text:
|Publication number
|Publication type
|Application number
|Publication date
|May 11, 2004
|Filing date
|Apr 10, 2000
|Priority date
|Apr 9, 1999
|Fee status
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|Publication number
|Tracy D. Wilkins, David M. Lyerly, J. Scott Moncrief, Limin Zheng, Carol Phelps
|Original Assignee
|Techlab, Inc.
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|Patent Citations (9), Non-Patent Citations (14), Referenced by (11), Classifications (21), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
This application claims priority under 35 U.S.C � 119(e) of provisional applications Ser. No. 60/190,111 filed Mar. 20, 2000; Ser. No. 60/186,201 filed Mar. 1, 2000; and 60/128,686 filed Apr. 9, 1999. The content of these applications are incorporated herein by reference.
The present invention relates to the field of medical immunology and further to pharmaceutical compositions, methods of making and methods of use of vaccines. More specifically this invention relates to recombinant proteins derived from the genes encoding Clostridium difficile toxin A and toxin B, and their use in an active vaccine against C. difficile.
Clostridium difficile, a Gram positive anaerobic spore-forming bacillus is an etiologic agent of antibiotic associated diarrhea (AAD) and colitis (AAC). The symptoms of the disease range from mild diarrhea to fulminant and life-threatening pseudomembranous colitis (PMC). Antibiotic therapy can disrupt the normal intestinal microflora. Destruction of the normal flora results in a condition in which C. difficile can spores of C. difficile can germinate and the organism can grow and produce disease causing toxins. C. difficile causes about 25% of antibiotic-associated diarrheas, however, it is almost always the causative agent of PMC (Lyerly, D. M. and T. D. Wilkins, in Infections of the Gastrointestinal Tract, Chapter 58, pages 867-891, (Raven Press, Ltd, New York 1995)). Additionally, C. difficile is frequently identified as a causative agent of nosocomial infectious diarrheas, particularly in older or immuno-compromised patients (U.S. Pat. No. 4,863,852 (Wilkins et al.) (1989)).
Disease caused by C. difficile is due to two enteric toxins A and B produced by toxigenic strains (U.S. Pat. No. 5,098,826 (Wilkins et al.) (1992)). Toxin A is an enterotoxin with minimal cytotoxic activity, whereas toxin B is a potent cytotoxin but has limited enterotoxic activity. The extensive damage to the intestinal mucosa is attributable to the action of toxin A, however, toxins A and B act synergistically in the intestine.
The genetic sequences encoding both toxigenic proteins A and B, the largest known bacterial toxins, with molecular weights of 308,000 and 269,000, respectively, have been elucidated (Moncrief et al., Infect. Immun. 65:1105-1108 (1997); Barroso et al., Nucl. Acids Res. 18:4004 (1990); Dove et al. Infect. Immun. 58:480-488 (1990)). Because of the degree of similarity when conserved substitutions are considered, these toxins are thought to have arisen from gene duplication. The proteins share a number of similar structural features with one another. For example, both proteins possess a putative nucleotide binding site, a central hydrophobic region, four conserved cysteines and a long series of repeating units at their carboxyl ends. The repeating units of toxin A, particularly, are immunodominant and are responsible for binding to type 2 core carbohydrate antigens on the surface of the intestinal epithelium (Krivan et al., Infect. Immun. 53:573-581 (1986); Tucker, K. and T. D. Wilkins, Infect. Immun. 59:73-78 (1991)).
The toxins share a similar molecular mechanism of action involving the covalent modification of Rho proteins. Rho proteins are small molecular weight effector proteins that have a number of cellular functions including maintaining the organization of the cytoskeleton. The covalent modification of Rho proteins is due to glucosyltransferase activity of the toxins. A glucose moiety is added to Rho using UDP-glucose as a co-substrate (Just et al. Nature 375:500-503 (1995), Just et al. J Biol. Chem 270:13932-13939 (1995)). The glucosyltransferase activity has been localized to approximately the initial 25% of the amino acid sequence of each of these toxins (Hofmann et al. J Biol. Chem. 272:11074-11078 (1997), Faust and Song, Biochem. Biophys. Res. Commun. 251:100-105 (1998)) leaving a large portion of the toxins, including the repeating units, that do not participate in the enzymatic activity responsible for cytotoxicity.
The toxin A protein comprises 31 contiguous repeating units (rARU) and may contain multiple T cell epitopes (Dove et al. Infect. Immun. 58:480-488 (1990). The repeating units are defined as class I repeats and class II. rARU may be uniquely suited for use in inducing T cell-dependent response to an antigen. The sequence of each unit is similar but not identical. These features along with its usefulness in eliciting toxin A neutralizing antibodies make rARU a novel candidate as a carrier protein.
The toxin B repeating units have similar features to those of rARU. Like rARU, the recombinant toxin B repeating units (rBRU) are relatively large (˜70 kDa) and are composed of contiguous repeats of similar amino acid sequences (Barroso et al. Nucleic Acids Res. 18:4004 (1990); Eichel-Streiber et al. Gene 96:107-113 (1992)). Less is known about this portion of toxin B than the binding domain of toxin A.
Thomas et al (U.S. Pat. No. 5,919,463 (1999)) disclose C. difficile toxin A or toxin B or certain fragments thereof as mucosal adjuvants intranasally administered to stimulate an immune response to an antigen (e.g., Helicobacter pylori urease, ovalbumin (OVA), or keyhole limpet hemocyanin (KLH)). However, Thomas does not teach the use of such adjuvant for protection against strains of C. difficile. Lyerly et al. Current Microbiology 21:29-32 (1990) considered at a smaller recombinant fragment from the toxin A repeats in hamster protection assays. However, these data suggest at best only a very weak or partial protection from strains of C. difficile, whereas the present invention demonstrates the use of C. difficile toxin repeating units that provide a clear immunogenic response and at higher levels, which afford protection against C. difficile.
Even were one to consider rARU and rBRU as candidate proteins for conjugate vaccines, the production of such proteins presents certain challenges. There are methods for the production of toxin A and antibodies elicited thereto (U.S. Pat. No. 4,530,833 (Wilkins et al.) (1985); U.S. Pat. No. 4,533,630 (Wilkins et al.) (1985); and U.S. Pat. No. 4,879,218 (Wilkins et al.) (1989)). There are significant difficulties in producing sufficient quantities of the C. difficile toxin A and toxin B proteins. These methods are generally cumbersome and expensive. However, the present invention provides for the construction and recombinant expression of a nontoxic truncated portions or fragments of C. difficile toxin A and toxin B in strains of E. coli. Such methods are more effective and commercially feasible for the production of sufficient quantities of a protein molecule for raising humoral immunogenicity to antigens.
Part of the difficulty that the present invention overcomes concerns the fact that large proteins are difficult to express at high levels in E. coli. Further, an unusually high content of AT in these clostridial gene sequences (i.e., AT-rich) makes them particularly difficult to express at high levels (Makoff et al. Bio/Technology 7:1043-1046 (1989)). It has been reported that expression difficulties are often encountered when large (i.e., greater than 100 kd) fragments are expressed in E. coli. A number of expression constructs containing smaller fragments of the toxin A gene have been constructed, to determine if small regions of the gene can be expressed to high levels without extensive protein degradation. In all cases, it was reported that higher levels of intact, full length fusion proteins were observed rather than the larger recombinant fragments (Kink et al., U.S. Pat. No. 5,736,139; see: Example 11(c)). It has been further reported that AT-rich genes contain rare codons that are thought to interfere with their high-level expression in E. coli (Makoffet al. Nucleic Acids Research 17:10191-10202). The present invention provides for methods to produce genes that are both large and AT-rich and immunogenic compositions thereof. For example, the toxin A repeating units are approximately 98 kDa and the gene sequence has an AT content of approximately 70% that is far above the approximately 50% AT content of the E. coli geneome. The present invention provides for methods of expressing AT-rich genes (including very large ones) at high levels in E. coli without changing the rare codons or supplying rare tRNA.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. Further, all documents referred to throughout this application are incorporated in their entirety by reference herein. Specifically, the present application claims benefit of priority to U.S. provisional patent application serial No. 60/190,111, which was filed on Mar. 20, 2000; and U.S. provisional patent application serial No. 60/186,201, which was filed on Mar. 1, 2000; and U.S. provisional patent application serial No. 60/128,686, which was filed on Apr. 9, 1999, and which provisional patent applications are incorporated in their entirety by reference herein.
The present invention is drawn to an immunogenic composition that includes recombinant proteins. The genes encoding the proteins are isolated from a strain of C. difficile. A preferred embodiment of this invention provides that at least one protein is a toxin or a toxin fragment. A further preferred embodiment provides that the toxin is C. difficile toxin A or toxin B. A more preferred embodiment of the present invention provides that the recombinant protein components are nontoxic and comprise a portion of both toxins including all of the amino acid sequence of the C. difficile toxin A or toxin B repeating units (RARU or rBRU) or fragment thereof. The immunogenic composition may further include a carbohydrate moiety as well as a pharmaceutically acceptable carrier or other compositions in a formulation suitable for injection in a mammal.
Another embodiment of the invention is that the rARU and rBRU components are combined, preferably in a manner that results in high levels of neutralizing antibodies to toxins A and B when the immunogenic composition is used in vaccine. The components may be admixed at different ratios. Further, the rARU and rBRU components may be chemically or physically linked to form a complex. Another preferred embodiment is that the rARU and rBRU sequences, or fragments thereof, may be genetically fused in a manner that results in the production of a hybrid molecule. A further embodiment is that the immunogenic composition elicits antibodies that precipitate the native C. difficile toxins and neutralize their cytotoxic activity thus providing protection against C. difficile associated disease.
FIG. 1. Schematic of toxins A and B. The repeating units of the toxins function in binding to the cell surface. Antibodies to the repeating units of the toxins neutralize
FIG. 2 shows the nucleotide sequence (SEQ ID NO: 1) (numbers 5690-8293, GenBank accession number M30307, Dove et al. 1993) of the toxin A gene region that encodes rARU and the toxin A stop codon. The sequence encodes for the entire repeating units of toxin A from C. difficile strain VPI 10463 as defined by Dove et al. (Dove et al., Infect Immun. 58:480-488 (1990)). In addition it encodes for 4 amino acids upstream of the beginning of the repeating units and a small stretch of hydrophobic amino acids at the end of toxin A. The Sau3A site (underlined) at the beginning of the sequence was used to subclone the gene fragment to an expression vector. The stop codon at the end of the sequence is italicized.
FIG. 3 shows the amino acid sequence (SEQ ID NO:2) (GenBank accession number M303307) of rARU. The invention contemplates the use of any recombinant protein containing this amino acid sequence, any fragment therein, any fusion protein containing rARU or a fragment therein, and any larger fragment from toxin A carrying all or part of rARU, as a carrier for conjugate vaccine compositions.
FIG. 4 shows the expression vector pRSETB-ARU-Kmr used for expression of rARU. A Sau3A/HindIII gene fragment of approximately 2.7 kb containing the entire nucleotide sequence encoding rARU, stop codon, and a small region downstream of the toxin A stop codon, was subcloned to the vector pRSETB digested with BarrHI and HindIII. In a subsequent step the kanamycin resistance gene was subcloned at the HindIII site located downstream of the rARU gene fragment. The 1.2 kb fragment encoding the Kmr gene was derived from pUC4K (GenBank accession number X06404) by digestion with EcoRI and subcloned at the HindIll site after blunt ending of the vector and Kmr cassette with Klenow fragment. Expression vector pRSETB-ARU-Kmr was transformed into BL21(DE3) for expression of rARU under control of the T7 promoter.
HindIII/EcoRI sites were eliminated by blunt ending.
FIG. 5 shows an SDS-PAGE gel (15% acrylamide) of rARU expression and purification steps. Lanes: 1) 4 μl of 10X BL21(DE3) E. coli/pRSETB-ARU-Kmr lysate 2) 4 μl of dialyzed 40% ammonium sulfate fraction at 10X relative to the original culture volume 3) 5 μl rARU (0.88 mg/ml) purified by CL-6B Sepharose anion exchange chromatography.
FIG. 6 shows the nucleotide sequence (SEQ ID NO:3) (GenBank accession number X531138, Wilkins et al. 1990) of the toxin B gene region that encodes rBRU and a small portion upstream. The Sau3a restriction sites used for subcloning are underlined. The sequence of the repeating units of toxin B from C. difficile strain VPI was defined previously (Eichel-Streiber et al. Mol. Gen. Gen. 233:260-268).
FIG. 7 shows the amino acid sequence (SEQ ID NO:4) (GenBank accession number X53138) of rBRU and a small upstream region. The invention contemplates the use of any recombinant protein containing this amino acid sequence, any fragment therein, any fusion protein containing rBRU or a fragment therein, and any larger fragment from toxin B carrying all or part of rBRU, as a component in a vaccine against C. difficile.
FIG. 8 shows the expression vector pRSETC-BRU-Kmr used for expression of rBRU. A gene fragment of approximately 1.8 kb containing nearly the entire nucleotide sequence encoding rBRU (final 10 amino acids of toxin B are eliminated) was subcloned from the toxin B gene (Phelps et al. Infect. Immun. 59:150-153 (1991)) to pGEX-3X. A BamHI/EcoRI from pGEX-3X-BRU was subcloned to pRSETC. In a subsequent step the kanamycin resistance gene was subcloned at the EcoRI site located downstream of the rBRU gene fragment. The 1.2 kb fragment encoding the Kmr gene was derived from pUC4K (GenBank accession number X06404) by digestion with EcoRI. Expression vector pRSETC-BRU-Kmr was transformed into BL21 (DE3) for expression of rBRU under control of the T7 promoter.
FIG. 9. SDS-PAGE of purified rARU and partially purified rBRU. Lanes; 1) rARU purified by sequential ammonium sulfate precipitation and Sepharose CL-6B anion exchange chromatography, 2) rBRU partially purified by ammonium sulfate precipitation and hydrophobic interaction chromatography on phenyl Sepharose, 3) lysate (10X concentration) of Escherichia coli BL21(DE3)/pRSETC-BRU-Kmr.
FIG. 10. Crossed-immunoelectrophoresis of (A) C. difficile culture filtrate and (B) partially purified rBRU. C. difficile goat antisera was used as the precipitating antibody.
FIG. 11. shows an example of a genetic fusion of rARU and rBRU. A Sau3A/EcoRI toxin A gene fragment (nucleotides 5530 through 6577) may be fused to an ApoI toxin B gene fragment (nucleotides 5464 through 6180) to create an in-frame 1,763 bp gene fusion expressing a 588 amino acid rARU′/′rBRU′ fusion protein of approximately 68 kDa containing a significant portion of the repeating units from both toxin genes. The rARU′ fragment encodes an epitope for PCG-4 represented by the open bar in the rARU′ portion of the gene fusion.
The present invention is drawn to an immunogenic composition that includes at least one-recombinant protein component, wherein the gene encoding the protein component is isolated from a strain of Clostridium difficile. A preferred embodiment of this invention provides that the protein is a toxin or a toxin fragment. An even further preferred embodiment provides that the toxin is toxin A, with yet a further preferred embodiment being a portion of the toxin containing all of the amino acid sequence of the toxin A repeating units (rARU) or fragment thereof. Another preferred embodiment is that the toxin is toxin B, with yet another preferred embodiment being a portion of the toxin containing all of the amino acid sequence of the repeating units (rBRU) or a fragment thereof. The immunogenic composition may further include a pharmaceutically acceptable carrier or other compositions in a formulation suitable for injection in a mammal.
These immunogenic compositions of the present invention elicit an immune response in a mammalian host, including humans and other animals. The immune response may be either a cellular dependent response or an antibody dependent response or both and further the response may provide immunological memory or a booster effect or both in the mammalian host. These immunogenic compositions are useful as vaccines and may provide a protective response by the mammalian subject or host to infection by strains of Clostridium difficile.
The present invention further includes methods for producing an immunogenic composition by: constructing a genetic sequence encoding a recombinant protein component, where the gene encoding the protein component is isolated from a strain of Clostridium difficile, expressing the recombinant protein component in a microbial host; recovering the recombinant protein from a culture of the host; conjugating the protein to a second protein component, and recovering the conjugated protein and polysaccharide component. The protein component may also consist of a fusion protein, whereby a portion of said recombinant protein is genetically fused to a second protein component. Preferably the expression of the genetic sequence is regulated by an inducible promoter that is operatively positioned upstream of the sequence and is functional in the host. Even further, said genetic sequence is maintained throughout the growth of the host by constant and stable selective pressure. Maintenance of the expression vector may be conferred by incorporation in the expression vector of a genetic sequence that encodes a selective genotype, the expression of which in the microbial host cell results in a selective phenotype. Such selective genotypes, include a gene encoding resistance to antibiotics, such as kanamycin. The expression of this selective genotypic sequence on the expression vector in the presence of a selective agent or condition, such as the presence of kanamycin, results in stable maintenance of the vector throughout growth of the host. A selective genotype sequence could also include a gene complementing a conditional lethal mutation.
Other genetic sequences may be incorporated in the expression vector, such as other drug resistance genes or genes that complement lethal mutations.
Microbial hosts of this invention may include: Gram positive bacteria; Gram negative bacteria, preferably E. coli; yeasts; filamentous fungi; mammalian cells; insect cells; or plant cells.
The methods of the present invention also provide for a level of expression of the recombinant protein in the host at a level greater than about 10 mg/liter of the culture, more preferably greater than about 50 mg/liter and even more preferably at 100 mg/liter or greater than about 100 mg/liter. The molecular weight of the protein is greater than about 30 kDa, preferably greater than about 50 kDa and even more preferably greater than about 90 kDa. This invention also provides that the protein may be recovered by any number of methods known to those in the art for the isolation and recovery of proteins, but preferably the recovery is by ammonium sulfate precipitation followed by ion exchange chromatography.
The present invention further includes methods for preparing the immunogeniccomposition that provides that the protein component is conjugated to a second protein component by one of a number of means known to those in the art, particularly an amidization reaction.
Also, high yields of recombinant protein may be dependent on the growth conditions, the rate of expression, and the length of time used to express AT-rich gene sequences. In general, AT-rich genes appear to be expressed at a higher level in E. coli during a post-exponential or slowed phase of growth. High-level production of the encoded protein requires moderate levels of expression over an extended period (e.g. 20-24 h) of post-exponential growth rather than the typical approach of high-level expression during exponential growth for shorter periods (e.g. 4-6 h). In this regard, it is more efficient to maintain plasmids carrying the gene of interest by maintaining constant selective pressure for the gene or its expression vector during the extended period of growth. One aspect of the present invention is using an antibiotic that is not inactivated or degraded during growth of the expression host cell as is found with ampicillin. One such preferred embodiment involves the expression of genes encoding resistance to kanamycin as the selective phenotype for maintaining the expression vector which comprises such kanamycin resistance genetic sequences. Expression of large AT-rich clostridial genes in E. coli at levels (>100 mg/liter) provided for by methods of the present invention was hitherto unknown.
Terms as used herein are based upon their art recognized meaning and should be clearly understood by the ordinary skilled artisan.
An immunogenic composition is any composition of material that elicits an immune response in a mammalian host when the immunogenic composition is injected or otherwise introduced. The immune response may be humoral, cellular, or both.
A fusion protein is a recombinant protein encoded by a gene or fragment of a gene, genetically fused to another gene or fragment of a gene.
A booster effect refers to an increased immune response to an immunogenic composition upon subsequent exposure of the mammalian host to the same immunogenic composition. A humoral response results in the production of antibodies by the mammalian host upon exposure to the immunogenic composition.
rARU is a recombinant protein containing the repeating units of Clostridium difficile toxin A as defined by Dove et al. (Dove et al. Infect. Immun. 58:480-488 (1990)). The nucleotide sequence encoding rARU and the amino acid sequence of rARU are shown in FIGS. 2 and 3, respectively. The rARU expressed by pRSETB-ARU-Kmr contains the entire repeating units region of toxin A. The invention further contemplates the use of this recombinant protein component, or any other protein component containing the entire repeating units of toxin A or any fragment therein, whether expressed alone or as a fusion protein.
Similar methods may be used to isolate, clone and express a recombinant protein component comprising the repeating units of Clostridium difficile toxin B (rBRU). The nucleotide sequence encoding rBRU and the amino acid sequence of rBRU are shown in FIGS. 6 and 7, respectively. The rBRU expressed by pRSETC-BRU-Kmr contains the entire repeating units region of toxin B (see FIG. 8).
The present methods provide for preparation of immunogenic compositions comprising rARU or rBRU or both, which are useful as vaccines. Immunogenic compositions may be formulated as vaccines in a pharmaceutically acceptable carrier or diluent (e.g., water, a saline solution (e.g., phosphate-buffered saline), a bicarbonate solution (e.g., 0.24 M NaHCO.sub.3), a suppository, cream, or jelly), which are selected on the basis of the mode and route of administration, and standard pharmaceutical practice, see: U.S. Pat. No. 5,919,463 Thomas, et al., (1999), which is incorporated in its entirety by reference herein. Suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences (Alfonso Gennaro et al., eds., 17th edn., Mack Publishing Co., Easton Pa., 1985), a standard reference text in this field, in the USP/NF, and by Lachman et al. (The Theory & Practice of Industrial Pharmacy, 2nd edn., Lea & Febiger, Philadelphia Pa., 1976). In the case of rectal and vaginal administration, the vaccines are administered using methods and carriers standardly used in administering pharmaceutical materials to these regions. For example, suppositories, creams (e.g., cocoa butter), or jellies, as well as standard vaginal applicators, droppers, syringes, or enemas may be used, as determined to be appropriate by one skilled in the art.
The vaccine compositions of the invention may be administered by any route clinically indicated, such as by application to the surface of mucosal membranes (including: intranasal, oral, ocular, gastrointestinal, rectal, vaginal, or genito-urinary). Alternatively, parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular) modes of administration may also be used. The amounts of vaccine administered depend on the particular vaccine antigen and any adjuvant employed; the mode and frequency of administration; and the desired effect (e.g., protection and/or treatment), as determined by one skilled in the art. In general, the vaccines of the invention will be administered in amounts ranging between 1 μg and 100 mg. Administration is repeated as is determined to be necessary by one skilled in the art. For example, a priming dose may be followed by 3 booster doses at weekly intervals.
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.
Construction of rARU Expression Vector.
The vector pRSETB-ARU-Kmr used for expression and purification was constructed using standard techniques for cloning (Sambrook et al., Molecular Cloning: A Laboratory Manual (1989)). The nucleotide sequence of the toxin A gene fragment encoding rARU was derived from the cloned toxin A gene (Dove et al., Infect. Immun. 58:480-488 (1990); Phelps et al., Infect Immun. 59:150-153 (1991)) and is shown in FIG. 2. The gene fragment encodes a protein 867 amino acids in length (FIG. 3) with a calculated molecular weight of 98 kDa. The gene fragment was subcloned to the expression vector pRSETB. A kanamycin resistance gene was subsequently subcloned to the vector. The resulting vector pRSETB-ARU-Kmr expresses rARU. An additional 31 amino acids at the N-terminus of the recombinant protein are contributed by the expression vector pRSETB. The final calculated molecular weight of the recombinant protein is 102 kDa.
Expression and Purification of rARU.
Escherichia coli T7 expression host strain BL21 (DE3) was transformed with pRSETB-ARU-Kmr as described (Sambrook et al. Molecular Cloning: A Laboratory Manual (1989)). One liter cultures were inoculated with 10 ml of overnight growth of Escherichia coli BL21(DE3) containing pRSETB-ARU-Kmr and grown at 37� C. in Terrific broth (Sigma, St. Louis, Mo.) containing 25 μg/ml of kanamycin to an O.D. 600 of 1.8-2.0 and isopropyl B-D-thiogalactopyranoside (IPTG) was added to a final concentration of 40 μM. Cells were harvested after 22 h of induction, suspended in 0.1 liter of standard phosphate buffered saline, pH 7.4, containing 0.2% casanino acids, and disrupted by sonication. Cellular debris was removed from the lysate by centrifugation. Lysates typically contained a titer (reciprocal of the highest dilution with an A450 greater than 0.2) of 106 in the TOX-A test EIA (TechLab, Inc., Blacksburg, Va.). Lysates were saturated with 40% ammonium sulfate, stirred at 4� C. overnight and precipitating proteins were harvested by centrifugation. The ammonium sulfate fraction was suspended in 0.1 liters of 5 mM K2PO4, 0.1 M NaCl2, pH 8.0 and dialyzed extensively against the same buffer at 4� C. Insoluble material was removed by centrifugation. The dialyzed solution was passed through a column containing Sepharose CL-6B chromatography media (50 ml media/100 ml solution). Fractions were collected and monitored for the presence of rARU by EIA using the TOX-A test. Fractions containing EIA activity were analyzed by SDS-PAGE for the presence of rARU at a molecular weight of approximately 102 kDa. Fractions containing a single band of rARU were pooled. To further ensure purity the pooled solution was again passed over a Sepharose CL-6B column (25 ml media/100 ml protein solution). The solution containing purified rARU was filtered sterilized by passage through a 22 μ filter and stored at 4� C. Purified rARU along with samples from the steps of purification (lysate and dialyzed ammonium sulfate fraction) are shown in FIG. 5. The procedure typically yields approximately 100 mg rARU per liter of E. coli/pRSETB-ARU-Kmr culture. A combined 6-liter batch yielded 0.850 liters of rARU at 0.88 mg/ml for a total of 748 mg of rARU or 125 mg/liter of culture. The amount of rARU recovered represented 23% of the total soluble protein.
Construction of rBRU Expression Vector.
The vector pRSETC-BRU-Kmr used for expression and purification was constructed using standard techniques for cloning (Sambrook et al., Molecular Cloning: A Laboratory Manual (1989)). The nucleotide sequence of the toxin B gene fragment encoding rBRU was derived from the cloned toxin B gene (Barroso et al., Nucleic Acids Res 18:4004 (1990)) and is shown in FIG. 6. The gene fragment encodes a protein 622 amino acids in length with a molecular weight of approximately 70 kDa. The gene fragment was subcloned to the expression vector pRSETC. A kanamycin resistance gene was subsequently subcloned to the vector. The resulting vector pRSETC-BRU-Kmr expresses rBRU.
High-level Expression and Partial Purification of rBRU.
One liter of Escherichia coli pRSETC-BRU-Kmr was grown for 25 h at 37� C. in a shaking incubator. Cells were harvested by centrifugation and resuspended in 0.1 liter phosphate buffered saline with 0.2% casamino acids. Supernatant of the culture at harvest had a pH of 6.2. Cells were disrupted by sonication and cellular debri was removed by centrifugation. The 10X lysate is shown in FIG. 9, lane 3.
Immune Response to the rARU Component of the Conjugates.
Antibodies to C. difficile toxin A (CDTA). Antibodies to native toxin A were measured by ELISA, with toxin A isolated from C. difficile as the coating antigen, and by in-vitro neutralization of cytotoxicity (Lyerly et al. Infect. Immun. 35:1147-1150 (1982)). Human intestinal epithelial HT-29 cells (ATCC HTB 38) were maintained in 96 well plates with McCoy's 5A medium supplemented with 10% fetal calf serum in a 5% CO2 atmosphere. HT-29 cells were chosen because of their high sensitivity to CDTA probably because of the high density of the carbohydrate receptor on their surface. Serial 2-fold dilutions of sera were incubated with 0.4 μg/ml of CDTA for 30 min at room temperature. CDTA-serum mixtures were added to the wells at a final concentration of 20 ng of toxin A per well (about 200 times the minimal cytotoxic dose for HT-29 cells) in a final volume of 0.2 ml. The neutralization titer is expressed as the reciprocal of the
Serum antibodies (μg/ml) to Clostridium difficile
toxin A (CDTA) elicited in mice by recombinant
enterotoxin A (rARU) or polysaccharides bound to
rARU alone or succinylated (rARUsucc)
ELISA (Geometric mean and 25-75 centiles)
717 (621-863)
80.1 (69.8-131)
194 (113-236)
183 (146-175)
371 (274-463)
433 (258-609)
613 (485-778)
191 (118-291)
518 (366-615)
437 (372-547)
242 (172-443)
84.9 (72.5-131)
390 (279-470)
183 vs 7.94 p = 0.0001, 3.71 vs 183 p = 0.0005, 80.1 vs 3.70 p = 0.0001, 194 vs 80.1 p = 0.007, 7.94 vs 3.70 p = 0.01, 183 vs 80.1 p = 0.004, 371 vs 194 p = 0.01
*hsd/ICR mice. Remainder were NIH SA mice. ND (not done).
6 wks-old mice were injected SC with 2.5 μg of polysaccharide as a conjugate at 2 wk intervals. Groups of mice (n = 10) were exsanguinated 7 days after each injection and their sera assayed for anti-CDTA by ELISA.
highest dilution that completely neutralized cytotoxicity.
All 5 conjugates elicited high levels of anti-CDTA (194-613 μg/ml) (Table 1). Since the 2.5 μg immunizing dose of the conjugates was based on its polysaccharide content, the amount of rARU injected was different for each conjugate. For example, on a protein weight basis, Pn14-rARU, with 1.29 μg of rARU, elicited 194 μg CDTA antibody/ml (150.3 μg Ab/μg rARU injected). In contrast, Pn14-rARUsucc, that contained 7.3 μg of rARU per dose, elicited 371 μg CDTA antibody/ml (50.8 μg Ab/μg rARUsucc injected). Pn14-rARU induced more anti-CDTA per μg rARU than Pn14-rARUsucc, however, the total amount of anti-CDTA elicited by Pn14-rARUsucc was greater due to its higher content of rARU. The difference between the levels of anti-CDTA elicited by Pn14-rARU (194 μg CDTA antibody/ml) compared with Pn14-rARUsucc (371 μg CDTA antibody/ml) was significant.
SF-rARU, containing 3.9 μg of rARU, elicited 437 μg CDTA antibody/ml (112.0 μg Ab/μg rARU injected) compared to 518 μg CDTA antibody/ml for SF-rARUsucc (34.9 μg Ab/μg rARUsucc injected). Although the specific immunogenic activity for the rARUsucc was lower than that of the rARU in the SF conjugates, there was no statistical difference between the levels of CDTA antibody elicited by the two conjugates (437 μg Ab/ml for SF-rARUsucc vs 242 μg Ab/ml for SF-rARU).
K1-rARUsucc, that elicited 390 μg CDTA antibody/ml, had comparable specific immunogenic activity of its rARU component (48 μg Ab/ml per μg rARUsucc).
CDTA Neutralizing Antibodies.
Individual sera obtained 7 days after the third injection of the conjugates were assayed individually for their neutralization of approximately 200 times the cytotoxic dose of CDTA on human intestinal epithelial HT-29 cells. All sera from the mice immunized with the conjugates had a neutralizing titer greater than or equal to 64. The geometric mean and range of neutralizing titers for each conjugate is shown in Table 2.
Serum neutralizing activity against the in vitro
cytotocicity for HT-29 cells of Clostridium difficile
toxin A (CDTA)
μg Ab/ml
neutra;ization titer
(GM and range)
Neutralizing titers were the highest serum dilution that completely inhibited the cytotoxicity of CDTA (20 ng/well) on HT-29 cells. The titers represent the geometric mean of sera from general purpose Swiss Albino mice (n = 10) obtained 7 days after the 3rd injection. Anti-CDTA was measured by ELISA and the mean value expressed as μg Ab/ml serum.
*Affinity purified goat antibody
Protection Against Lethal Challenge with CDTA (Table 3).
Hsd/ICR mice were injected with SF-rARU, SF-rARUsucc or rARU as described in EXAMPLE 4 above. One week after the third injection, the mice were challenged intraperitoneally with a lethal dose (150 ng) of CDTA. Almost all mice vaccinated with either conjugate or rARU were protected. Based upon the amount of rARU injected, rARU and SF-rARU elicited similar levels of anti-CDTA. As expected, SF-rARUsucc elicited lower levels of anti-CDTA than the other two immunogens but the recipients were comparably protected.
Conjugate-induced antibody levels approached or surpassed the neutralizing activity of an affinity-purified goat antibody, containing 0.5 mg/ml, that was raised against formalin inactivated CDTA.
Protection of mice against lethal challenge
with 150 ng of Clostridium difficile
toxin A (CDTA)a induced by vaccination with
polysaccharide-rARU conjugates
μg rARU
717 (621-863)
437 (372-547)
242 (172-443)
Not determined
aMice (hsd/ICR) injected I.P. with 150 ng of CDTA 7 days after the 3rd injection of rARU or conjugate.
bMean antibody level (25-75 centiles) of sera used for pool (n = 10 from each group bled 4 h before challenge with CDTA.
cHighest dilutions of sera (range) that completely neutralized the cytotoxicity of CDTA (20 ng/well) on HT-29 cells.
This invention has been described by a direct description and by examples. As noted above, the examples are meant to be only examples and not to limit the invention in any meaningful way. Additionally, one having ordinary skill in the art to which this invention pertains in reviewing the specification and claims which follow would appreciate that there are equivalents to those claimed aspects of the invention. The inventors intend to encompass those equivalents within the reasonable scope of the claimed invention.
U.S. Pat. No. 5,098,826 (Wilkins et al.) (1992).
U.S. Pat. No. 5,736,139 (Kink et al.) (1998)
U.S. Pat. No. 5,919,463 (Thomas et al.) (1999)
Lyerly, D. M. and T. D. Wilkins, in Infections of the Gastrointestinal Tract, Chapter 58,
pages 867-891, Raven Press, Ltd, New York 1995
Moncrief et al., Infect. Immun. 65:1105-1108 (1997);
Barroso et al., Nucl. Acids Res. 18:4004 (1990);
Dove et al. Infect. Immun. 58:480-488 (1990)). (
Krivan et al., Infect. Immun. 53:573-581 (1986);
Tucker, K. and T. D. Wilkins, Infect. Immun. 59:73-78 (1991)).
Just et al. Nature 375:500-503 (1995),
Just et al. J. Biol. Chem 270:13932-13939 (1995)).
Hofmann et al. J. Biol. Chem. 272:11074-11078 (1997),
Faust and Song, Biochem. Biophys. Res. Commun. 251:100-105 (1998))
Lyerly et al. Current Microbiology 21:29-32 (1990)
|Cited Patent
|Filing date
|Publication date
|Sep 13, 1982
|Jul 23, 1985
|Wilkins Tracy D
|Toxins and antibodies of C. difficile
|Sep 13, 1982
|Aug 6, 1985
|Wilkins Tracy D
|Toxins and antibodies of C. difficile
|Feb 11, 1986
|Sep 5, 1989
|Virginia Tech Intellectual Properties, Inc.
|Method of detecting, isolating and purifying clostridium difficile toxin A and its receptors
|Jun 20, 1988
|Nov 7, 1989
|Virginia Tech Intellectual Properties, Inc.
|Antibody for C.difficile
|Mar 9, 1990
|Mar 24, 1992
|Virginia Tech Intellectual Properties, Inc.
|Detection, isolation and purification of Clostridium difficile toxin A with toxin receptors
|Jun 7, 1995
|Apr 7, 1998
|Ochidian Pharmaceuticals, Inc.
|Treatment of Clostridium difficile induced disease
|Oct 16, 1995
|Jul 6, 1999
|Oravax, Inc.
|Clostridium difficle toxins as mucosal adjuvants
|Oct 23, 1995
|May 2, 1996
|Joseph R Firca
|Vaccine and antitoxin for treatment and prevention of c. difficile disease
|Jul 1, 1996
|Jan 30, 1997
|Oravax Inc
|Clostridium difficile toxins as mucosal adjuvants
|Barroso et al., 1990, "Nucleotide Sequence of Clostridium Difficile Toxin B Gene" Nucl. Acids Res. 18:4004.
|Dove et al., 1990, "Molecular Characterization of the Clostridium Difficle Toxin A Gene" Infect. Immun. 58:480-488.
|Eichel-Streiber et al., 1992, "Clostridium Difficile Toxin A Carries a C-Terminal Repetitive Structure Homologous to the Carbohydrate Binding Region of Streptococcal Glycosyltransferases" Gene 96:107-113.
|Faust et al., 1998, "The Enzymatic Domain of Clostridium Difficile Toxin A is Located within its N-Terminal Region" Biochem. Biophys. Res. Commun. 251:100-105.
|Hofmann et al., 1997, "Localization of the Glucosyltransferase Activity of Clostridium Difficile Toxin B to the N-terminal Part of the Holotoxin" J. Biol. Chem. 272:11074-11078.
|Just et al., 1995, "Glucosylation of Rho Proteins by Clostridium Difficile Toxin B"Nature 375:500-503.
|Just et al., 1995, The Enterotoxin from Clostridium Difficile (ToxA) Monoglucosylates the Rho Proteins J. Biol. Chem. 270:13932-13939..
|Krivan et al., 1986, "Cell Surface Binding Site for Clostridium difficile Enterotoxin: Evidence for a Glycoconjugate Containing the Sequence . . . " Infect. Immun. 53:573-581.
|Lyerly et al., 1990, "Vaccination against Lethal Clostridium difficile Enterocolitis with a Nontoxic Recombinant Peptide of Toxin A" Current Microbiology 21:29-32.
|Lyerly et al., 1995 "Infections of the Gastrointestinal Tract" Chapter 58, pp. 867-891.
|Makoff et al., 1989, "Expression of Tetanus Toxin Fragment C in E coli: High Level Expression by Removing Rare Codons" Nucleic Acids Res. 17:10191-10202.
|Makoff et al., 1989, "Expression of Tetanus Toxin Fragment C in E. Coli: Its Purification and potential use as a Vaccine" Bio/Technology 7:1043-1046.
|Moncrief et al., 1997, "Positive Regulation of Clostridium difficile Toxins" Infect. Immun. 65:1105-1108.
|Tucker et al., 1991, "Toxin A of Clostridium difficile Binds to the Human Carbohydrate Antigens I, X and Y" Infect. Immun. 59:73-78.
|Citing Patent
|Filing date
|Publication date
|Jul 31, 2009
|Aug 7, 2012
|University Of Massachusetts
|Antibodies against clostridium difficile toxins and uses thereof
|Jul 31, 2009
|Sep 4, 2012
|University Of Massachusetts
|Antibodies against Clostridium difficile toxins and uses thereof
|Apr 20, 2012
|Jul 9, 2013
|Wyeth Llc
|Compositions relating to a mutant Clostridium difficile toxin and methods thereof
|Mar 22, 2013
|Oct 15, 2013
|Wyeth Llc
|Compositions relating to a mutant Clostridium difficile toxin and methods thereof
|Jun 7, 2012
|Dec 17, 2013
|University Of Massachusetts
|Antibodies against clostridium difficile toxins and uses thereof
|Jun 7, 2007
|Oct 7, 2014
|The Rockefeller University
|Codon-optimized DNA molecules encoding the receptor binding domains of Clostridium difficile toxins A and B, and methods of use thereof
|Aug 19, 2013
|Dec 2, 2014
|Wyeth Llc
|Compositions relating to a mutant Clostridium difficile toxin and methods thereof
|Jul 26, 2013
|Dec 9, 2014
|Pfenex Inc.
|High level expression of recombinant toxin proteins
|May 30, 2012
|Jun 30, 2015
|Johannes Gutenberg-Universitaet Mainz
|Medicament for LCT poisoning
|Jun 7, 2007
|Nov 4, 2010
|Cornell University
|Codon-Optimized Dna Molecules Encoding the Receptor Binding Domains of Clostridium Difficile Toxins A and B, and Methods of Use Thereof
|Sep 5, 2011
|Mar 8, 2012
|Intercell Ag
|Isolated polypeptide of the toxin a and toxin b proteins of c. difficile and uses thereof
|U.S. Classification
|424/247.1, 424/9.1, 530/300, 424/200.1, 424/9.2, 530/350, 424/184.1, 424/185.1, 424/192.1, 424/234.1
|International Classification
|Cooperative Classification
|European Classification
|Jul 2, 2002
Owner name: TECHLAB, INC., VIRGINIA
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Triple Jewel
A selection of articles related to triple jewel.
Original articles from our library related to the Triple Jewel. See Table of Contents for further available material (downloadable resources) on Triple Jewel.
- Brigid - The Goddess of Imbolc and Celtic Europe
- Celtic Myth | Ireland | Britain | Christianity | A Myth The Pagan goddess Brigid is perhaps one of the oldest goddesses of Celtic Europe still recognized and worshipped. In fact, until the mid-twentieth century in Scotland, she was still welcomed in at Imbolc...
Deities & Heros >> Celtic, Welsh, Irish & Brittish
- Baba Yaga: A Demon or A Goddess?
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Deities & Heros >> Slavic
- The Paths of the Qabalah
- I must confess, I've been trying to use the book "The Talking Tree" by William Gray and I admit that I've been having a hard time. Most Tree of Life glyphs start the eleventh path with the Fool, as it is with most tarot decks. Gray begins with the...
Magick >> Qabalah
- The Ancient Goddess Barbi: Historical Views from the Year 5000
- (Author's Note: Read this only if you consider laughter a sincere form of worship!) In the year 5000, historians will seek to patch together traces of the past, to discover what life was like in today's current era. Here's one humorous view of what they might...
Paganism & Wicca >> Humor
- Morrison, Dorothy
- Dorothy is a Wiccan High Priestess of the Georgian tradition and an avid practitioner of the ancient arts for over 20 years. She teaches the Craft to students throughout the US and in Australia. Her interests include archery and bowhunting, magical herbalism,...
Real Interviews >> Authors
- The Skeptical Witch
- The Choir finished the last notes of the worship service. I sit down in unison with 3000 other people preparing to hear the word of the Lord from Apostle Benjamin. The worship service does not touch me. The words of the Apostle are empty for me. My mind is in...
Paganism & Wicca >> Daily Life
- Nyx
- Nyx is Night, a powerful goddess whose dark light falls from the stars and who dictates not only to men but also to gods. Even Zeus does not wish to upset Night: It happened that Hera bribed Hypnos in order to make Zeus fall asleep, so that she could have it...
Deities & Heros >> Greek
Triple Jewel is described in multiple online sources, as addition to our editors' articles, see section below for printable documents, Triple Jewel books and related discussion.
Suggested Pdf Resources
- Great Compassion Ritual The Dharma of Repentance of the Great
- the Triple Jewel of the Dharma Realm of the ten directions, my body everywhere makes offerings.
- kaladarshan.arts.ohio-state.edu
- The Shurangama Mantra:An Explanation
- Take refuge with Proper Enlightenment—the Triple Jewel.
- www.drbachinese.org
- To Understand Buddhism
- www.buddhanet.net
- The Bodhisattva Precepts for Laypeople:
- (23rd Minor) The Precept against Failure to Offer New Food to the Triple Jewel First.
- www.drby.net
Suggested News Resources
- My $10000 storage unit mistake
- This is why we're urged to ground our happiness in things beyond change and why Buddhists vow to take refuge in the Buddha, the Dharma (teachings) and the Sangha (community) -- the so-called Triple Jewel. The concept of refuge struck a chord with me.
Suggested Web Resources
- Three Jewels - Wikipedia, the free encyclopedia
- This article is about the "Three Jewels", "Three Treasures", "Three Refuges or " Triple Gem" in Buddhism. For other uses, see Three Treasures (disambiguation).
- en.wikipedia.org
- Taking Refuge in the Triple Jewels, by Venerable Master Chin Kung
- www.dharmaweb.org
- Triple-Jewel
- www.magicaldancer.com
- Amazon.com: Complete Vanguard Recordings (Triple Jewel Case
- Amazon.com: Complete Vanguard Recordings (Triple Jewel Case): Big Mama Thornton: Music.
- www.amazon.com
- The Triple Jewels. THE BUDDHA JEWEL. "Buddha" is a sanskrit word which means 'Awareness and Understanding'.
- www.amtb.org.tw
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Our washer died (11 or 1 2 years old)didnt actually go out to buy this model , found it as a display model at a home inprovement store.It is HE ,very little water use,very little detergent needed to do a load of clothes,many setting, it is a great washer.
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concept art and set photos!
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June 13 2013, 09:42 PM
Crazy Eddie
Rear Admiral
Location: I'm in your ___, ___ing your ___
Re: concept art and set photos!
trevanian wrote:
Crazy Eddie wrote:
trevanian wrote:
It's photographically corrupt.
To use lens flares on occasion for effect can be a superb creative choice (I'd say the first DIE HARD is an example of going almost to the brink of too far with them, but they are well-orchestrated, run out for key moments); to overuse them, and do so arbitrarily, is just bad photography.
And you, with your illustrious and respected career directing major motion pictures, would be the one to know, right?
I have a pair of eyes, an appreciation of what has gone before, knowledge on the subject, and have been hands-on with equipment -- and might possibly even possess a modicum of taste.
Same for me -- in all respects -- and I disagree.
Now what?
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You will love this location! Step outside your sliding glass doors for direct access to the pools and courtyard. This gorgeous King Room suite is loaded with luxury. Granite kitchen with eating bar and stainless steel appliances, granite in the bathrooms, King Master Suite with private ensuite with glassed shower. The second bedroom/*den is equipped with 2 twin beds, and also have a tv and playstation 2 console. This gorgeous condo sleeps 6. (Max of 4 adults please)Discovery Bay offers an unbeatable location on the waterfront of Downtown Kelowna. Just a short stroll from our condo you'll find a great beach, a bird sanctuary, and beautiful ...Read more
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text:
Gwendolen Jane (née Armstrong-Jones), Lady Buckley
1 portrait of
© National Portrait Gallery, London
Gwendolen Jane (née Armstrong-Jones), Lady Buckley
by Bassano Ltd
whole-plate glass negative, 24 June 1920
Given by Bassano & Vandyk Studios, 1974
Photographs Collection
NPG x74883
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- Gwendolen Jane (née Armstrong-Jones), Lady Buckley (died 1985), Wife of Sir Denys Burton Buckley; daughter of Sir Robert Armstrong-Jones. Sitter in 9 portraits.
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- Bassano Ltd (active 1901-1962). Artist associated with 42717 portraits.
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You should not plan a pregnancy if
Posted by Anonymous + Show Post
Finish this sentence....
Posted by Anonymous on Sep. 29, 2012 at 9:49 AM
Replies (21-30):
2 moms liked this
You are an addict.
1 mom liked this
you can't provide for your kids
by Anonymous 6 on Sep. 29, 2012 at 9:55 AM
2 moms liked this
that's not true. there's plenty of well off single women who don't need nor want to wait to find the right man.
shit if they got the money to blow on ivf treatments, I can't imagine their poor needing food stamps and shit.
but I get your point. lol
shit if they got the money to blow on ivf treatments, I can't imagine their poor needing food stamps and shit.
but I get your point. lol
Quoting UgtaBkdnMe:
you're single
3 moms liked this
If you're sleeping with someone else's husband.
1 mom liked this
if you're with a low life...
by *JuStRITa* on Sep. 29, 2012 at 9:57 AM
If you don't know how to raise/care for the ones you have, are not mentally/emotionally mature, are not financially stable, don't know/want to know how to manage your finances and just want to keep having kids just to get the gender you desire etc.
by the sauce is boss on Sep. 29, 2012 at 9:58 AM
1 mom liked this
You do drugs.
you are not financially stable.
you have a disease that your baby would have too.
by Anonymous 5 on Sep. 29, 2012 at 9:59 AM
2 moms liked this
I had my son at 19 too. But it doesnt change the fact that I wasnt mature enough to have a kid. I dont know many 18,19, or hell 20, 21 and up to 25 year olds that are.
Quoting Anonymous:
I don't agree with this. I married at 18 and had my son at 19. Why should I have waited?
Quoting Anonymous:
Under the age of 20.
Add your quick reply below:
You must be a member to reply to this post.


text:
Howard Johnson Missoula MT
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New details are emerging Monday into the events leading up to the death of a Long Island doctor who was found in the doorway of a Chelsea apartment building early Sunday morning. Sources are saying that her death does not seem to be a criminal act.
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text:
In mybook®: Food
4601 E Main St, Farmington, NM 87402
Family Style, American
Closed Now
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- 10:45 am - 10:00 pm
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- 10:45 am - 10:00 pm
Regular Hours
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- 10:45 am - 9:00 pm
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Animas Valley Mall
MenuTop ▲
Mozzarella Sticks
Tricked Out Nachos
Firehouse Boneless Wings
All Salads (except Taco Salad) Include Grilled Or Crispy Chicken Breast
Chicken Caesara traditional favorite with caesar dressing and parmesan cheese
Market Tossmonterrey jack and cheddar cheeses, tomatoes, eggs, almonds and bacon
Taco Saladgrilled chicken or ground beef, cheddar cheese, pico de gallo, guacamole and cour cream served in an edible taco shell
Napa Valleyromaine lettuce, bleu cheese, apples, dried cranberries and almonds
Specialty Burgers
Southwestsmokehouse bacon, guacamole and pepper jack cheese
The Workssmokehouse bacon, american cheese and grilled mushrooms
Three Cheeseamerican, cheddar and swiss
Infernospicy jalapenos, caramelized onions, pepper jack cheese
Swiss Meltsauteed mushrooms, grilled onions and swiss cheese
Bacon Cheddarsmokehouse bacon and cheddar cheese
Fudd's Exotic Burgers
Buffalo Burgera tender and flavorful classic burger served on our wheat bun. leaner than most traditional beef burgers, it has 0 g. of artificial trans fats and is just as juicy when grilled to medium. try it with cheddar cheese and your fair share of our free market fresh produce.
Elk Burgera lean and savory burger that has 0 g. of artificial trans fats and is low in cholesterol when ordered with our wheat bun and piled high with fuddruckers market fresh produce. or add swiss cheese, grilled onions and mushrooms for a totally different world's greatest elk burger experience.
Wild Boar Burgermild, sweet, smoky and delicious. our wild boar is served with a wheat bun, has 0 g. of artificial trans fats, and is low in saturated fat and cholesterol. keep it simple or make it yours by adding pepper jack cheese, bacon and sauteed mushrooms.
World's Greatest Hamburgers - The Original Fuddsfresh never frozen 1/3, 1/2, 2/3 or 1 lb. 100% all-american, premium-cut, vegetarian-fed beef born and raised on a handful of ranches only in the u.s.a. grilled to order* and placed atop our fresh, sesame-topped scratch baked bun ready for you to pile it high at our market fresh produce bar.
Chicken Sandwiches
Crispy Chickenbeer-battered skinless, boneless chicken breast
Grilled Chickengrilled skinless, boneless chicken breast
Bacon & Swissa crispy or grilled chicken breast with smokehouse bacon and swiss cheese
Firehouse Buffaloa crispy chicken breast dipped in fudds red hot buffalo sauce
Chipotle Bbqa crispy or grilled chicken breast with bbq sauce, smokehouse bacon and cheddar cheese
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text:
Van Dusen v. Barrack
From Wikipedia, the free encyclopedia
|Van Dusen v. Barrack
|Argued January 8–9, 1964
Decided March 30, 1964
|Full case name
|Van Dusen, U.S. District Judge, et al. v. Barrack, Administratrix, et al.
|376 U.S. 612 (more)
84 S. Ct. 805; 11 L. Ed. 2d 945; 1964 U.S. LEXIS 1537
|Court membership
|Case opinions
|Goldberg, joined by Warren, Douglas, Clark, Harlan, Brennan, Stewart, White
Van Dusen v. Barrack, 376 U.S. 612 (1964), was a United States Supreme Court case in which the Court held that when a case is transferred from a federal court in one state to a federal court in another, the choice of law should be that of the state in which the case was originally filed.[1]
- ^ Yeazell, S.C. Civil Procedure, Seventh Edition. Aspen Publishers, New York, NY: 2008, p. 231


text: Arrived here late for an overnight stay. We were greeting by very friendly staff. They informed me that they upgraded one of our rooms for free so that our two rooms would be close together. The rooms were large and very clean. The bathroom was spotless. it was a great say. We would definitely make it a point to stay... More


text:
Granville's Hunter Rogerson approaches the final hurdle in the 110 hurdles this past Friday during the Division I regional meet at Pickerington North. Rogerson placed third in the event to qualify for the state meet.
- Filed Under
Granville boys track coach Bob Hollen goes back a decade and shakes his head in amazement.
Ten years ago, Granville won a district title in Division III. This season, the Blue Aces remarkably won a Division I district title and earned a regional runner-up trophy this past weekend at Pickerington North.
Granville sends two relays and athletes in five individual events to this weekend's state meet at Ohio State's Jesse Owens Memorial Stadium in just the Blue Aces' second year in the highest division. Granville has quickly placed itself on the map. ...


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2 of 4 BOD members are orig developers exempt from the rental rule that applies to other unit owners. They have been buying up foreclosures and renting them out. I'm next. What can I do to stop them? BOD president agrees with me, but says he can't do anything.
Lawsuit / Dispute Attorney
Get the Board to change the exempt rule on rent. Rely therefore on the political process
Licensing Attorney
Per your statement, the two "orig developers" are doing exactly what they are permitted to do - rent units. As these are foreclosures that would likely be vacant otherwise, that may not be a bad thing.
At any rate, unless and until the BoD changes that rule, you have no cause of action.
The foregoing is for general information purposes and does not establish an attorney-client relationship.
Landlord / Tenant Lawyer
In the vast majority of condominium disputes, the cheapest and most effective resolution is a political one. Get yourself and other like minded owners elected and change the rules/bylaws. Litigation over these matters is extremely expensive and nasty.
I'm not sure what you mean by "I'm next." Does that mean you are going to be foreclosed on? If so, having a bidder in addition to your mortgage-holder isn't always a bad thing.
Though we strive to provide accurate legal information in our answers on AVVO, our answer should not be construed as legal advice and it does not create an attorney-client relationship. Our firm only forms attorney-client relationships by written agreement signed by both our firm and the client. Please seek an in-person consultation with an attorney immediately as almost all legal matters are time sensitive and failing to meet deadlines can result in adverse consequences.
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text:
The Montbeliard-based outfit have been dealt a heavy blow as two of their most influential players have confirmed that they will switch clubs in the summerMarvin Martin and Ryad Boudebouz have announced that they both intend to leave Sochaux in the summer. The midfield duo confirmed their decisions on Sunday following their club's 1-0 victory over Ligue 1 rivals Marseille.
Martin, 24, has been strongly linked with a move abroad, but fresh speculation in France suggests that he is now closing in on a switch to Lille. However, he was unable to shed any light on his next destination.
"I honestly do not know where I will go," the France international is quoted as saying by AFP. "I really do not know, otherwise I would tell you. And I do not know when I will make my decision.
"My goal was just to leave Sochaux with my head held high and the club still in Ligue 1.
"As for my future, we'll see. There is also selection for the Euros, which is important because I really want to be in the final 23-man squad."
As for Boudebouz, a move to the Premier League has been mooted in recent weeks but he, too, was remaining coy on his future plans.
"It's not a done deal [that he will leave] and I still have a two-year contract, but my wish is to go," the Algeria international confirmed.
Sochaux finished 14th in the Ligue 1 standings, 40 points adrift of champions Montpellier.


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Laura Linney to Join Ian McKellen in Sherlock Holmes Film A SLIGHT TRICK OF THE MIND
According to The Hollywood Reporter, Laura Linney has signed on to join Ian McKellen in Bill Condon's upcoming Sherlock Holmes film A SLIGHT TRICK OF THE MIND. 'MIND' is based on Mitch Cullin's novel, and features a script by Jeffrey Hatcher.
The film, set in 1947, follows "an aging Holmes (Ian McKellen) returns from a journey to Japan, where, in search of a rare plant with powerful restorative qualities, he has witnessed the devastation of nuclear warfare. Now, in his remote seaside Sussex farmhouse, Holmes faces the end of his days with only the company of his housekeeper (Linney) and her young son, Roger. Grappling with the diminishing powers of his mind, Holmes comes to rely upon the boy as he revisits the circumstances of the unsolved case that forced him into retirement."
'MIND' marks the fourth time Condon and Linney have worked together.
Linney's extensive stage credits on Broadway and elsewhere include Hedda Gabler (for which she won a 1994 Joe A. Callaway Award), Holiday (based on the 1938 movie starringKatharine Hepburn), and her Best Actress Tony Award-nominated 2002 role in The Crucible (where she appeared alongside future The Exorcism Of Emily Rose co-star Jennifer Carpenter). She was nominated again in 2005 for Sight Unseen.
Linney most recently wrapped showtime's hit series THE BIG C, and has scored four Emmy Awards, two Golden Globe Awards and a Screen Actors Guild Award over the course of her career.
Photo by © RD/Jackson/ Retna Digital
About Author
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text:
WestBow Press, a division of Thomas Nelson, signed six Author Agreements with us on May 13, 2015 to publish all books highlighted on this website. In addition, on June 3, 2015, WestBow Press signed a seventh agreement to publish our new fiction book titled U're Warned.
We are excited by WestBow's interest in our books and their commitment to excellence. Our books should be available at most retail booksellers and Internet locations in the near future. As soon as they are, we will add a blog sharing where you can review and if interested purchase them. We will also post it on Facebook and Twitter.
Thanks for your patience and interest.
God bless,
Dave and Lorraine Plante
WestBow Press?
"WestBow Press opened its doors in October 2009. Its legacy taps into the deep heritage of Thomas Nelson. Founded in 1798 in Edinburgh, Scotland, Thomas Nelson got its start in a building located at 7 West Bow Street, publishing early editions of many top fiction titles, including Pilgrim's Progress and Robinson Crusoe and later releasing books by such authors as Jane Austen, Charles Dickens, and Arthur Conan Doyle."
A close friend who lives out of town has three young sons. He and his wife visit about once a year and sometimes stay for a few days. During one of their visits, they needed to attend a wedding so we babysat their boys.
It was fun. At bedtime, I focused on the older one who, his dad had told me, had a hard time going to bed at night. After we played games and wrestled for a while, he nodded off to sleep without any conflict. My friend refers to that visit as "Dave's boot camp," because the boys had no problem going to sleep. I was complimented and reminded of a saying from my childhood relative to the subject.
As a young boy, I wasn't interested in going to sleep at bedtime. While my dad was fighting World War II, my mother, sister, and I lived with my grandparents. I loved sitting in their living room during the evening and listening to the adults chat. Eventually, my grandmother would say, "David, it's time for you to go to bed." She had to be up early to go to work in the Gonic Woolen Mill.
Mom and I headed upstairs, and I got into bed. However, as soon as she walked back downstairs, I climbed out of bed and quietly sat on the stairs and through the railings watched them in the living room until I got bored. Then I got up and ran to my grandmother and asked her to tell me a story. She said, "I'll tell you a story about Johnny Borey, and now my story has begun. I'll tell you another about his brother, and now my story is done. And now, David, it's time for you to go back to bed."
At that point, I knew the fun was over, and that it was my turn to do the right thing. I kissed her and Mom goodnight and headed upstairs on my own. She and Mom always made me feel that I was responsible enough to do what was right, and that made me feel good about myself. I never met Johnny Borey and don't believe he exists, but you never know. In a subtle and funny way, the story was a good way to tell children that it was bedtime.
Rules are amazing and can be beneficial in many ways. I can't say I have ever been a lover of rules, but some have kept me from danger or out of trouble. To me, the source or originator of the rule has a lot to do with the value of the rule, and I assume that is true with most other people.
As a young boy and later as a teenager, I enjoyed riding my bike throughout our community. I had many part-time jobs until I could secure a work permit at age fourteen, so I was always out looking for work after school or during the summer.
When I couldn't find work, one week I visited a classmate after school. She was one of the prettier and exciting girls in our class. She was social and invited me in and offered me cookies and a soda. I enjoyed the visit and decided I would do it again. The next day after school I biked to East Rochester and visited another good looking girl in our class. She was just as social; we had a wonderful time. I biked home thinking both girls were classy and a pleasure to spend time with. On the third day, I decided to bike to Rochester Hill and visited the third special girl in our class. She had beautiful brown hair and a bubbly personality. I again enjoyed the visit and felt I had discovered a new routine and planned to repeat it the following week.
However, the following Monday afternoon one of the three girls I had visited stopped me in the corridor at school. Her name was Lorraine. She asked if we could talk. I said, "Sure."
We stepped out of the main stream of traffic so our words would not be heard by others. "The two girls you visited last week and I talked and decided that one of us needed to talk to you. What you are doing is wrong. Visiting three girls three days in a row breaks all the rules. You need to decide which girl you like the best and visit just her."
I was fine with that and told her so. But I also wasn't stupid. There was no way I was going to select one of them and send negative signals to the other two. I liked all three of them too much. I just moved my visits to the other side of town.
I did learn a new rule: smart ladies talk! It was a good lesson. Lorraine and I became close friends in our senior year. We eventually went steady, got engaged and married after I finished my first year of college. She now edits everything I write and has an editing guide book on her shelf titled, "Lori's Rules." I love her but have to admit that although I have not memorized every rule in her book, we have been happily married for fifty-four years.


text:
- 136Promote
Canvas Print
Portland = Beardsby Rachel Caldwell
Fine art print on bright white, fine poly-cotton blend, matte canvas using latest generation Epson archival inks. Individually trimmed and hand stretched museum wrap over 1-1/2" deep wood stretcher bars. Includes wall hanging hardware.
Portland is full of bearded humans who are extremely friendly. What a lovely, warm and fuzzy place to be.
haha good point!
ha ha! love beards!
So awesome! I love this piece Rachel. I'm working on my beard right now ;)
I figured there would be more beards. That is awesome, I can't wait to see yours. This could have been a beard-themed show altogether!
My face wasn't very good at growing beards so to punish it I made it live in Seattle.
So true. Beards all around!
Congratulations on the Mercury cover! It turned out great.
this line work makes me swoon!
a t-shirt please!
Haha that's great. Love Portland. Love bearded humans.
great work
digs your beard.
love it!
muy buen trabajo!
So cool
i have this shirt. love it.
Love this!!
nice!. Following!
Love it!
See More Comments


text:
Preston City Wrestling have announced a big match for their August 1st show, 'I Want To Break Three' at Evoque, Preston.
It has been confirmed by Preston City Wrestling that former PCW Champion, Kris Travis will face Ring Of Honor & Pro Wrestling Guerilla star, Michael Elgin at Evoque on August 1st at 'I Want To Break Three'.
The announced match card is as follows:
Match Card
Michael Elgin vs Kris Travis
Tickets are due to go on sale in the near future.
The Official Facebook event page can be viewed here.
Source: Preston City Wrestling Facebook
Tagged: 2014, august 1, brit, british, Facebook, i want to break three, indie, kris travis, Match Card, michael elgin, news, PCW, preston city, pro, tickets, UK, united kingdom, wrestle ropes, wrestler indy, wrestleropes, wrestling


text:
Imagine That receives $63,362 theater grant
Published: Sunday, January 9, 2011 at 3:15 a.m.
Last Modified: Saturday, January 8, 2011 at 11:04 p.m.
Imagine That, a program of the Spartanburg Little Theatre recently announced that it had received a grant of $63,362 from the Mary Black Foundation as part of the Community Health Fund that will be distributed over the next three years.
The grant will go toward covering general operating expenses, implement the Imagine That program, and provide direct services to volunteer cast members, youth and adult audience members who are impacted by these performances.
"I am so grateful for Imagine That to be the recipient of this generous grant," program coordinator Sterling Kenny said. "Increasing community capacity for youth development leads to more
strategies to enhance support and opportunities for youth.
"I am encouraged to know that the community is choosing to support the positive work of young people in our community."
Spartanburg junior wins Poetry Out Loud contest
McKenzie Wilson, a junior at Spartanburg High School, has been named the school winner in the Poetry Out Loud Contest, a national recitation contest created by the National Endowment for the Arts, for her recitation of "Jabberwocky" by Lewis Carroll and "One Art" by Elizabeth Bishop. Jordan Fox placed second in the competition, and Travis Trojan placed third.
Wilson will go on to compete in the Upstate Finals on Jan. 22 at The Showroom in Spartanburg. Each champion at the state level will receive $200 and an all-expenses-paid trip to Washington, D.C., to compete for the national championship in April.
St. Paul duo win foreign language medals
Two eighth-grade students at St. Paul the Apostle Catholic School in Spartanburg recently were awarded medals at Clemson University's 38th Foreign Language Declamation.
Isaac Drummond-Henking, son of Melissa Drummond and Eric Henking, won a gold medal in the Spanish Outside Experience category.
Sue Alex Wright, daughter of Mr. and Mrs. Mark Wright, was awarded a silver medal in the Regular Spanish category.
Isaac and Sue competed against high school students from South Carolina and Georgia.
Reader comments posted to this article may be published in our print edition. All rights reserved. This copyrighted material may not be re-published without permission. Links are encouraged.


text:
The Raiders ugly Monday night divisional matchup with the Broncos may sting longer than just this week. Terrelle Pryor left the game early in the forth with what is now being diagnosed as a concussion
Official now. Terrelle has been formally diagnosed with a concussion. Tracy porter too. #raiders
— Scott Bair (@BairCSN) September 24, 2013
Matt Flynn will get the start next week if Pryor can't go. Big loss for the Raiders as Pryor has been steadily improving.


text:
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AES E-Library
Time-quantized Frequency Modulation with Time Dispersive Codes for the Generation of Sigma-delta Modulation
Time quantization and noise shaping applied to linear frequency modulation (LFM) can form an alternative although unconventional means of generating 1-bit uniformly sampled code that is similar in structure to a feedback sigma-delta modulator (SDM). Fundamental insight into the SDM process and base-line coding spectrum emerges, where specifically linearity of signal conversion is studied and compared to that of linear pulse code modulation (LPCM). Time dispersive limiters both within and outside the noise shaper are investigated and their consequence on linearity explored. A noise averaging simulation reveals intrinsic distortion and noise modulation to be low when appropriate dither is used.
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text: http://www.linuxquestions.org – (yes, I am cross-posting this from the gentoo forums. so sue me) I've begun putting together a new machine to replace my 7-year-old main rig. So far I've got the motherboard (a Tyan S4985), CPUs, RAM and power supply. The rig is up and running, and seems to perform quite a bit better than the old box as far as compile times are concerned. Until I can get a decent video card, I am going to use the motherboard's integrated video - an XGI Volari Z7. I have tried a couple of different drivers and haven't yet gotten adequate results. sis: fails to detect the card Code: [ 79888.486] (--) PCI:* (HowTos)


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and coming out of spring woolfolk is starting opposite either mike williams or vlad emilien. No one is really competing with Woolfolk at this point.
Niko Porikos grew up in an NTDP billet home. Cool story.
and coming out of spring woolfolk is starting opposite either mike williams or vlad emilien. No one is really competing with Woolfolk at this point.
Not enough points to start a thread, but he can make a diary. Close out that loophole Brian.
Dont be so anal dude ... just wanted to ask a couple questions
Not being anal to you. If Brian wants posters to have a waiting period before starting Threads, it makes sense to extend the waiting period to Diaries.
The long and short of it, To much non diary material is ending up in diaries because antsy new posters, such as yourself, want to contribute right away. I appreciate your enthusiasm, all you have to do is wait about 20 posts then you can start a Thread.
I wonder if there is anything telling new folks what these ground rules are? There should be an e-mail or similar notice sent to new accounts describing the points system and how you need 20 to start a thread.
A better post than most of what's been up recently.
Mike Williams and Vlad are both intelligent players, Williams is a bit more experienced and Vlad is returning from an injury. But it will be a (good) battle for the other safety spot in the fall.
I too have wondered how the said injuries are healing.
PS - Since you are new, don't be disheartened by all the bitching. They are just grumpy old men with small penises.
Would that make you a young man with a large San Diego? (Whale's Vagina!)
Whats the scoop on him. I would think he would be in the running for this position also.
Brandon should right in the mix for that as well. But look for Stevie Brown to play back there before we go too deep into our lineup. We have more people that can play the spinner position than we have safeties.
He was moved to linebacker due to a lack of speed.
Some of the best grammar I have ever read/heard. Where are the grammar nazis when you need them..
for full rcovery by training camp
That the diary waiting period should be higher than the board waiting period. I'd like to go back to where diaries were by respected posters and the board was for the trash (myself included)


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