative to tICAM(185) is specific to ICAM-1/rhinovirus interaction.
While the present invention has been described in terms of specific methods and compositions, it is understood that variation and modifications will occur to those skilled in the art upon consideration of the present invention.
For example, it is anticipated that smaller protein fragments and peptides derived from ICAM-1 that still contain the virus-binding site would also be effective.
Further, it is anticipated that the general method of the invention of preparing soluble protein forms from insoluble, normally membrane bound receptor proteins can be used to prepare soluble multimeric forms of other receptor proteins useful for binding to and decreasing infectivity of viruses other than those that bind to the "major group" receptor. Such other viruses include Herpes simplex and Epstein-Barr viruses.
Accordingly, it is intended in the appended claims to cover all such equivalent variations which come within the scope of the invention as claimed, and consequently only such limitations as appear in the appended claims should be placed thereon.
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text:
Bibb And Grapefruit Salad
23 Oct 2010
|Torn bibb lettuce
|1 1⁄2 Cup (24 tbs)
|Pink grapefruit
|1 Medium, peeled
|Sliced scallion
|2 Tablespoon (green onion)
|Finely chopped cilantro/Italian flat leaf parsley
|1 Teaspoon (Chinese parsley)
|Peanut oil
|1 Teaspoon
|White wine vinegar
|1 Teaspoon
|Dijon style mustard
|1 Teaspoon
|Crushed red pepper
|1 Dash
1. Line serving platter with lettuce; decoratively arrange grapefruit over lettuce and sprinkle with scallion.
2. Using a wire whisk, in small mixing bowl beat together reserved grapefruit juice, cilantro, oil, vinegar, mustard, and pepper. Pour over salad.
Recipe Summary
Difficulty Level:Easy
Course:Side Dish
Rate It
0 Comments
Bibb And Grapefruit Salad Recipe


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Select a date to view five days of filings.
There were no filings accepted on this day.
EFS Energy LLC
Application for Certification as an Installer of Distributed Generation Facilities under Section 16-128A of the Public Utilities Act.
Filed: August 22, 2014
ALJ: Katina H. Baker
Ameren Transmission Company of Illinois
Petition for a Certificate of Public Convenience and Necessity, pursuant to Section 8-406 of the Illinois Public Utilities Act, and an Order pursuant to Section 8-503 of the Public Utilities Act, to Construct, Operate and Maintain a New High Voltage Electric Service Line in the Counties of Peoria and Knox, Illinois.
Filed: August 21, 2014
ALJ: Janis Von Qualen; John D. Albers
August 1, 2015 5:00 PM - No deadline, but REQUESTED by August 1, 2015
Chart House Energy LLC
BNSF Railway Company, Village of Table Grove, and the State of Illinois, Department of Transportation.
Stipulated Agreement regarding improving public safety at the Main Street and Laurel Street highway-rail grade crossings of the Company's track in Table Grove, Fulton County, Illinois, designated as crossings AAR/DOT #078 463R, railroad milepost 145.79-LS12, and AAR/DOT #078 468A, railroad milepost 145.89-LS12, respectively.
Vodafone US Inc.
Application for a certificate of authority to operate as a provider of facilities-based and resold local exchange, facilities-based interexchange services and resold wireless services in the State of Illinois.
Filed: August 20, 2014
ALJ: Bonita Benn
BNSF Railway Company,
County of Ogle, Illinois,
Petition for an Order of the Illinois Commerce Commission authorizing the installation of an additional railroad track at the grade crossing inventoried as DOT #069 848H (M.P. 114.75) at what is commonly known as Freeport Road near Polo, IL for determination of suitable and appropriate warning and traffic devices at or near the crossing, and for determination by the Illinois Commerce Commission of the division of costs between the parties.
ALJ: Latrice Kirkland-Montaque
CCG Communications LLC
d/b/a Lightower Fiber Networks
Petition for Withdrawal of its Certificates of Service Authority.
Filed: August 19, 2014
Illinois Commerce Commission
On It's Own Motion
Major Energy Electric Services LLC
Citation for alleged violations of Sections 16-115A(b) and 16-115A(e) of the Public Utilities Act and of 83 Ill. Admin. Code Parts 412.110, 412.130.
ALJ: Claudia Sainsot
FutureGen Industrial Alliance, Inc.
Application for a Certificate Authorizing the Construction and Operation of a Carbon Dioxide Pipeline.
ALJ: Janis Von Qualen


text:
M&M's World New York Coupon - 15% Off Any $30+ PurchaseDecember 20, 2013 - by CG Discounts
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24th April 2013
Comment: We have the power to halt the spread of HIV within a generation 63
11:20 AM — Will Harris from the Terrence Higgins Trust says gay and bisexual men should not wait for a cure when it comes to halting the spread of HIV.
Department of Health launches new HIV prevention campaign for gay and bisexual men in England 42
10:36 AM — Gay and bisexual men in England have the power to halt the spread of HIV in their community within a generation, according to a major new campaign launched today by the Department of Health.
23rd April 2013
Comment: The story of a gay man and his dad 7
3:28 PM — Paul Burgess shares the powerful story of the journey his late father Cyril went on when it came to accepting his son's sexuality.
Marc Almond: 'Why would gay couples want to get married in a church that thinks you are a sinner?' 58
1:09 PM — Marc Almond has questioned same-sex couples who wish to get married in churches that refuse to welcome them "with open arms," the singer also thinks it's a mistake to expect religion to modernise.
22nd April 2013
JLS, Bananarama and Paloma Faith set to headline Birmingham Pride 3
7:07 PM — Boy band JLS will be heading this year's Birmingham Pride as DJs along with Bananarama and a DJ set by Paloma Faith.
World's only transgender MP to give Kaleidoscope Trust lecture 5
1:20 PM — Polish MP Anna Grodzka is to give the annual Kaleidoscope Trust lecture to mark International Day Against Homophobia and Transphobia.
Kent: No further action to be taken over former youth crime commissioner's anti-gay tweets 6
11:17 AM — Former youth police and crime commissioner Paris Brown will not face prosecution over homophobic and racist comments she made on Twitter.
UK: Deputy Lib Dem leader 'really disappointed' at IPS rejection of trans and intersex inclusive passports 19
10:30 AM — Lib Dem MP Simon Hughes says he is "really disappointed" by the Identity and Passport Service's decision to reject non gender-specific passports for trans and intersex people in Britain.
19th April 2013
Cleo Rocos: We imagined 'Future Queen Dies In Gay Bar Brawl' headlines over Princess Diana drag disguise 9
6:30 PM — Comedian Cleo Rocos has elaborated on her claims that, along with Freddie Mercury and Kenny Everett, she snuck Princess Diana into a London gay bar disguised as an "eccentrically dressed gay male model".
UK: Thames Valley sees rise in recorded homophobic crime 5
12:58 PM — Thames Valley Police have recorded an increase in homophobic crime across Berkshire, Buckinghamshire and Oxfordshire.
Duncan James: 'People were shocked when they heard I was gay on The Big Reunion' 23
12:20 PM — Even though Duncan James came out three years ago, some of his fans only learnt about the singer's sexuality from watching The Big Reunion last month.
18th April 2013
Brighton: English Defence League to march near city's gay bars 37
5:48 PM — LGBT businesses in Brighton are concerned at the prospect of an English Defence League (EDL) march taking place on their doorstep this Sunday.
UK: Court of Appeal to review sentence of man who burnt gay teenager to death 12
3:50 PM — The Attorney General's Office has told PinkNews.co.uk that a decision of a judge to hand down a three and a half year sentence for the killing of a gay teenager has now been referred to the Court of Appeal.
Gloucestershire: British Transport Police seek to identify youth over homophobic incident 7
1:41 PM — Police are appealing for help in identifying a youth after a train manager was the victim of homophobic abuse in Gloucestershire.
17th April 2013
Bishop of Chichester to attend Christian LGBT conference in Brighton 8
6:42 PM — The leader of the Church of England in Sussex will attend an LGBT conference in Brighton this weekend.
UK: Thatcher opponents stage funeral protests 40
12:51 PM — A small group of protesters of Margaret Thatcher's LGBT legacy have lined the route of the former PM's funeral in central London.
16th April 2013
Amal Fashanu: 'I was getting ready to visit Elton John's studio when I learnt Justin Fashanu had died' 6
4:57 PM — The niece of gay footballer Justin Fashanu has spoken about the moment she was told her uncle had died.
Jeremy Irons: 'I don't know the difference between a same-sex marriage and a civil partnership' 37
3:10 PM — Jeremy Irons says he should have "buttoned" his lip when he joked equal marriage could lead to fathers marrying their sons, although he now admits that he doesn't know the difference between a same-sex marriage and a civil partnership.
Ian McKellen: 'Margaret Thatcher misjudged the future with Section 28′ 23
1:46 PM — Actor Sir Ian McKellen says Margaret Thatcher "misjudged the future" with her support for Section 28.
Archbishop of Canterbury to discuss gay rights with Peter Tatchell on Thursday 50
12:33 PM — The Archbishop of Canterbury, Justin Welby, is set to discuss gay rights with Peter Tatchell on Thursday at Lambeth Palace.


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Teen Mom
Pregnant Leah Messer Spotted Out and About With Her Twins! (PHOTO)
Stop what you're doing and take a look at these brand-spankin' new pictures of Teen Mom 2's Leah Messer, who is expecting baby no. 3 this winter!
We couldn't be more thrilled to report that Leah — who appears to be about 6 - 7 months preggo in this shot on her Facebook fan page — and her twins girls, Ali and Aleeah, look happy and healthy! The MTV star and the girls spent the day frolicking at a park near their home on Oct. 28.
Leah and her second hubby, Jeremy Calvert, announced the pregnancy on Oct. 23.
"Jeremy and I are excited to be adding a new addition to our family," the 20-year-old told Us Magazine. "Being a teen mom was difficult, but I'm older and in a different place now — married to Jeremy and excited for our family to grow."
Congrats to the happy family, and looking forward to seeing Leah on our small screens this season on Teen Mom 2! Catch Teen Mom 2 Season 3 on Monday nights at 10 p.m. ET on MTV.
Wanna chat with fellow Teen Mom fans? Join the discussion on our Teen Mom Facebook page!
Follow @TeenMomWetpaint on Twitter
More About Teen Mom
Teen Mom News
Teen Mom 2 News
Teen Mom 3 News


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|Hawthorn Suites Goose Creek properties are listed below. Search for cheap and discount Hawthorn Suites hotel rooms in Goose Creek, SC for your upcoming meeting or individual travels. We list the best Hawthorn Suites Goose Creek properties so you can review the Goose Creek Hawthorn Suites hotel list below to find the perfect place.
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Laparoscopy in Gynecology
Laparoscopy is the term that refers to minimally invasive surgery of the abdomen and pelvis and this article will review the details of this procedure when used in gynecology
Management of Birth Canal Cancers
A malignancy of the birth canal (BC) can be difficult to detect. The symptoms can be non-specific but any abnormal bleeding, unusual discharge or genital mass warrants further evaluation and prompt treatment. Read this article to learn more about the management of this cancer.
Management of VIN and VAIN
Precancerous lesions of the genital tract are the precursors to cancer. A large percentage of these lesions will resolve spontaneously but it is impossible to know which ones will progress. Read this article to learn how these lesions are treated.
Medical Management of Obesity
Obesity leads to many serious medical problems and premature death but can be reversed. Diet, regular exercise and behavior modifications are effective in select groups but for those with obesity other interventions maybe required. This article will review the medical treatments.
Medical Treatment of Endometriosis
Endometriosis is a common problem in menstruating women. It causes chronic pelvic pain and infertility. The pain can affect the quality of life, interfere with intimate relationships and can be quite debilitating. Read this article to learn about medical treatment.
Medical Treatment of Fibroids
Fibroids or leiomyomata are benign smooth muscle tumors of the uterus. The most common presentation of fibroids is irregular or heavy uterine bleeding or prolonged heavy periods. Not all fibroids need to be treated but medical options are available if needed. Read this article to learn more.
Medications for Hypertension
High blood pressure is the most common reason for office visits in non-pregnant adults. Approximately 30% of adults in the U.S. suffer from this problem. Different categories of medications have been developed to treat high blood pressure. This article will review the various categories.
Medications to Prevent Breast Cancer
All women and some men are at risk of developing breast cancer. There are medications available to prevent breast cancer but these should only be prescribed to those who are at high risk. Read this article to learn more about these medications.
Medications Used for Mild Endometriosis
Endometriosis is a chronic condition, which can recur and progress. A large percentage of women require long-term treatment. Many hormonal type medications are utilized in the initial and long-term management of endometriosis. Read this article to learn more.
Medications Used for Severe Endometriosis
Endometriosis may plaque some women their entire reproductive life. There is a wide range of analgesics and hormonal medication that are effective but in some cases, this treatment is insufficient and other treatment options should be explored. Read this article to learn more.
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CBS' 2008-09 Schedule Expands Comedies, Adds Drama
As expected, CBS has ordered the new dramas "
With 18 shows returning to the strongly performing network, CBS has a fair amount of scheduling stability, so the biggest shift may be in returning comedies to Wednesday night after a season of keeping laughter as a Monday-only thing.
Monday will still feature a two-hour comedy block on the network, with "The Big Bang Theory,"
The addition of "Worst Week" to Mondays has allowed CBS to move
"We've been looking to expand our comedy lineup, and we now have the depth and development to make the move," says CBS Entertainment President Nina Tassler in a statement. "Monday is already television's signature night for comedy and we think we can make the case that CBS is the signature network for comedy overall."
CBS' schedule hasn't had many problem spots in recent years, but the Tuesday 10 p.m. hour has been a show-killer lately. To that end, CBS is transplanting "Without a Trace," a proven hit with a track record on both Thursday and Sunday nights, into the hour.
The network has also had difficulties on Friday nights at 9 p.m. where "Close to Home" was cancelled despite solid ratings two years ago and where
One place CBS doesn't need to worry about ratings is the Thursday night 10 p.m. slot after "
In addition to "Without a Trace" and "The New Adventures of Old Christine," the other CBS show to switch time slots is "The Unit," which leaves Tuesday for Sundays, where it will have under-the-radar hit
Beyond "Harper's Island," CBS has also ordered
CBS' Fall 2008-09 Schedule (New shows in bold, all times ET):
8:30-9:00 PM "How I Met Your Mother"
9:00-9:30 PM "Two and a Half Men"
9:30-10:00 PM "Worst Week" 10:00-11:00 PM "CSI: Miami"
9:00-10:00 PM "The Mentalist 10:00-11:00 PM "Without a Trace" (New time)
8:00-8:30 PM "The New Adventures of Old Christine" (NT)
8:30-9:00 PM "Project Gary"
9:00-10:00 PM "Criminal Minds" 10:00-11:00 PM "CSI: NY"
9:00-10:00 PM "CSI: Crime Scene Investigation"
10:00-11:00 PM "Eleventh Hour"
8:00-9:00 PM "Ghost Whisperer"
9:00-10:00 PM "The Ex List" 10:00-11:00 PM
8:00-9:00 PM Crime drama repeats
9:00-10:00 PM Crime drama repeats 10:00-11:00 PM
9:00-10:00 PM "Cold Case" 10:00-11:00 PM


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If it makes the injury heal stronger, what's the big deal? It hurts for, like.. what, a few hours? It's usually only skin-deep.. it's not like they're burning to the bone. (It doesn't look like they use *acid* by the way.. just heat.. acid would pretty much destroy the tendon.)
Don't people stick needles into their skin all the time(acupuncture) to help their health?
Look at it this way-- if your horse hadn't been pinfired, do you think you would have that horse, with him being sound with his bowed tendon?


text:
2013 - October Dodge Ram Member Of The Month
Winner will receive a code good for 10% off the purchase of a Supercharger kit for your 2009-2010 Ram!
We are pleased to sponsor the MOTM by offering $100 off a minimum order of $125!!!
Any other discounts will not apply to this prize. On Canadian orders duties/taxes/fees will be paid by the recipient on the original order amount prior to the MOTM discount.
*Click our banner for our 'Deal of the month'*
In addition to the $100 savings we are proud to announce every MOTM winner from here on out until further notice will also receive a Bully's Performance sponsorship!!
Get your nominations in!
Voting begins on the 15th!
It is that time again!! Please take the time to review the rules below!
1: Nominations will last for the first half of the month and voting will go on for the second half of the month.
2. You can nominate up to two members for the contest. Don't nominate yourself!
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4: The 5 nominees and ties with the most votes at nomination closing will be moved into the voting gallery for a final vote count.
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Should a member receive nominations or votes in the contest, and the member's name has been mentioned on any other forum in a thread/post mentioning this contest, both the person mentioning the name and the member mentioned will be disqualified from the contest that month as well as all future months. No member is allowed to make a post, profile or private message that would indicate they are currently in an active contest. If staff deems the act as a malicious attempt on the part of the poster to cause a disqualification we will allow the contestant to remain in the running and the offending member will be banned.
8: The winner will a special MOTM badge to sport by their user name. The winner must write a minimum 500+ word essay about their Dodge Ram, (include an image of themselves) what it means to them, and what they think about the forum, its members, and how much fun they are having here!:Banana01::999::pepper: (Or not having :( ) In which case we will have to :LOL:
9: MOTM winners are not eligible to run for one year after their win.
10: In the event of a tie, the nominee with the most nominations of the tied contestants will be declared the winner.
(Please Read Carefully Before Making Your Nominations! *ALL NON-ELIGIBLE ENTRIES WILL BE IGNORED*)
Not Eligible For This MonthGrubrunner
Arth 11-2013
Asur 12-2013
bighemi04 1-2014
jhinkle1989 2-2014
Excoastie 3-2014
Chris b 4-2014
ShawnC13 5-2014
CajunDodge 6-2014
gonefishin776 7-2014
Hermes1 8-2014
brandonjansen 9-2014
Canadian Hemi Pride
primo spaghetti
Non-Eligible Nominees Per Member Requests
WarlockIII and PaulMys
WarlockIII and PaulMys
PaulMys and RogerRamJet
WarlockIII and RogerRamJet
Warlock III and Chunkem
PaulMys and Chunkem.
- Cajun
Dang, so Grubrunner still not eligible. I would like to nominate PaulMys and Roger RamRamJet
PaulMys and RogerRamJet get my nominations as well.
PaulMys & RamRamJet
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We Did It! A Victory for VAWA
Recent Victories
Oleg Savvin, Mikhail Feldman and Dmitriy Fonarev, who have spent more than a year in detention for raising the German flag over an official building in Kaliningrad on 4 March 2014, were released on 17 June.
Foudama Ousmane, a detainee who was at risk of blindness, was examined by a doctor on the morning of 20 April and prescribed medication for his eyes. This was as a direct result of the mobilization of Amnesty International's members around the world who called and sent emails to authorities in Cameroon, after an Urgent Action was issued on 16 April.
Sandara Farouq Kadouda was released on 15 April. She had been abducted on 12 April by a group of men in civilian clothes, believed to belong to the National Intelligence Security Service (NISS).
Celia Ismalej and her 18-month-old son have been released from detention after being granted asylum protection in the USA.


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Team tool: Practice challenging veterinary client conversations
Use these practice cases to rehearse challenging client conversations.
Sep 01, 2013
Practice these challenging veterinary client interactions to prepare for some of the tough conversations you face with clients each day.
Hot topics on dvm360
22 stress-hacks that will change your life
May 27, 2015
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Apr 28, 2015


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Search Results for: Furque
Alberto Furque: 2005 Tempranillo, 2009 Malbec and 2009 Syrah
IMAGINE living in a place where over 80% of the year you are basking in sunshine, and yet, have at your disposal elevations of over 3000 feet, immense diurnal temperature swings, and a river from which to source water (otherwise … Continue reading
Filed under Alberto Furque, Argentina, Emily Crichton, Malbec, Mendoza, Syrah, Tempranillo, Uco Valley
2005 Alberto Furque Tempranillo
The more I try new wines, the more I realize that I really like wine. Love wine, actually. If you were a fly on the wall here at TWH this week, you may have heard me mumbling something about … Continue reading
Filed under Argentina, Peter Zavialoff
Malbec from Alberto Furque
I am going to "alaverdi" my praise of the 2007 Malbec from Alberto Furque onto the 2008. Ala-ve…what? In Georgia, the country and not the state, celebrations at the table are epic, lasting hours and even days. Eating and drinking … Continue reading
Filed under Anya Balistreri
The May 2015 Dirty Dozen
Ah, May … It's the only month of spring with 31 days in it. And that's plenty of time to fit a little fun in. Speaking of fun, check out the May 2015 Dirty Dozen: 12 wines, all different, all … Continue reading
Filed under The Dirty Dozen, Wine Clubs/Samplers
November 2014 Dirty Dozen
And just like that, it's November. It's getting chilly out. It's getting darker earlier and earlier. No need to fear, we've arrived at that time of year where people gather indoors and enjoy one another's company. The Holidays are around … Continue reading
Filed under Anya Balistreri, Peter Zavialoff, The Dirty Dozen
August 2014 Dirty Dozen
How lucky the calendar makers decided to give summer not one, but two months with 31 days in them. Welcome to August! 'Tis the month of leisure, holiday, and relaxation. Ah, but there are also picnics, trips to the beach, … Continue reading
Filed under Peter Zavialoff, The Dirty Dozen, Wine Clubs/Samplers
August 2012 Dirty Dozen
How fortunate that summer gives us not 1, but 2 months with 31 days in them. Let's revel in that! That leaves plenty of time for more picnicking and barbecuing, among other fun summer endeavors. Whether you're on vacation, a … Continue reading
Filed under Alberto Furque, Argentina, Malbec, Mendoza, New Zealand, Peter Zavialoff, Portugal, Rose, Tempranillo, The Dirty Dozen, Touraine, Washington State
April 2012 Dirty Dozen
Days get longer, the nights grow short, our Easter baskets are getting filled up, and what's this? Baseball season? Yep, it's April and it's time for opening the windows and doors, getting some fresh air, and maybe a picnic or … Continue reading
January 2012 Dirty Dozen
Happy New Year! As the whirr of the holi-daze shrinks away in our rear view mirrors, we look forward to many more vinous discoveries coming in 2012! The new year brings hope and optimism, resolutions, and the NFL playoffs! There's … Continue reading
Filed under Alberto Furque, Chianti, Malbec, Monastrell, Peter Zavialoff, Rose, Southwest France, The Dirty Dozen, Touraine
July 2011 Dirty Dozen
Be a fan on Facebook and follow us on Twitter! Check out our Dirty Dozen archive HERE on our blog Yay! Summer's here … finally. To get you going this July, may we suggest the Dirty Dozen. That's 12 different … Continue reading


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Exclusive: how the Mazda Furai died
Back in 2008, Mazda's incredible race concept disappeared without trace. Here's what happened
Posted: 19 Feb 2015
Over a period of three years between 2005 and 2008, Mazda produced some stunning design concepts showcasing its 'Nagare' design language. And although Mazda subsequently evolved Nagare into the 'Kodo' theme in 2011 with the Shinari concept (translated into road-going metal like the CX-5 and Mazda6) the Nagare dynasty remains a glimpse into a world where Mazda designers were set free to push boundaries. There were no limits or restrictions. The Furai? Basically the poster boy of Nagare, the last and most eye-popping vehicle to be born of that philosophy. Created as a concept to show how a racecar for the road might look, it was the brainchild of a design department deeply proud of its motorsport heritage, the most extreme concept of its generation. It won global acclaim, but was more than that: the Furai was possibly the defining concept car of its era. Which makes what happens next all the more tragic.
So what did actually happen that summer?


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Black bear spotted again Wednesday in Arvada, police say
ARVADA, Colo. - Arvada police say several bear sightings have been reported in the city on Wednesday.
There were reports of a bear near West 72nd Avenue and Wadsworth Boulevard. A bear was also spotted nearby at the Arvada Center.
This comes one day after a bear was seen in a tree near at West 66th Lane and Union Street Trail.
The bear was spotted again in the same area about 5 p.m. Tuesday, Arvada police said via Twitter.
Police said Arvada Animal Management officers were monitoring the situation and asked residents to "remain alert and keep pets inside."
"Please don't approach [the bear]," Police tweeted. "Please take your pets inside and call 911 if you spot him," police said on Facebook.