text:
A hybrid molecule including a first part and a second part connected by a covalent bond,
(a) the first part including a portion of the binding domain of a cell-binding ligand, which portion is able to cause the hybrid molecule of the invention to bind to an animal cell; and
(b) the second part including a portion of a translocation domain of a protein, provided that (i) the hybrid molecule does not include an enzymatically-active portion of the protein, (ii) the first part and the second part are not segments of the same naturally-occurring polypeptide toxin, and (iii) the portion of the translocation domain, when covalently bonded to the enzymatically-active effector region of a toxin selected from diphtheria toxin, Pseudomonas exotoxin A, cholera toxin, ricin toxin, and Shiga-like toxin, is capable of translocating such effector region across the cytoplasmic membrane of the cell.
1. A hybrid molecule comprising a first part and a second part connected via a covalent bond,
(a) said first part comprising a portion of the binding domain of a cell binding ligand effective to cause said hybrid molecule to bind to a cell of an animal; and
(b) said second part comprising a portion of the translocation domain of a protein, provided that said hybrid molecule does not include an enzymatically active portion of said protein, and wherein said first part and said second part are not segments of the same naturally occurring protein.
2. The hybrid molecule of claim 1, wherein said cell-binding ligand is a polypeptide.
3. The hybrid molecule of claim 1, wherein said protein of (b) is a naturally-occurring toxin.
4. The hybrid molecule of claim 2, wherein said first part comprises the binding domain of said polypeptide cell-binding ligand.
5. The hybrid molecule of claim 2, wherein said first part comprises the cell-binding polypeptide ligand.
6. The hybrid molecule of claim 2, wherein said cell-binding polypeptide binding ligand is a hormone or growth factor.
7. The hybrid molecule of claim 2, wherein said cell-binding polypeptide ligand is an antigen-binding, single-chain analog of a monoclonal antibody.
8. The hybrid molecule of claim 2, wherein said first part comprises a monoclonal antibody.
9. The hybrid molecule of claim 2, wherein said first part comprises a portion of the binding domain of a polypeptide toxin.
10. The hybrid molecule of claim 6, wherein said hormone is selected from the group consisting of insulin, interleukin II, interleukin IV, interleukin VI and EGF.
11. The hybrid molecule of claim 10, wherein said hormone is interleukin II.
12. The hybrid molecule of claim 10, wherein said hormone is EGF.
13. The hybrid molecule of claim 9, wherein said toxin is diphtheria toxin.
14. The hybrid molecule of claim 3, wherein said toxin is diphtheria toxin.
15. The hybrid molecule of claim 3, wherein said toxin is botulinum neurotoxin.
16. The hybrid molecule of claim 3, wherein said toxin is cholera toxin.
17. The hybrid molecule of claim 3, wherein said toxin is ricin toxin.
18. The hybrid molecule of claim 3, wherein said toxin is Shiga toxin.
19. The hybrid molecule of claim 3, wherein said toxin is Shiga-like toxin.
20. The hybrid molecule of claim 3, wherein said toxin is Pseudomonas exotoxin A.
This is a continuation of application Ser. No. 07/722,484, filed Jun. 27, 1991, now abandoned, which is a CIP application Ser. No. 07/538,276, filed Jun. 14, 1990, now abandoned, which is a CIP of application Ser. No. 07/456,095, filed Dec. 22, 1989, now abandoned, which is a CIP of application Ser. No. 06/742,554, filed Jun. 7, 1985, now abandoned, which is a CIP of application Ser. No. 06/726,808, filed Apr. 25, 1985, now abandoned, which is a continuation of application Ser. No. 06/618,199, filed Jun. 7, 1984, now abandoned.
This invention relates to hybrid molecules having a cell-binding part and a translocation part.
The literature contains many examples of fused genes which code for hybrid proteins. For example, Villa-Komaroff et al., Proc. Natl. Acad. Sci. U.S.A. 75: 3727-3731, 1978, describes a fused gene made up of a eukaryotic structural gene fused to a non-cytoplasmic bacterial gene. The fused gene codes for a hybrid protein which is transported out of the cytoplasm.
Hybrid proteins also have been made by other methods (e.g., the coupling of two different protein molecules) which do not involve recombinant DNA techniques. For example, it has been proposed to form, by coupling, therapeutic hybrid proteins consisting of portions of toxin molecules coupled to a ligand capable of binding specifically to a selected class of cells. One attempt to make such a hybrid protein, reported in Chang et al., J. Biol. Chem. 252: 1515-1522, 1977, resulted in a hybrid consisting of the diphtheria toxin A chain coupled to human placental lactogen hormone by cross-linking through a disulfide bond. The hybrid protein, although it bound to cells containing lactogen receptors, did not inhibit protein synthesis in those cells.
A hybrid protein consisting of the ricin toxin A chain coupled to the B chain of human chorionic gonadotropin hormone by similarly cross-linking through a disulfide bond has also been reported; although said to have specificity, its binding capacity has not been reported. Furthermore, extremely high concentrations were required to significantly inhibit protein synthesis in rat Leydig tumor cells, making it difficult to distinguish between "non-specific" entry caused by endocytosis and "specific" entry caused by transport of the toxic portion of the hybrid across the cytoplasmic membrane of the target cells (Oeltman et al., J. Biol. Chem. 254: 1028-1032, 1979). The same shortcoming was found in a hybrid protein consisting of diphtheria A coupled to insulin using cystamine as the cross-linking agent (Miskimins et al., Biochem. Biophys. Res. Commun. 91: 143-151, 1979). A hybrid consisting of ricin A coupled to epidermal growth factor (EGF) by means of a heterobifunctional cross-linker has also been made; the binding characteristics provided by the EGF are not limited to specific cells, but rather encompass a wide variety of cell types (Cawley et al., Cell 22: 563-570, 1980).
As illustrated in FIG. 1, the natural diphtheria toxin molecule consists of several functional "domains" which can be characterized, starting at the amino terminal end of the molecule, as a hydrophobic leader signal sequence s (amino acids Val-25 -Ala-1); enzymatically-active Fragment A (amino acids Gly1 -Arg193); the proteolytically-sensitive disulfide loop l1 (amino acids Cys186 -Cys201), containing a cleavage domain; and Fragment B (amino acids Ser194 -Ser535), which includes a translocation domain and a generalized binding domain flanking a second disulfide loop (l2, amino acids Cys461 -Cys471).
The process by which diphtheria toxin intoxicates sensitive eukaryotic cells involves at least the following steps: (i) the binding domain of diphtheria toxin binds to specific receptors on the surface of a sensitive cell; (ii) while bound to its receptor, the toxin molecule is internalized into an endocytic vesicle; (iii) either prior to internalization, or within the endocytic vesicle, the toxin molecule undergoes a proteolytic cleavage in l1 between fragments A and B; (iv) as the pH of the endocytic vesicle decreases to below 6, the toxin spontaneously inserts into the endosomal membrane; (v) once embedded in the membrane, the translocation domain of the toxin facilitates the delivery of Fragment A into the cytosol; (vi) the catalytic activity of Fragment A (i.e., the nicotinamide adenine dinucleotide-dependent adenosine diphosphate (ADP) ribosylation of the eukaryotic protein synthesis factor termed "Elongation Factor 2") causes the death of the intoxicated cell. It is apparent that a single molecule of Fragment A introduced into the cytosol is sufficient to shut down the cell's protein synthesis machinery and kill the cell. The mechanism of cell killing by Pseudomonas exotoxin A, and possibly by certain other naturally-occurring toxins, is very similar.
In general, the invention features, in one aspect, a hybrid molecule including a first part and a second part connected by a covalent bond,
(a) the first part including a portion of the binding domain of a cell-binding ligand, which portion is able to cause the hybrid molecule of the invention to bind to an animal cell; and
(b) the second part including a portion of a translocation domain of a protein, provided that (i) the hybrid molecule does not include an enzymatically-active portion of such protein, (ii) the first part and the second part are not segments of the same naturally-occurring polypeptide toxin, and (iii) the portion of the translocation domain, when covalently bonded to an enzymatically-active portion (i.e., the "effector region") of a toxin selected from diphtheria toxin, Pseudomonas exotoxin A, cholera toxin, ricin toxin, and Shiga-like toxin, is capable of translocating such effector region across the cytoplasmic membrane of the cell. "Translocation" here means the facilitation of movement of a chemical entity from the exterior surface of a cellular membrane (or what constituted the exterior surface prior to formation of an endocytic vesicle), through the membrane, and into the cytosol at the interior of the cell. A "translocation domain" is a segment of a protein which, when the protein is bound to the exterior surface of a cellular membrane, is capable of translocating some portion of that protein through the membrane.
In another aspect, the invention features a hybrid molecule including a first part, a second part, and a third part connected by covalent bonds,
(a) such first part including a portion of the binding domain of a cell-binding ligand, which portion is effective to cause the hybrid molecule to bind to a cell of an animal;
(b) such second part including a portion of a translocation domain of a protein capable of translocating such third part across the cytoplasmic membrane of the cell; and
(c) such third part including a chemical entity to be introduced into the cell, provided that (i) the second part and the third part are not segments of the same naturally-occurring polypeptide toxin; (ii) the second part is connected to the third part by at least one covalent bond (such as a disulfide bond, a peptide bond that is specifically susceptible to endocytic proteases, or a thioether bond) which is substantially cleaved under the chemical and enzymatic conditions that are likely to exist within endocytic vesicles of the targeted cell (such bond being herein specifically termed a "cleavable bond"); and either (iii) the hybrid molecule is a polypeptide produced by expression of a recombinant DNA molecule, or (iv) the first part and the second part are not segments of the same naturally-occurring polypeptide toxin, or both (iii) and (iv).
In preferred embodiments, the second part comprises at least a portion of the translocation domain of a naturally-occurring toxin (e.g. diphtheria toxin or Pseudomonas exotoxin A), and the ligand comprises a hormone (e.g. a polypeptide hormone such as insulin, Interleukin II (also termed "IL2"), Interleukin IV, Interleukin VI or EGF, or, alternatively, asteroid hormone); an antigen-binding, single-chain analog of a monoclonal antibody; or a polypeptide toxin capable of binding to the desired class of cells (more preferably, both the first and the second parts are derived from diphtheria toxin); where both the first and second parts are polypeptides, the hybrid molecule is preferably a recombinant protein; the hybrid molecule preferably additionally comprises a third part which is connected to the second part by at least one covalent bond and which is a chemical entity to be introduced into the cell (provided that where the third part is a polypeptide, the cleavable bond is a disulfide bond). More preferably, all three parts are polypeptides and the hybrid molecule is a recombinant protein (that is, a protein produced by recombinant DNA techniques); the third part and the second part are linked through a proteolytically-sensitive disulfide loop (defined below); the third part is an antigen-binding, single-chain analog of a monoclonal antibody (where such antigen is, for example, a viral protein such as the human immunodeficiency virus (HIV) protease), or alternatively, the enzymatically active portion of an enzyme (e.g., hexosaminidase A; α-1,4-glucosidase; phenylalanine hydroxylase; a protease; a nuclease; or a toxin such as cholera toxin, LT toxin, C3 toxin, Shiga toxin, E.coli Shiga-like toxin, ricin toxin, pertussis toxin, tetanus toxin, diphtheria toxin or Pseudomonas exotoxin A), and most preferably it supplies an enzymatic activity in which the cell is deficient, as, for example, in the case of a genetic deficiency. Where the enzyme is cholera toxin, the resulting hybrid molecule may be used to raise the cyclic AMP level within an animal cell: preferably, the cell so treated is a T-cell and the hybrid molecule includes at least a portion of the binding domain of IL2. By "proteolytically-sensitive disulfide loop" is meant a sequence of at least 5 amino acid residues (preferably from 6 to 30, and more preferably from 11 to 18) joined in series by peptide bonds, the first and last residues of which sequence are Cys residues which link to form a cystine disulfide bond. At least two of the remaining residues of the sequence together create a proteolytically-sensitive site: i.e., a peptide bond formed between two residues, the second (carboxyl side) of which may be, e.g., Arg, Lys, Phe, Tyr, or Trp. There is preferably also at least one Ser residue within the sequence of the loop. The loop, which may be a naturally-occurring feature of the second part or the third part, or may be engineered (e.g., from a synthetic DNA sequence) into the hybrid, joins the third part to the second part by two types of covalent linkages, peptide and sulfhydryl, ensuring that these two portions of the hybrid will remain associated with each other, even in the presence of extracellular proteases, until after the hybrid has bound to the target cell, but will separate at the appropriate stage. Both the proteolytically-sensitive peptide bond(s) within the disulfide loop and the disulfide bond itself are cleaved at some point prior to or during passage of the chemical entity through the cellular membrane of the endocytic vesicle, resulting in the release of the chemical entity into the cytosol, free of the receptor-bound cell-binding ligand portion (the first part), and translocation domain portion (the second part) of the hybrid.
The hybrid molecule of the invention includes the cholera toxin A/diphtheria toxin B'/IL2 hybrid polypeptide encoded by the plasmid illustrated in FIG. 6; the Shiga-like toxin A/diphtheria toxin B'/IL2 hybrid polypeptide encoded by the plasmid illustrated in FIG. 9; the ricin A/diphtheria toxin B'/IL2 hybrid polypeptide encoded by the plasmid illustrated in FIG. 12; the phenylalanine hydroxylase/diphtheria toxin fragment B hybrid polypeptide encoded by the plasmid illustrated in FIG. 14; an HIV protease-binding protein (HIVP-BP)/diphtheria toxin B'/IL2 hybrid polypeptide prepared as hereinafter described; and a Shiga-like toxin A/IL2 hybrid in which both the enzymatic activity and the translocation function are provided by the Shiga-like toxin A portion of the hybrid, and which contains a proteolytically-sensitive disulfide loop. Also included are biologically active mutational analogs of any of the above hybrid polypeptides. As used herein, a "biologically active mutational analog" is a polypeptide which exhibits the same type of cell-binding specificity and the same type of biological activity (e.g., a particular enzymatic or antigen-binding activity) as the listed hybrid polypeptide of which it is an analog, but which differs from such listed hybrid polypeptide by one or more deletions and/or one or more substitutions of one or more amino acid residues. Preferably, the amino acid sequence of the biologically active mutational analog shows at least a 70% (more preferably at least 80% and most preferably at least 90%) homology with the hybrid polypeptide of which it is an analog, and the analog exhibits at least 50% (more preferably, at least 75%) of a biological activity exhibited by the hybrid polypeptide of which it is an analog.
Also within the invention is a recombinant DNA molecule encoding any of the above hybrid polypeptide molecules (including biologically active mutational analogs), a vector including such a recombinant DNA molecule, a cell containing such a vector or recombinant DNA molecule (and which preferably is capable of expressing the recombinant DNA molecule to produce the hybrid polypeptide encoded by it), and a method of preparing the hybrid polypeptide molecule of the invention by permitting a cell containing a recombinant DNA molecule encoding the polypeptide (the "transformed cell") to express the recombinant DNA molecule.
In other preferred embodiments, the third part comprises a detectable label, more preferably a fluorescent moiety, a radioactive moiety, or an electron-dense moiety.
The invention also features a method of labeling a class of cells, which method involves contacting the cells with a hybrid molecule having a third part comprising a detectable label.
Also included in the invention are (1) a method of treating an animal having a deficiency in a certain enzyme, by administering to the animal an effective amount of a hybrid molecule comprising that enzyme; and (2) a method of treating a human patient infected with HIV, by administering to the patient an effective amount of a hybrid molecule having as its third part an HIV protease-binding, single-chain analog of a monoclonal antibody against HIV protease.
Based upon the observation that certain types of polypeptide toxins have three separate functional regions, one region which binds the molecule to particular receptors on the surface of a target cell, a second one which facilitates entry of the enzymatically-active region into the cytosol of the cell, and a third region which exhibits the enzymatic activity that characterizes the toxic effect of the molecule, the invention comprises bi- or tripartite hybrid molecules in which any of these regions may be replaced with functionally comparable regions from other sources. That is, the first functional region may be replaced with a particular binding moiety which binds the hybrid molecule to a selected class of cells, such as IL2 (which binds to high-affinity IL2 receptor-bearing T-cells), or α melanocyte stimulating hormone (αMSH, which binds to melanocytes), or a moiety which binds to a broad spectrum of cell types, as is characteristic of the binding domains of cholera toxin and diphtheria toxin; the second part may be taken from any type of polypeptide in which a translocation domain is identifiable, but will most likely be from a toxin molecule that translocates in a manner similar to diphtheria toxin and Pseudomonas exotoxin A. The optional third part may be any type of moiety that one wants to insert into the cell and that will fit through the channel in the membrane formed by the translocation domain: for example, a cell-killing enzyme such as Shiga toxin; a metabolic enzyme such as phenylalanine hydroxylase (the enzyme in which phenylketonurics are deficient); an antigen-binding, single-chain analog of a monoclonal antibody against an antigen that appears within the target cell; or a fluorescent label.
Although the medical community is rapidly expanding its understanding of the molecular bases of many diseases, one problem has particularly frustrated efforts to translate this understanding into rational protocols for treating the diseases: the problem of how to direct the appropriate therapy into the affected cells so that it can function properly to alleviate or cure the disease. By providing such a method, the present invention will have virtually unlimited applications: from treating genetic deficiency diseases by delivering to affected cells an enzyme supplying the missing function, to supplementing cellular levels of a particular enzyme or a scarce precursor or cofactor, to directing toxins or other poisons to destroy particular cells (such as adipocytes, cancer cells, or virus-infected cells), to counteracting viral infections such as HIV (which causes Acquired Immunodeficiency Syndrome ("AIDS")) by introducing into appropriate cells antibodies to viral proteins. The invention also provides a means for getting other, non-therapeutic substances, such as detectable labels, into targeted cells. The use of a translocation mechanism ensures that the hybrid will be effective in relatively low doses, since a high proportion of the substance of interest will be taken into the targeted cells.
To the extent that the two or three parts of the hybrids of the invention are polypeptides, they may be manufactured as a single hybrid recombinant protein, permitting reproducibility, consistency, and the precise control of composition which is desirable for any pharmaceutical product.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims.
The drawings will be briefly described.
FIG. 1 is a diagrammatic representation of the diphtheria toxin molecule.
FIG. 2 is a restriction map showing the location and orientation of the diphtheria tox gene on the 3.9 kb BamHI restriction fragment of corynephage βtox.
FIG. 3 is a representation of the diphtheria toxin gene and flanking regions, with the protein encoded shown above; the B' region is the region between the labeled Sau3A1-2 and SphI sites.
FIG. 4 is a diagrammatic representation of the strategy used to construct a plasmid encoding fragment B' of diphtheria toxin.
FIGS. 5A and 5B together are a representation of the nucleotide sequence of the Vibrio cholerae toxin gene, with amino acids shown below corresponding codons.
FIG. 6 is a diagrammatic representation of the cloning strategy followed to construct a plasmid encoding cholera toxin A1 -diphtheria toxin B'-IL2 hybrid.
FIG. 7 is a diagrammatic representation of the cloning strategy followed in order to construct the plasmid pPA123.
FIGS. 8A, 8B and 8C together are a representation of the nucleotide sequence of the E.coli bacteriophage H19B Shiga-like toxin gene, with amino acids shown below corresponding codons.
FIG. 9 is a diagrammatic representation of a cloning strategy proposed for constructing a plasmid encoding a Shiga-like toxin A-diphtheria toxin B'-IL2 hybrid.
FIG. 10 is a diagrammatic representation of an alternative cloning strategy for constructing a plasmid encoding a Shiga-like toxin A-diphtheria toxin B'-IL2 hybrid.
FIGS. 11A and 11B together are a representation of the nucleotide sequence of the Ricinus communis ricin gene, with amino acids shown above corresponding codons; this figure is adapted from FIG. 2 of Halling et al. (Nucl. Acids Res. 13: 8019-8033, 1985).
FIG. 12 is a diagrammatic representation of the cloning strategy proposed for constructing a plasmid encoding a ricin A-diphtheria toxin B'-IL2 hybrid.
FIGS. 13A, 13B and 13C together are a representation of the nucleotide sequence of human phenylalanine hydroxylase cDNA, with amino acids shown below corresponding codons.
FIG. 14 is a diagrammatic representation of a cloning strategy proposed for constructing a plasmid encoding a phenylalanine hydroxylase-diphtheria toxin B hybrid.
One embodiment of the hybrid molecule of the invention is a two-part hybrid protein, comprising (1) a translocation domain, such as that of diphtheria toxin, and (2) a cell-binding domain from the same or a different origin as the translocation domain sequence. The cell-binding domain may be generalized (i.e. it is capable of binding the hybrid molecule to a wide variety of cell types) or specific for one or a few types of cells. The hybrid molecule may also contain a third part, linked to the translocation domain through a cleavable bond (as defined above) such that the translocation domain is capable of translocating the third part into or across the membrane of the cell to which the cell-binding portion of the hybrid is bound. This third part may be, for example, an enzymatically active polypeptide, an antigen-binding portion of a monoclonal antibody, or a detectable label such as a fluorescent dye. It may not, however, be a fragment of the same naturally-occurring molecule from which the translocation domain originates.
Naturally-occurring proteins which are known to have a translocation domain include diphtheria toxin and Pseudomonas exotoxin A, and may include other toxins and non-toxin molecules, as well. The translocation domains of diphtheria toxin and Pseudomonas exotoxin A are well characterized (see, e.g., Hoch et al., Proc. Natl. Acad. Sci. USA 82: 1692-1696, 1985; Colombatti et al., J. Biol. Chem. 261: 3030-3035, 1986; and Deleers et al., FEBS 160: 82-86, 1983), and the existence and location of such a domain in other molecules may be determined by methods such as those employed by Hwang et al., Cell 48: 129-136, 1987; and Gray et al., Proc. Natl. Acad. Sci. USA 81: 2645-2649, 1984.
The segment of diphtheria toxin labelled "Fragment B" in FIG. 3 includes both the translocation domain and the generalized cell-binding domain of the naturally-occurring molecule. Truncation of Fragment B to the segment marked B' effectively eliminates the cell-binding function of diphtheria toxin while retaining the translocation function of the molecule. In the two-part hybrid of the invention, a portion of Fragment B encoded by a sequence ending at or downstream from the Sph1 restriction site may be used as long as it does not include sequences encoding a sufficient part of the diphtheria toxin receptor-binding domain to yield a functional receptor-binding domain.
The part of the hybrid protein contributed by the polypeptide ligand can consist of the entire ligand, or a portion of the ligand which includes the entire binding domain of the ligand, or an effective portion of the binding domain. When the ligand being used is large, it is desirable that as little of the non-binding portion as possible of the ligand be included, so that the binding domain of the molecule is positioned close to the translocation domain. It is also desirable to include all or most of the binding domain of the ligand molecule.
The polypeptide portions of the hybrids of the invention are conveniently made using recombinant DNA techniques involving forming the desired fused gene encoding the hybrid protein, and then expressing the fused gene. Chemical cross-linking is utilized only where one or more of the parts of the hybrid molecule are not polypeptides.
Standard procedures for DNA cloning, cell transformation and plasmid isolation (as described, for example, by Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982), and for oligodeoxynucleotide synthesis, could be employed to carry out the following constructions.
Referring to FIGS. 2 and 3, the location and orientation of the diphtheria tox operon on the 3.9 kb BamHI restriction fragment of corynephage βtox+ allows the tox operon to be cleaved at a desired location, and the desired portion of the operon to be fused with the desired portion of the gene for a selected polypeptide ligand.
Gene fusions of the invention may be made as follows. First, the NsiI-SphI fragment encoding most of Fragment B' (FIG. 3) is isolated from the tox gene of plasmid pABM508 (Murphy et al., Proc. Natl. Acad. Sci. USA 83: 8258-8262, 1986). The following linker is ligated into the 5' (NsiI) end: ##STR1## and the following linker is ligated into the 3' (SphI) end: ##STR2##
The resulting fragment is then cloned into NcoI+HindIII-digested pKK233-2 (Pharmacia, Piscataway, N.J.).
This modification allows the expression of Fragment B' to be driven off the trc promoter (Ptrc) in E.coli. The SphI site permits in-frame fusion with a gene sequence encoding the binding domain of a peptide ligand.
Generally, the manipulative operations are carried out using cloning vectors; e.g., phages or plasmids. The genetic material coding for the binding domain of the polypeptide ligand can be either DNA cloned from a natural source, or a synthetic oligonucleotide sequence. Generally the fused gene will reside on a cloning vector, e.g., a plasmid or a phage, which is used to transform cultured bacteria, yeast or tissue culture host cells. The hybrid protein is then harvested from the cells using conventional techniques. Purification of the hybrid proteins of the invention, regardless of the polypeptide ligand used, can be carried out via affinity chromatography, using a monoclonal antibody against diphtheria toxin Fragment B'.
The purified hybrid protein of the invention may be used as a transport system to carry a detectable label into specific cells. The label which is attached to the hybrid protein molecule can be any conventional atom or molecule used for diagnostic labeling; examples are radioactive labels such as 125 I-compounds, technetium isotopes, NMR reporter groups, and fluorescent dyes. The most preferred labels are hydrophobic labels such as fluorescent dyes (most conventional fluorescent dyes happen to be hydrophobic) which are incorporated into the cytoplasmic membrane of target cells, as will be explained in more detail below. Labels can be attached to the hybrid protein according to conventional labeling techniques. Labels are used in an amount (e.g., one or two label molecules per protein molecule) which does not interfere with the binding or cell penetration functions of the hybrid protein molecule.
The labeled hybrid proteins of the invention can be used diagnostically to label a desired class of target cells, a class determined by the specific polypeptide ligand which imparts the binding domain of the hybrid molecule. The specific binding domain of the polypeptide ligand portion selectively binds to those cells; the labeled molecule is then taken up by the cells via receptor-mediated endocytosis, and the label subsequently is delivered to the cell membrane and/or the cytoplasm of the target cells.
The process by which a labeled hybrid protein of the invention can be incorporated into cells can be summarized as follows. The labeled hybrid protein is taken up by target cells via receptor-mediated endocytosis into an endocytic vesicle; thereafter a pH differential across the membrane of the endocytic vesicle is established as a result of the cell's ATP-dependent proton pump. The pH differential across the membrane causes the hybrid protein, including its lipid-associating portion and its label, to be inserted into the plane of the membrane of the endocytic vesicle. The hydrophobic nature of the hybrid protein causes it to remain in the membrane, protected from the rapid enzymatic degradation which would occur were the labeled protein to reside in the cytoplasm or in the lumen of the endocytic vesicle.
After insertion into the plane of the membrane of the endocytic vesicle, the labeled hybrid protein can "traffic", as follows. The endocytic vesicle buds off from the cytoplasmic membrane and enters the cytoplasm of the cell, where it can merge with a lysosome into which the labeled hybrid protein is then incorporated. Alternatively, the endocytic vesicle can recycle to the cytoplasmic membrane of the cell. In either case, the label remains trapped in the target cell.
As is mentioned above, a major diagnostic use of the labeled hybrid proteins will be the in vivo and in vitro detection of metastatic loci, using conventional cell staining and labeling techniques. Such detection could be of particular value in surgery, by providing the surgeon with information needed to know how much tissue to excise when removing, e.g., metastatic melanoma cells.
Plasmid pCVD2 containing the coding sequence for the enzymatically-active A1 fragment of cholera toxin (see FIG. 5) was prepared from a Vibrio cholera DNA library as described by Mekalanos et al. (Nature 306: 551-557, 1983). FIG. 6 outlines the strategy employed in engineering a cholera toxin A1 -diphtheria toxin B'-IL2 gene. Briefly, pCVD2 was cleaved with the restriction enzyme XbaI at the unique XbaI site. The following synthetic linker, which has 1/2 of an XbaI site at each end, was ligated to the linearized plasmid in order to introduce a NcoI site upstream from the XbaI site: ##STR3## The appropriate construct was selected by restriction site mapping and sequence determinations, and then was digested with Nco1 and ClaI to produce a Nco1-ClaI fragment. This in turn was digested with ScrFI. The 3' end of the resulting Nco1-ScrFI fragment was ligated to the following synthetic linker: ##STR4##
The polypeptide encoded by the resulting NcoI-ApaI fragment lacks the natural cholera toxin signal sequence, having instead fmet-Gly followed by the mature A1 region of cholera toxin, followed by Gly-Ser-Gly-Pro. This construct can be cloned into a plasmid that encodes diphtheria toxin fragment B' fused to the human interleukin-2 gene (plasmid pPA123, FIG. 7). Plasmid pPA123 was constructed from plasmid pDW24 (Diane Williams, Ph.D. dissertation, Boston University School of Medicine, Department of Microbiology, Boston, Mass., 02118, 1989) as outlined in FIG. 7. Plasmid pDW24 encodes a diphtheria toxin fragment A-fragment B'-IL2 fusion protein that is expressed off the trc promoter in E.coli. The sequences encoding fragment A were deleted by digestion with the restriction endonucleases NcoI and NsiI. The following oligonucleotides were used to rebuild the fragment A/B disulfide loop (l1) sequence, introduce an ApaI site on the 5' end of the loop, and recreate the NcoI site encoding the translation-initiating ATG codon: ##STR5##
Plasmid pPA123 resulted from ligating the above oligonucleotide fragment onto the NcoI-NsiI-digested pDW124 vector fragment. Plasmid pPA123 can now be used to fuse sequences encoding cholera toxin fragment A1 to diphtheria toxin B'-IL2 as shown in FIG. 6. Plasmid pPA123 is digested with restriction enzymes NcoI and ApaI, and the resulting vector fragment is ligated to the modified cholera toxin fragment A1 described above, to yield a plasmid encoding a cholera toxin A1 -diphtheria toxin B'-IL2 hybrid ("CTA/DTB'/IL2 hybrid"), which is expressed from the trc promoter on the plasmid.
Following expression of the recombinant gene in E.coli, the CTA/DTB'/IL2 hybrid protein can be isolated and used in appropriate treatment regimens: for example, as an adjunct to treatment with diphtheria toxin-IL2 hybrid. Diphtheria toxin-IL2 hybrid effectively targets the cell-killing ability of diphtheria toxin to cells bearing the IL2 receptor, such as certain leukemic T-cells. However, the pharmacological effectiveness of diphtheria toxin-IL2 hybrid is diluted by circulating endogenous IL2, which is naturally synthesized by activated T-cells and which competes with diphtheria toxin-IL2 hybrid for IL2 receptors on T-cells. By first exposing the target cells to CTA/DTB'/IL2 hybrid, the biological activity of cholera toxin can be harnessed to alleviate this problem. The A1 subunit of natural cholera toxin enzymatically catalyzes the ADP-ribosylation of a GTP-binding regulatory component of the adenylate cyclase complex, resulting in the accumulation of cyclic AMP within the affected cell and thereby disrupting a multitude of cellular functions without killing the cell. Targeting the cholera toxin A1 activity specifically to cells bearing the IL2 receptor will result in the temporary inhibition of IL2 synthesis within those cells. This permits depletion of the amount of circulating IL2 available to compete with diphtheria toxin-IL2 for IL2 receptors, without interfering with expression of IL2 receptors on the surfaces of the T-cells and without injuring non-targeted cells. Subsequent treatment with diphtheria toxin-IL2 will thus be more effective at killing T-cells than if CTA/DTB'/IL2 hybrid had not been used.
The DNA sequence and corresponding amino acid sequence for the A subunit of Shiga-like toxin ("SLT-A") are shown in FIG. 8. Bacteriophage H19B DNA from a strain of E.coli that produces SLT-A is prepared as described by Calderwood et al. (Proc. Natl. Acad. Sci. USA 84: 4364-4368, 1987) and digested with TagI and XmnI. A TagI-XmnI fragment (approx. 650 bp) corresponding to most of the coding sequence for SLT-A (the "sltA gene") is isolated therefrom (see FIG. 9); the following oligonucleotide is then ligated onto the 5'(TagI) end of the fragment: ##STR6##
This oligonucleotide sequence provides an fmet-Gly coding sequence followed by a sequence coding for the first eight amino acids of the mature SLT A subunit, to replace the section of the natural gene (coding for the toxin signal peptide and same eight amino acids of the mature SLT A subunit) which was cleaved off during TagI digestion of the gene. Also provided by the oligonucleotide linker is a 1/2 NcoI site at the 5' end of the construct, to permit expression from the trc promoter of the hybrid plasmid.
The following oligonucleotide sequence, which regenerates the coding region (cleaved off by XmnI digestion) for the carboxyl end of the SLT A subunit up to the initial Cys codon, and introduces an ApaI restriction site, is ligated to the 3' (XmnI) end of the sltA gene fragment: ##STR7##
The NcoI-ApaI sltA gene sequence can be ligated into a NcoI+ApaI-digested plasmid pPA123 (FIG. 7) to yield a SLTA-diphtheria toxin B'-IL2 ("SLTA/DTB'/IL2 hybrid") gene that can be expressed in E.coli from the trc promoter on the plasmid (see FIG. 9).
An alternative cloning strategy for constructing a plasmid encoding a Shiga-like toxin A-diphtheria toxin B'-IL2 hybrid is illustrated in FIG. 10.
Purified SLTA/DTB'/IL2 hybrid protein would be useful as a treatment for conditions involving overproduction of cells bearing IL2 receptors, such as certain T-cell lymphomas and organ transplant rejection crises. As is the case for diphtheria toxin-IL2, the IL2 portion of the hybrid causes the hybrid to attach specifically to IL2-receptor-bearing cells, and the diphtheria toxin B' portion acts to insert the enzymatic portion of the hybrid into the targeted cell; the enzymatic portions of both types of hybrid toxins then act on the protein synthesis machinery in the cell to shut down protein synthesis, thus killing the cell. The difference between these two types of hybrid toxins is the nature of their enzymatic activities: the enzymatic portion of diphtheria toxin-IL2 hybrid catalyzes the ADP-ribosylation by nicotinamide adenine dinucleotide of Elongation Factor 2, thereby inactivating this factor which is necessary for protein synthesis, while the enzymatic portion of SLTA/DTB'/IL2 hybrid is a ribonuclease capable of cleaving ribosomal RNA at a critical site, thereby inactivating the ribosome. SLTA/DTB'/IL2 hybrid would therefore be useful as a treatment for the same indications as diphtheria toxin-IL2 hybrid, and could be substituted if, for example, the proliferating T-cells develop a resistance to the latter hybrid toxin.
A genomic clone bank of castor bean (Ricinus communis) DNA is prepared as described in Halling et al., Nucl. Acids Res. 13: 8019-8033, 1985, and a ˜780 bp BanI fragment of the ricin gene, corresponding to most of the ricin A domain (the enzymatic domain) and a portion of the ricin A-to-B linker peptide, is isolated therefrom (see FIG. 11). The following synthetic oligonucleotide is ligated onto both ends of the fragment, phosphorylating only the bottom strand of DNA shown: ##STR8## The resulting ligated fragment (illustrated in FIG. 11) is partially digested with FspI, and the ˜780 bp band corresponding to a BamI-FspI ricin A gene fragment with a NcoI-BaMI linker at the 5' end is isolated (see FIG. 11). The NcoI-BamI linker supplies the mature ricin A N-terminal amino acid codons which were cleaved from the fragment during BamI digestion, as well as the codons for fmet-Ala to replace the natural ricin A signal peptide.
The following oligonucleotide is ligated onto the 3' (FspI) blunt end of the fragment, phosphorylating only the top strand shown: ##STR9## This linker supplies the ricin A coding sequence cleaved from the 3' end of the ricin A fragment during the FspI digest, plus a 1/2 ApaI site for fusion to plasmid pPA123.
The completed construct is then cloned into NcoI/ApaI-digested pPA123 to yield a ricin A-diphtheria toxin B'-IL2 gene that can be expressed in E.coli from the trc promoter on the plasmid (see FIG. 12).
Purified ricin A-diphtheria toxin B'-IL2 hybrid, like the SLTA/DTB'/IL2 hybrid of Example 3, inactivates ribosomes in cells bearing IL2 receptors, resulting in cessation of protein synthesis and death of the targeted cells. The ricin A hybrid would thus have the same applications as SLTA/DTB'/IL2 hybrid, as discussed in Example 3.
A human liver cDNA library is screened for phenylalanine hydroxylase ("PH") cDNA as described by Kwok et al., Biochem. 24: 556-561, 1985. The approximately 1160-bp EcoRII-AflII fragment that encodes most of the PH protein is isolated (see FIGS. 13 and 14). The following linkers are ligated onto the 5' EcoRII end in order to recreate the 5' coding sequences and incorporate an NcoI site: ##STR10##
The following linkers are ligated onto the 3' AflII end to complete the PH coding sequence and to include an ApaI restriction site in the correct translational reading frame for fusion to diphtheria toxin fragment B sequences (FIG. 14): ##STR11##
This fragment is then ligated onto the NcoI-ApaI digested pPA123 vector (FIG. 14) resulting in a plasmid that encodes phenylalanine hydroxylase fused to diphtheria toxin B'-IL-2. Finally, this plasmid is digested with EcoRI and SphI to remove IL-2 encoding sequences, which are replaced by the approximately 230 bp SphI-EcoRI fragment of corynebacteriophage β that encodes the 3' end of diphtheria toxin fragment B (FIG. 14). This completed construct codes for a PH-diphtheria toxin B hybrid protein that can be expressed in E.coli from the trc promoter on the plasmid (see FIG. 14).
The inherited disorder phenylketonuria, in which the inability to metabolize phenylalanine leads to an accumulation of excess phenylalanine and possible brain damage in affected individuals, has been attributed to a genetic deficiency of the enzyme PH. By constructing a molecule in which active PH enzyme is linked to the cell-binding and translocation domains of diphtheria toxin Fragment B, the enzyme can be targeted to and incorporated into the broad range of cells which native diphtheria toxin normally attacks, achieving the widespread therapy that is called for by a defect such as phenylketonuria. This cloning strategy would be applicable to the construction of other hybrids useful in the treatment of other genetic defects.
A recombinant gene expressing a novel protein, an antigen-binding, single-polypeptide-chain analog of a monoclonal antibody composed of an antibody variable light-chain amino acid sequence (VL) linked to a variable heavy-chain sequence (VH) by a linker peptide, is constructed by the method of Bird et al., Science 242: 423-426, 1988, based upon the VL and VH sequences of a monoclonal antibody specific for and able to inactivate HIV protease (Hansen et al., Embo J. 7: 1785-1791, 1988) and a linker peptide designed by the method of Bird et al. The ends of the VL -linker-VH gene are modified with appropriate restriction enzymes and synthetic DNA linkers in order to produce an intact VL -linker-VH gene having 1/2 of a NcoI site at the 5' end and 1/2 of an ApaI site at the 3' end. The gene is then cloned into NcoI+ApaI-digested pPA123 to produce a plasmid expressing, from the trc promoter, an HIV protease-binding protein-diphtheria toxin B'-IL2 hybrid protein ("HIVP-BP/DTB'/IL2 hybrid").
Following expression of the recombinant gene in E.coli, the HIVP-BP/DTB'/IL2 hybrid protein can be isolated and used to treat an HIV infection in a human patient. The HIV virus infects and proliferates within T-cells, commandeering the cellular protein synthesis machinery to produce multiple copies of its own proteins. One viral protein in particular, the HIV protease, plays a critical role in the processing of other viral proteins; identifying a way to inactivate this protease within the infected cell has been the focus of much recent effort toward developing an effective AIDS therapy (see, e.g., Hansen et al.). The HIVP-BP/DTB'/IL2 hybrid delivers a viral protease-specific inhibitor specifically to activated T-cells bearing the IL2 receptor, and thus can be effective at low dosages, with little or no toxicity to other types of cells. This technology could be applied as well to other viral infections or genetic disorders.
Other embodiments are within the following claims. For example, any cell-specific polypeptide ligand can be used which has a binding domain specific for the particular class of cells which are to be labeled. Polypeptide hormones are useful such ligands. Hybrid protein made using the binding domain of α or β MSH, for example, can selectively bind to melanocytes, rendering hybrids, once labelled with a detectable label, useful in the diagnosis of melanoma and the in vivo and in vitro detection of metastic melanoma loci. Such a hybrid, when attached to an enzymatically-active portion of a toxin molecule instead of to a detectable label, could be utilized to deliver that toxic activity specifically to the target melanoma cells. Other ligands provide different specificities: e.g., the binding domain of substance P recognizes receptors on the surfaces of neurons involved in the transmission of pain, so that labeled hybrids made using substance P can be used to map areas of the nervous system containing substance P receptors. Other specific-binding ligands which can be used include insulin, somatostatin, EGF, and Interleukins I, II, III, IV and VI. Interleukin II is of particular importance because of its role in allergic reactions and autoimmune diseases such as Systemic Lupus Erythmatosis (SLE), involving activated T cells. Other useful polypeptide ligands having cell-specific binding domains are follicle stimulating hormone (specific for ovarian cells), luteinizing hormone (specific for ovarian cells), thyroid stimulating hormone (specific for thyroid cells), vasopressin (specific for uterine cells, as well as bladder and intestinal cells), prolactin (specific for breast cells), and growth hormone (specific for certain bone cells). Alternatively, a relatively indiscriminate cell-binding ligand (such as that of diphtheria toxin or ricin toxin) capable of binding to a wide variety of cell types in an organism can be used to effect widespread introduction of a specific chemical entity into cells of that organism, where more specific targeting is not the goal.
For a number of cell-specific ligands, the region within each such ligand in which the binding domain is located is now known. Furthermore, recent advances in solid phase polypeptide synthesis enable those skilled in this technology to determine the binding domain of practically any such ligand, by synthesizing various fragments of the ligand and testing them for the ability to bind to the class of cells to be labeled. Thus, the hybrid molecules of the invention need not include an entire ligand, but rather may include only a fragment of a ligand which exhibits the desired cell-binding capacity. Likewise, analogs of the ligand or its cell-binding region having minor sequence variations may be synthesized, tested for their ability to bind to cells, and incorporated into the hybrid molecules of the invention. Other potential ligands include monoclonal antibodies or antigen-binding, single-chain analogs of monoclonal antibodies, where the antigen is a receptor or other moiety expressed on the surface of the target cell membrane.
The translocation function of the hybrid molecule may be supplied by an appropriate piece of a polypeptide other than diphtheria toxin, but which is capable of translocating in a manner analogous to that of diphtheria toxin (e.g., Pseudomonas exotoxin A, botulinum, neurotoxin, or ricin), or in any other manner which accomplishes the objective of translocating the functional "third part" of the hybrid molecule into the cell's cytoplasm.
The chemical entity to be inserted into the cell can vary widely and still be within the invention. For example, the enzyme which is genetically deficient in Tay-Sachs disease, hexosaminidase A, could be attached to a hybrid having a cell-binding domain specific for the cells most affected by the disease, nerve cells. Patients suffering from type 2 glycogenosis might be treated with a hybrid comprising α-1,4-glucosidase linked to the translocation segment of diphtheria toxin linked to insulin, which would largely target muscle cells, hepatocytes, and lymphocytes. (See Poznansky et al., Science 223: 1304-1306, 1984.) These are simply examples: any other enzyme deficiency disease for which the natural enzyme or its gene has been sequenced (or is amenable to sequencing by one skilled in the art, without undue experimentation) could be treated with a hybrid comprising the active enzyme linked to a translocation domain linked to an appropriate cell-binding ligand.
Intracellular viral and bacterial infections could be treated by an appropriate hybrid: for example, a hybrid which delivers into the cell a potent antibiotic, or a recombinant VL -linker-VH antigen-binding polypeptide which specifically binds viral particles or proteins.
Likewise, the hybrid of the invention will be useful for specifically destroying certain cells. Besides the cholera toxin A1 -hybrid, ricin A-hybrid and Shiga-like toxin A-hybrid exemplified above, a cell-killing function may be provided by the enzymatically-active portion of any polypeptide toxin, including but not limited to LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin and Pseudomonas exotoxin A. Cells to be targeted might include cancer cells, virus-infected cells, or adipocytes.
The invention includes biologically active mutational analogs of hybrid polypeptides described above. By manipulating the recombinant DNA sequence encoding the subject hybrid polypeptide using methods well known to those of ordinary skill in the art of genetic engineering, a series of mutations involving deletions and/or substitutions of individual or multiple base pairs in such recombinant DNA sequence is first created. Each such mutated sequence is then inserted into an expression vector and expressed in an appropriate expression system. The biological activity of the mutational analog so produced can then be compared to that exhibited by the hybrid molecule of which it is an analog (the "parent polypeptide"). The particular assay used will depend upon the particular enzymatic activity and cell-binding specificity of the parent polypeptide. For example, mutational analogs of the Shiga-like toxin A/diphtheria toxin B'/IL2 (SLTA/DTB'/IL2) hybrid, the cholera toxin A1 /diphtheria toxin B'/IL2 (CTA/DTB'/IL2) hybrid, and the ricin A/diphtheria toxin B'/IL2 hybrid may be tested and compared to their respective parent polypeptides in the following cell cytotoxicity assay, which is specific for toxins capable of binding to IL2 receptor-bearing cells.
Cultured HUT 102/6TG (Tsudo et al., Proc. Natl. Acad. Sci. USA 83: 9694, 1986) or YT2C2 (Teshigawari et al., J. Exp. Med. 165: 223, 1987) cells are maintained in RPMI 1640 medium (Gibco, Grand Island, N.Y.) supplemented with 10% fetal bovine serum (Cellect, GIBCO), 2 mM glutamine, and penicillin and streptomycin to 50 IU and 50 μg/ml, respectively. Cells are seeded in 96-well V-bottomed plates (Linbro-Flow Laboratories, McLean, Va.) at a concentration of 5�104 per well in complete medium. Putative toxins are added to varying concentrations (10-12 M to 10-6 M) and the cultures are incubated for 18 hrs. at 37� C. in a 5% CO2 atmosphere. Following incubation, the plates are centrifuged for 5 min. at 170� g, and the medium removed and replaced with 200 μl leucine-free medium (MEM, Gibco) containing 1.0 μCi/ml [14 C]-leucine (New England Nuclear, Boston, Mass.). After an additional 90 min. at 37� C., the plates are centrifuged for 5 min. at 170� g, the medium is removed and the cells are lysed by the addition of 4M KOH. Protein is precipitated by the addition of 10% trichloroacetic acid and the insoluble material is then collected on glass fiber filters using a cell harvester (Skatron, Sterling, Va.). Filters are washed, dried, and counted according to standard methods. Cells cultured with medium alone serve as the control.
Where IL4 replaces IL2 as the cell-binding portion of the resulting hybrid, the hybrid and its mutational analogs may be tested by a similar assay utilizing CT4R cells (William E. Paul, NIH), P815 cells (ATCC), or CTLL2 (ATCC), seeded at 1�104 cells per well and incubated for 40 hours.
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Vulvodynia is defined as "vulvar discomfort, most often described as burning pain, occurring in the absence of relevant visible findings or a specific, clinically identifiable, neurologic disorder." Vulvodynia affects an estimated 16% of women in the general population. There are two major types of vulvodynia that are based on pain location. The first is localized vulvodynia, in which pain is restricted to a portion of the vulva, such as the vestibule, as in provoked vestibulodynia (PVD). The second is generalized vulvodynia (GVD), in which the pain is more diffuse, involving the whole vulva.
PVD is the most common cause of dyspareunia (i.e., painful intercourse) in women of child-bearing age. An epidemiological study estimated that PVD affects 12% of pre-menopausal women in the general population. Women with PVD experience a highly localized, burning and/or cutting pain at the entrance of the vagina (called the vulvar vestibule) during sexual intercourse, as well as during other activities that involve applying pressure to the vestibule (e.g., tampon insertion, gynecological exams). Although the pain of PVD typically disappears after pressure to the vestibule is removed, many women report lasting pain or discomfort after sexual intercourse or similar activities.
Approximately 50% of women who suffer from PVD have what is called primary PVD, indicating that the pain has been present since their first intercourse or vaginal penetration attempt. The other half has secondary or acquired PVD, which develops after a period of pain-free intercourse or vaginal penetration activities, and in many cases, after an aggravating factor (e.g., repeated vaginal infections, sexually transmitted infections). Not all women with secondary PVD, however, can identify a trigger for the onset of their pain. Little is known about the causes of PVD; most health professionals agree that it is caused by a combination of factors.
How is PVD Treated?
There is scientific evidence that the following treatments are effective for PVD:
It is generally recommended to begin treatment with either psychotherapy or pelvic floor physiotherapy (PFP), or both. One study found that psychotherapy and PFP are equally successful in treating the pain, physical, and emotional components of PVD. Not surprisingly, psychotherapy is a little better at treating the sexual component of PVD, and PFP is a little better at treating pelvic floor muscle tone and post-contraction relaxation of the muscles; both treatments complement each other well. As well, another study indicated that 77% of women who underwent 8 sessions of PFP reported significant improvements in their pain and sexual functioning, and cognitive and emotional function. Although less commonly offered, a combined treatment of PFP and psychotherapy was reported to be beneficial in 19/24 (79%) of women with PVD.
If there is no significant improvement with psychotherapy or physiotherapy, a vestibulectomy may be indicated. This is a relatively minor day procedure carried out under general or spinal anesthesia. Following the operation, women will typically experience some discomfort in the genital region. Neither intercourse nor any other penetrative activity should be attempted for 6-8 weeks post-surgery. Seventy percent of women with PVD who underwent this surgery in one study reported a great decrease in their pain or complete pain relief. For a small percentage of women, however, there can be no benefit or worsening of symptoms.
You may have come across information about other forms of treatment for PVD, such as vaginal creams, diets, and laser surgery. There is no evidence for their effectiveness, and in fact, some of these treatments may have unintended negative side effects. Reports have suggested that alternative treatments, such as hypnosis for pain control, mindfulness, and acupuncture, have been successful in some women with PVD. However, more research is needed to fully understand the effects of these treatments.
GVD is a common form of vulvar pain, affecting 6-7% of women in the general population, with a higher prevalence in women over the age of 30. In GVD, the pain is present on a constant or almost constant basis and affects the entire vulvar region. Like PVD, the pain of GVD is described predominantly as burning; in fact, the original term for GVD was "the burning vulva syndrome." GVD not only affects sexual functioning in most women, it also affects daily activities (e.g., sitting for long periods of time at work, bicycle riding) due to the constant nature of the pain.
How is GVD treated?
There is little research on the treatment of GVD. A few small studies showed that women who were treated with a low dose antidepressant medication (i.e., amitriptyline; commonly used for the treatment of neuropathic pain conditions which share the "burning" and "constant" qualities of pain with GVD) or gabapentin (also used for the treatment of neuropathic pain) reported pain reduction. While psychotherapy that combines a pain management and sexuality component might be helpful for women with GVD, there is no direct evidence to support this. Surgery, however, should be avoided.
*If you do not reside in the Kingston area and would like help finding resources in your local area, please contact the Sexual Health Research Lab at 613.533.3276 or email@example.com
Contact your family doctor for a referral to a local gynecologist.
Contact your family doctor for a referral to a local neurologist.
Pelvic floor physiotherapy
Elizabeth Tata, Melanie Law, & Cindy Auchincloss at K-TOWN Physiotherapy (two locations)
368 King Street East
Kingston, ON K7K 2Y2
(613) 542-5878
West End:
598 Cataraqui Woods Dr,
Kingston, ON K7P 1T8
(613) 634-5878
Please note that if you would like to make an insurance claim after your appointment with one of the pelvic floor physiotherapists, you will need a referral from your family doctor.
Psychology (focusing on sex and couples therapy)
Sex Therapy Service, Psychology Clinic, Queen's University
Dr. Caroline Pukall and student therapists
184 Barrie Street
Phone: (613) 533-6021
Fax: (613) 533-3282
Psychology (focusing on couples therapy)
Dr. Debra Kowalik
797 Princess Street, Kingston
Phone: (613) 544-1065
Dr. Gisele Pharand
55 Sunny Acres Road, Kingston
Phone: (613) 384-1014
Francoise Mathieu, M.Ed, CCC
847 Princess Street, Kingston
Phone: (613) 547-3247
Dr. Vince Caccamo
221 King Street East, Kingston
Phone: (613) 547-9814
Sexual Health Research Laboratory
Phone: (613) 533-3276
Email: firstname.lastname@example.com

subset: commoncrawl


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$12.95 104 pages
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Diamond: APR06 2846
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Graphic Novel, Action Adventure, Science Fiction
- - -
Find any AiT/Planet Lar publication at your nearest comic store.
Can't find it at your local retailer? Get it online at Khepri.com
Alicia's life as a typical suburban misfit takes a horrific turn as her dreams begin to alter reality. She quickly finds herself orphaned, pregnant and on the run from a pharmaceutical police state. Now she's fighting to stay awake and restore the world she once knew. But when a lonely doctor offers Alicia redemption, will she take it? Or will her dreams tear reality apart?
"This upcoming graphic novel from AIT/Planet Lar focuses on Alicia, a pregnant teenager who ran away to New York. The problem is, she's never had sex and no one's heard of New York. And every time she goes to sleep, the whole world changes. But in this story, what could have been a sci-fi cliche becomes an examination of teen loneliness and anxiety. Alicia is quite literally no longer in control of her life, as her dreams continually blur the boundaries between fantasy and reality. And she never quite manages to take control, despite forming new attachments in a family of choice, of sorts, as she still spends all her time and energy focusing on avoiding her central dilemma. Only at the end does she reach a resolution which manages to be both touching and tragic.
"Talbert's art is excellent. It's very expressive and he handles both action and quiet conversation well. He also does an exceptional job in showing, incrementally, Alicia's deterioration as she runs away, lives on the streets, and goes without sleep for nine months.
"Continuity has an emotional depth and resonance that you don't often find in AIT's more action-orientated works, coupled with very strong and dramatic art. It will be released in June 2006."
—Dorian Wright, PostModernBarney.com


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MARSHALL COUNTY, Ala. (WHNT)–Tuesday, the 12th day of March, marks the 20th anniversary of the start of the Blizzard of 1993. Meteorologists called it "The Storm of the Century," because of its power and size. It dumped more than a foot of snow in many of areas of North Alabama.
In the days that followed the storm, a news crew from WHNT News 19 visited the Folsoms in Marshall County. 20 years later we caught up with them. Below you'll see the story where we interviewed them in 2013 and below that, our original story from 1993.


text:
Rate this Game:
In this fun game you get to drive a milk truck and deliver milk to your customers. The challenge is not to break any bottles as you drive about! Can you make all your deliveries without spilling milk? Play this fun game today and find out!
Play our free online games. Play games online. We've got good games: 2 Player games, Action and Adventure games, Arcade games, Board and Card games, Cartoon Character games, Cooking games, Dress Up games, Educational games, Puzzle games, Racing games, Sports games and Time Management games. Play them today! Our good games are suitable for all ages! We also have Autumn games, Christmas games, Easter Games, Halloween Games, Spring Games, Summer Games, Valentine Games and Winter Games.


text:
Letters, we get letters . . ..
August 21, 2008 | 4:01 am
Yeah one should not type out of anger. You are gods people and i am a gentile. I see you and the others fighting its crazy. I have no faith and... More.
August 21, 2008 | 4:01 am
August 14, 2008 | 4:36 pm
August 12, 2008 | 10:20 pm
August 12, 2008 | 1:18 am
As web surfers from around the globe continue their perennial quest for the secret, hidden Jews who control all sorts things here on Olam HaZeh, some, inevitably, find their way to JewishJournal.com.
Here's a list of recent searches. Click on the name of the suspected Jew to see the article they found:
August 10, 2008 | 3:58 pm
August 5, 2008 | 5:16 pm
So I was sitting in "holding" barely shaded by the hot sun in Long Beach where they were filming the TV show Dexter. I had woken up at 5 a.m. again,... More.
July 17, 2008 | 1:03 pm
The 94th annual Hadassah convention recently traveled to Los Angeles and JewishJournal.com VideoJew Jay Firestone was all over it, like jelly on gefilte fish.
To get a feel for the convention, VideoJew submerged himself in many of the activities. On his journey, he made new friends and even visited with some old friends. But does he find his 'eshet chayil?'
Watch and find out. Your rabbi would want you to.
Video by VideoJew Jay Firestone and the amazing VideoJew Assistant, Sari Thayer.
July 15, 2008 | 7:16 pm
VideoJew Jay Firestone leaps out of hibernation with this teaser.


text:
- BlackBerry® Torch™ 9800 smartphone
The following error appears after taking a picture on the BlackBerry® Torch™ smartphone while Geotagging is enabled:
File Not Found
Update the BlackBerry Torch smartphone to the latest available BlackBerry Device Software. For instructions, see KB16071 .
Perform one of the following workarounds:
Workaround 1 - Manually save the image
- Click OK in the File Not Found dialog box.
- In the Save File dialog box that opens, provide an alternate path and filename for the image.
Workaround 2 - Disable Geotagging
- Open the camera application on the BlackBerry smartphone.
- Click the cross-hair icon to the left of the camera icon to disable Geotagging.
By downloading, accessing or otherwise using the Knowledge Base documents you agree:
(a) that the terms of use for the documents found at www.blackberry.com/legal/knowledgebase apply to your use or reference to these documents; and
(b) not to copy, distribute, disclose or reproduce, in full or in part any of the documents without the express written consent of RIM.
Visit the BlackBerry Technical Solution Center at www.blackberry.com/btsc.


text:
859 O'Farrell St.
San Francisco, CA 94109
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Wed. May 14, 2014An evening with Failure (wed) | San Francisco, CA |Presented By Great American Music Hall
Venue Information
Great American Music Hall (MAP)859 OFarrell StSan Francisco, CAUS 94109
Other Information
Event Information:415-885-0750
At this time Great American Music Hall does not have any tickets available for purchase for this event. Note: This does not mean this event is sold out. Contact the venue to purchase tickets.
More Information
Failure, the influential LA trio who recently reunited for their first shows in sixteen years, have announced the Tree of Stars North American 2014 Tour.
Failure's first outing of the new millennium was a sold out February show at the El Rey Theatre in Los Angeles. The mesmerizing performance, both captivating in an aural and visual sense, was described by The Orange County Register as "an almost spiritual experience," summarizing the special evening as "no rust, no fluff, just one influential rock favorite after another." The band recently joined Tool, a partnership with an extensive history, on their March tour.
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text:
Juneau police are looking for a man and a gun allegedly used in an assault and robbery earlier this week in the Costco parking lot.
One man is in custody for the New Year's Day incident in which a 20-year-old man was reportedly stunned by a taser, punched and robbed.
Twenty-nine-year old Dustin Daley has been jailed on charges of 1st degree robbery, two counts of 4th degree assault and one count of theft in the 3rd degree.
Police are still looking for another man, who was reportedly wearing a dark hooded sweatshirt, a black mask with the skull on it, and had a handgun.
Police say Daley and the unidentified man were in a four-door silver Dodge pickup with a broken driver's side window covered in plastic. Police later found the truck and Daley as well as a Crown Royal bag containing what may be heroin, a white powder that may be another illegal drug, a marijuana pipe and marijuana. The mask was also recovered from the pickup.
Police did not release any information about the incident until Friday. They are asking for assistance in identifying the man with Daley.
They say anyone with information should log onto the Juneau Crime Line website and report their tip.


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Giesen Sauvignon Blanc 2013
Sauvignon Blanc from Marlborough, New Zealand
Winemaker's Notes
The fruit and aroma spectrum shows a greater depth of tropical notes with a distinct greener profile running through as well. This is an aromatic blend displaying aromas of elderflower, kaffir lime leaf, pink grapefruit and dried herb. The palate has a vibrant citrus focus with considerable fruit concentration finishing crisp and bright.
Critical Acclaim
James Suckling - "Again, this is staggering value and quality for the volume made of this wine. Plenty of bright, ripe fruit here: passion fruit, lime and ripe melon, smooth and soft. Drink now. "
Tasting Panel - "Bright, racy and juicy with grapefruit and citron; fresh, tangy and balanced with good length. "
Giesen Winery
Established in 1981, the Giesen Wine Estate comprises some 75 acres at Burnham, just south of Christchurch. In Marlborough 360 acres are now in production. The Giesen Wine Estate is a family affair. The three brothers, Theo, Alex and Marcel run the winery that has grown into an export oriented business.
Low yielding grape vines grown in a cool climate is what makes Giesen wine unique. Complex fruit aromas make them enjoyable young, but refreshing acidity gives the wines backbone for long aging in the bottle. Wines with "vintage character" - something found only in the traditional viticultural areas of Europe - give a great variety of unique wines, year after year. View all Giesen Wines
About MarlboroughView a map of Marlborough wineries (mahrl-bore-oh)
Sitting pretty on the northern tip of New Zealand's south island, Marlborough has become synonymous with New Zealand Sauvignon Blanc. As well it should be – Marlborough is the primary region for those delicious, citrusy, summer-lovin' wines with vibrant acidity and pungent, grassy, grapefruit flavors. Sauvignon Blanc is the main grape here; Chardonnay, Pinot Noir and Riesling are also made.
Notable FactsThe region has well-drained alluvial loam soils, which are perfect for grape growing. The grapes receive a good deal of sunshine during the day, but recovers in the cool evenings. Marlborough's growing season is long, which helps foster the gradual, even ripening of the grapes. Not made for much aging, the Sauvignon Blancs of Marlborough are of the buy 'em and drink 'em class of wine. Expect little vintage variation here - quantity differs more than quality.
About New ZealandThe country of New Zealand is about 1000 miles from the coast of Australia. It consists of two long islands, end to end, that are approximately the same length as California. Most of the country's climate is maritime due to the abundant coastline. The northern island is warmer and wetter, while the southern island is cooler and dryer. The most popular grapes of New Zealand are Sauvignon Blanc (made most famous by the bright, crisp wines coming out of Marlborough), Chardonnay and the ever-growing Pinot Noir.
Customer ReviewsSign In to Add Your Review3.5 }div>3.5 out of 5 stars
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2 ratings, 2 with reviewsWilfred Wong (of Wine.com) - San Francisco, CA48/1/2014
One of the amazingly consistent Sauvignon Blancs in the global market, the nifty 2013 Giesen Sauvignon Blanc exhibits grapefruti peel and dried leaves; brisk yet satisfying on the palate; crisp in the finish.38/25/2014
- Style
- Light & Crisp
- Pair With
- steamed mussels
This is ok for the price, but there are Sauvignon Blanc's within a few dollar price range that are better. If you're looking for a Sauvignon Blanc that is smooth but still has a slight citrus taste, this is a good choice. If you want the strong citrus taste of the New Zealand Sauvignon Blanc's, I would skip this one.
- Style
- Light & Crisp
Alcohol By Volume GuideMost wine ranges from 10-16% alcohol by volume. Some varietals tend to have higher (for example Zinfandel and Cabernet Sauvignon) or lower alcohol levels (Pinot Noir and many white varietals), but there is always some variation from producer to producer. Some wine falls outside of this range, for instance Port weighs in closer to 20%, while Muscat and Riesling are usually a bit below 10%.
Wine Style Guide
Light & Crisp
- Light to medium bodied wines that are high in acid and light to medium fruit. Typically no oak.
Fruity & Smooth
- Light to medium bodied wines with lots of juicy fruit, typically medium acid and medium oak.
Rich & Creamy
- Full bodied wines that have typically undergone malo-lactic fermentation and/or spent time in oak.
- 5 Stars:


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August 4, 2015
On Tuesday, January 15, 2013 at 8:26pm, the Bryan Police Department responded to Walmart located at 2200 Briarcrest for a shoplifting in progress. Three subjects were placing baby formula, clothing, and various other items in a shopping cart. The three subjects then left the store without paying for the merchandise which was valued at $2004.31. The Bryan Police detained all three subjects. During the course of the investigation it was discovered that they had previously taken similar items from the College Station Walmart totaling $1,652.91.
The Bryan Police Department arrested and charged the following with Theft Property >=$1500 < 20K:
Darren Lee Brown 28 year old male from Houston
Latrice Denise Randle 28 year old female from Houston
Blanche Yvonne Buckner 54 year old female from Houston
The College Station Police Department also charged all three subjects with another count of Theft Property >=$1500 < 20K. According to Walmart, these individuals may also be responsible for similar thefts in the Austin, Houston, and other area Walmarts.
The safety of our community does not have borders. It is the goal of every law enforcement agency to protect the citizens that we serve. Be the eyes and ears of your police department. When you see suspicious activity contact the College Station Police Department at 979-764-3600, the Texas A&M University Police Department at 979-845-2345, or the Bryan Police Department and the Brazos County Sheriff's Office at 979-361-3888. Together we can make a difference in our community.
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Geek Speak
Geek Speak is where the world of Geekdom comes alive.
Yearly Archive for 2006
ChimaeraCon pics
Finally, here are pictures from ChimaeraCon '06. Also, look through the blog as we went back and inserted pictures. Most were taken by random people Quinn asked for assistance. [Read More]
Chris Quinn: Science and Super Heroes merge in a multimedia exhibition
I wish I could go to this. It looks tasty. It will be at the California Science Center. The exhibit will explore the Hulk's transformation process, the various powers of Spider-man and other heroes as well as the science behind Marvel's Universe Yes, It's all Marvel, so no in-depth examination of the physics and effects […] [Read More]
Bye! It was fun
Well it has been a darn fun day! I met a lot of new people and played some hardcore games. There were laughs, victories and some defeats. I want to thank the ChimaeraCon folks for allowing me to be here all day getting into people's business and games. And the gamers! Man, I love these […] [Read More]
Meeting more of the staff
Jose Avila with the con staff is here with me. We are the only ones on this side of the room. He is keeping an eye on the vendors' booths while the last of the games finish up. He is checking his email and reading the blog (Hey Jose what's up?) and just hanging out […] [Read More]
Winding down – sort of
That was fun. I took a quick walk around the floor. Only the dedicated remain. I did not want to intrude much, so I tried to keep my distance. There were some intense-looking games going on. The RPGers did not even look up when I entered into their cornered realm. They were bent over sheets […] [Read More]
It had to happen!
OK, some updates for Sunday. The con will open at 8 a.m. and stay open until 6:30 p.m. Cost at the gate is $10. You can still make it down for the last two hours of tonight's festivities. There are plenty of games still going on. Also, from 6 to 9 p.m. Monday, April 17, […] [Read More]
Star Wars miniatures battle strategy
Sorry I have been away for so long. I have been playing the Star Wars miniatures battle strategy game. It rocks. I was a squad of Imperial Storm Troopers. I wrought all kinds of hell! Actually I got taken down pretty fast. But I did maneuver well. At least that is what the guy whose […] [Read More]
The Winner is
The costume contest went off well. The winner? The transformer Preceptor. Darth Vader and Han Solo came in second and third respectively. Innocent bystander #1 and #2 got honorable mention for doing such a great scream. I am going to go and try and track down the transformer to get some thoughts on her victory. […] [Read More]
More than Meets the Eye
It's official: I am back into HeroClix. I just played a match using DC comics figures against Rich Quini. I was Batman, Robin and Hawkgirl. He was Manbat, Joker and Harley Quinn. Rich kicked my ever-loving rear right off the table. I hung in there till the end, though. Rich is tricky. He lured me […] [Read More]
Friends from not too far away
I just met Alan Marsh who has brought a whole vanload of kids down from Houston for the con and to meet artist Howard Tayler. Alan, his two sons and some of their friends woke up at 4 a.m. this morning and got on the road. They have been playing Munchkin a card game and […] [Read More]
Show More


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JAVOedge's Cradle is a USB cradle that is specifically designed for your mobile handheld device. Charge and sync data with this elegant cradle today!
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Simple and convenient way to quickly charge your phone or tablet while you drive. Connects to the cigarette lighter in your car and has 2 USB ports to charge multiple devices simultaneously.
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Monster Energy/Graves/Yamaha's Hayes Earns Provisional Pole In Friday Qualifying At Barber Motorsports Park
NEWS June 21, 2013
Photo by Brian J Nelson
Courtesy of Yamaha-Motor USA:
Birmingham, AL June 21, 2013 – With points to make up in the 2013 AMA Pro National Guard SuperBike Championship, Josh Hayes--the three-time defending title holder and rider of the #1 Monster Energy/Graves/Yamaha YZF-R1--got to work quickly at Barber Motorsports Park and put himself on the provisional pole in Friday's first qualifying session. Meanwhile, his teammate and rider of the #2 R1 Josh Herrin improved his laptimes from the morning's practice and is currently fifth on the grid. He's looking forward to improving even more in tomorrow's final qualifying.
After provisional qualifying, Josh Hayes said, "I started the session on a used tire, and I was working to make the bike a little bit easier to ride. I didn't really feel like I went a whole lot faster than in practice, so it felt pretty effortless, which is a good thing. My Monster Energy/Graves/Yamaha crew always gives me a good bike to start the weekend with, so we usually come off the truck already in good shape. We made some adjustments after practice, and those few clicks here and there really helped me in this first qualifying."
Commenting on his provisional qualifying session, Josh Herrin said, "We rode our backup bike in this morning's practice, and we tried running the same shock as last year for most of that session. We also ran a race simulation on the tire. And then, in the provisional qualifying, the first intent was to go out and get a good time in. On the last two laps, we put on a new tire to try to move up, but we got held up a little bit. Tomorrow morning, track temperatures will be cooler, so the laptimes should drop for everybody. Obviously, tomorrow will be all about doing fast laps and moving up on the starting grid."
Final qualifying for SuperBike is scheduled for tomorrow at 9:40 AM CST, and Race 1 will go green later tomorrow at 3:00 PM CST.
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NNSA's Office of Acquisition and Project Management (APM), the community manager for acquisition professionals, hosted NNSA Contracting Officers in Albuquerque this week to align NNSA acquisition policies and procedures with Departmental goals and priorities, and to clarify roles and responsibilities among the various COs across the Nuclear Security Enterprise.
A key discussion topic was how NNSA was implementing the Deputy Secretary's policy for improving acquisition planning and contract management for capital asset projects. The primary principle behind this policy is that the Department must align contract incentives and vehicles with taxpayer interests. Each party must share the risk by bearing responsibility for its own actions. The policy includes guidance to ensure proper project planning so that requirements are clearly defined before issuing a solicitation; to first consider the use of a firm-fixed-price contract to complete work requirements; to establish objective performance measures when a fixed-price contract is not in the government's best interest; to utilize fee strategies that assure each party in the contract bears responsibility for its own actions; and to document and stay apprised of real-time, accurate, and reliable project performance data.
With this new organizational construct in place, NNSA has delivered its $725M project portfolio $50M - or 7% - under budget during the past the three years. In 2013, NNSA was removed from the GAO High Risk list for construction projects, with a Total Project Cost up to $750 million as a result of the improvements it is making in NNSA contract and project management.
Preparing the country to respond to a large scale nuclear event is the primary function of the NNSA Office of Emergency Response. This week, Consequence Management teams from that office participated in the Vibrant Response 14 exercise at Camp Atterbury in Muscatatuck, Ind. and surrounding areas.
Taking advantage of a U. S. Army North exercise, NNSA, alongside FEMA, Indiana Department of Homeland Security, and numerous other local, state, and federal agencies with civilian personnel from across the country responded to a simulated terrorist attack with a nuclear weapon. NNSA deployed personnel to federal and state operating facilities to coordinate the response to the radiological aspects of this scenario. Additionally, a Consequence Management Home Team provided support from Las Vegas, NV, Livermore, CA. and Albuquerque, NM. This team provided models of fallout deposition and analysis of environmental monitoring data.
This was the most extensive exercise of this type to date, involving not only 5000+ field deployed Department of defense Military personnel in the field but also a multitude of support and coordination facilities across the country. Lessons learned during this exercise will be used to improve NNSA and other national organizations response to nuclear and radiological incidents of all scales and inform national level policies guiding that response.
More photos are available here.
Preparing students for the technological skill sets that will be required in tomorrow's STEM related jobs in industry is an ongoing priority for the National Security Campus (NSC) in Kansas City, Mo. Last week, the NSC hosted a Model-Based Engineering Workshop for 32 college professors and students from Historically Black Colleges and Universities (HBCUs) to gain first-hand knowledge of the exciting opportunities and challenges engineering professionals tackle each day.
In 2012, the NNSA established a $4 million grant which launched the Minority Serving Institution Partnership Program (MSIPP). This consortium based program formed two advanced manufacturing consortiums which included 8 universities and two NNSA sites (NSC and Y-12).
This year's students and professors were from Hampton University, Clark Atlanta University, Alabama A&M, University of District of Columbia, North Carolina A&T, Southern University New Orleans, Lincoln University, and Howard University. The Model-Based Engineering workshop helps educators incorporate 3D modeling and advanced manufacturing into their curriculum through hands-on model-based application training and an exercise for a mock rocket assembly.
Through this public and private partnership, the HBCU teachers can better shape their curriculum and advise students on what skills employers expect from engineers and scientists and the NNSA learns about the technical strengths of the participating universities, which will help future recruiting and R&D initiatives.
Fourteen hazardous materials response teams from New Mexico, Missouri, Nebraska and Oklahoma will test their skills at the 18th annual Hazmat Challenge July 29 through Aug. 1 at Los Alamos National Laboratory.
The intent of the challenge is to provide hazardous materials responders the opportunity to test their skills, share best practices with other response agencies, and learn new techniques through realistic hazardous materials release scenarios in a safe, non-hazardous environment.
A video about the 2008 and 2012 Hazmat challenges is on the Laboratory's Web site and on LANL's YouTube channel.
See more.
Officials from NNSA's Uranium Processing Facility Project Office and Consolidated Nuclear Security recently signed an agreement to create a team dedicated to accomplishing the Uranium Processing Facility mission: a new UPF with Building 9212 capabilities by 2025 for under $6.5 billion.
The agreement also emphasizes a collaborative approach to problem solving and issue resolution focused on early identification and rapid communication. It was signed by 25 leaders of the project from both UPO and CNS, including UPF Federal Project Director John Eschenberg and CNS Project Director Brian Reilly.
Partnering is an industry best practice that has been used by DOE's Environmental Management Program, the U.S. Army Corps of Engineers and large commercial construction projects. UPO's partnering agreement with CNS represents the first such agreement for NNSA and serves as yet another example of the way in which NNSA is applying industry best practices to improve project management.
Shaking hands: On left, UPF Federal Project Director John Eschenberg; on right, CNS Project Director Brian Reilly
Watch coverage of an NNSA Nevada-based national emergency response capability, featured by a Las Vegas NBC affiliate.
The Y-12 National Security Complex recently was recognized by NNSA for achieving the highest savings rate in the NNSA enterprise for fiscal year 2013.
At the recent Supply Chain Management Center (SCMC) biannual operational meeting, NNSA presented Y‑12 Procurement Operations with the award for attaining the Highest Total Strategic Savings Rate among NNSA's seven management and operating contractors. Y‑12 achieved a total 6.51 percent savings rate of total strategic spending, as measured by the SCMC.
In FY 2014, the SCMC also accepted Y‑12's recommendation for a regional sourcing approach. This new localized approach will award regional supplier contracts to improve lead time, reduce freight cost, and increase use of local small businesses. Sites within the same region will be served by the contracted supplier in the area instead of a national, single-source supplier.
Sandia radiation effects researcher Jim Schwank has won the 2014 IEEE Nuclear and Plasma Sciences Society Merit Award, which recognizes outstanding technical contributions to the fields of nuclear and plasma sciences.
The award is based on the importance of individual technical contributions, importance of technical contributions made by teams the individual led, quality and significance of publications and patents, years of technical distinction and leadership, and service in the fields of nuclear and plasma sciences and related disciplines.
Read more.
NNSA this week showcased its twin-engine Bell 412 helicopter during the Health Physics Society's annual meeting in Baltimore. The helicopter is equipped with gamma radiation sensing technology and used to measure naturally occurring background radiation at various locations throughout the country.
Read more.
Thousands of employees at the Pantex Plant and the Y-12 National Security Complex came together recently to celebrate the formation of a new team joining the two sites under the leadership of Consolidated Nuclear Security, LLC. Both sites' luncheons and safety fairs delivered a strong emphasis on safety. Each employee received a CNS lanyard with the Pantex, Y-12 and CNS logos, along with the slogan "One Team, Better Together."
CNS assumed responsibility for management and operations at Pantex and Y-12 on July 1 after a four-month transition process. The two sites will be operated jointly under a unique arrangement that will allow the new contractor to focus on its main priorities of safety, security, quality, mission delivery and cost efficiency.


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Gisella Gisolo, the newly appointed GC director, traveled with the first-year GC students to Ghana over the winter break.
Gisella Gisolo (far right) and Tina Richardson (center left), associate professor of education and human services, met with the administrator (middle right) of the Maranatha clinic, a non-profit medical clinic in Ghana.
Posted on Monday, January 26, 2009
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