text
stringlengths
0
1.67M
<SOH> SUMMARY OF THE INVENTION <EOH>In one aspect the present invention relates to a surface exposed protein, which can be detected in Moraxella catarrhalis , having an amino acid sequence as described in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or an immunogenic or IgD-binding fragment of said protein or variants, or an immunogenic and adhesive fragment of said surface exposed protein. In another aspect the present invention relates to an immunogenic or IgD-binding fragment of a surface exposed protein as defined above, which fragment can be detected in Moraxella catarrhalis , having a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof. In a further aspect the present invention relates to an immunogenic or IgD-binding fragment as described above, having an amino acid sequence as described in SEQ ID NO 10. In still a further aspect the present invention relates to an immunogenic and adhesive fragment of a surface exposed protein as defined above, which fragment can be detected in Moraxella catarrhalis , having a capacity of binding erythrocytes and epithelial cells. In still another aspect the present invention relates to an immunogenic and adhesive fragment as defined above, having an amino acid sequence as described in SEQ ID NO 8. In one aspect the present invention relates to a DNA segment comprising a DNA sequence, as shown in SEQ ID NO 2, which DNA sequence codes for a surface exposed protein of Moraxella catarrhalis as defined above, or naturally occurring or artificially modified variants of said DNA sequence. In yet another aspect the present invention relates to a DNA segment comprising a DNA sequence which codes for an immunogenic or IgD-binding fragment as defined above. In a further aspect the present invention relates to a DNA segment as defined above, comprising a DNA sequence, as shown in SEQ ID NO 11, which DNA sequence codes for an immunogenic or IgD-binding fragment as defined above. In still a further aspect the present invention relates to a DNA segment comprising a DNA sequence, which codes for an immunogenic and adhesive fragment of a surface exposed protein as defined above. In another aspect the present invention relates to a DNA segment as above, comprising a DNA sequence, as shown in SEQ ID NO 9, which DNA sequence codes for an immunogenic and adhesive fragment as defined above. In a further aspect the present invention relates to a vaccine containing a surface exposed protein of Moraxella catarrhalis , said protein having an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, or an immunogenic or IgD-binding fragment of said protein or variants, or an immunogenic and adhesive fragment of said surface exposed protein. In another aspect the present invention relates to a vaccine containing an immunogenic or IgD-binding fragment of a surface exposed protein of Moraxella catarrhalis , which has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, preferably a vaccine containing an immunogenic or IgD-binding fragment having an amino acid sequence as described in SEQ ID NO 10. In still another aspect the present invention relates to a vaccine containing an immunogenic and adhesive fragment of a surface exposed protein of Moraxella catarrhalis as defined above, preferably a vaccine containing an immunogenic and adhesive fragment having an amino acid sequence as described in SEQ ID NO 8. In one preferred embodiment said vaccines are combined with another vaccine and in another preferred embodiment said vaccines are combined with an immunogenic portion of another molecule. In one aspect the present invention relates to a plasmid or phage comprising a DNA sequence, which codes for a surface exposed protein of Moraxella catarrhalis , said protein having an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or an immunogenic or IgD-binding fragment of said protein or variants. In another aspect the present invention relates to a plasmid or phage comprising a DNA sequence, which codes for a an immunogenic or IgD-binding fragment of a surface exposed protein, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, preferably a plasmid or phage comprising a DNA sequence, which codes for a an immunogenic or IgD-binding fragment having an amino acid sequence as described in SEQ ID NO 10. In still another aspect the present invention relates to a plasmid or phage comprising a DNA sequence, which codes for an immunogenic and adhesive fragment of a surface exposed protein as defined above, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding erythrocytes and epithelial cells or naturally occurring or artificially modified variants of said fragment, preferably a plasmid or phage comprising a DNA sequence, which codes for a an immunogenic and adhesive fragment having an amino acid sequence as described in SEQ ID NO 8. In yet another aspect the present invention relates to a non human host comprising at least one plasmid or phage as defined above, and capable of producing said protein or variants, or said immunogenic or IgD-binding fragment of said protein or variants, or said immunogenic and adhesive fragment of said protein, which host is chosen among bacteria, yeast and plants. In one embodiment the host is E. coli. In one aspect the present invention relates to a recombinant DNA molecule comprising a DNA sequence coding for a surface exposed protein of Moraxella catarrhalis , said protein having an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or for an immunogenic or IgD-binding fragment of said protein, or variants, which DNA sequence is fused to another gene. In another aspect the present invention relates to a recombinant DNA molecule comprising a DNA sequence coding for an immunogenic or IgD-binding fragment of a surface exposed protein, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, which DNA sequence is fused to another gene, preferably a recombinant DNA molecule comprising a DNA sequence coding for an immunogenic or IgD-binding fragment having an amino acid sequence as described in SEQ ID NO 10. In still another aspect the present invention relates to a recombinant DNA molecule comprising a DNA sequence coding for an immunogenic and adhesive fragment of a surface exposed protein as above, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding erythrocytes and epithelial cells, or naturally occurring or artificially modified variants of said fragment, which DNA sequence is fused to another gene, preferably a recombinant DNA molecule comprising a DNA sequence coding for an immunogenic and adhesive fragment having an amino acid sequence as described in SEQ ID NO 8. In yet another aspect the present invention relates to a plasmid or phage comprising said fused DNA sequence as defined above. In a further aspect the present invention relates to a non-human host comprising at least one plasmid or phage as defined above, which host is chosen among bacteria, yeast and plants. In one embodiment the host is E. coli. In one aspect the present invention relates to a fusion protein or polypeptide, in which a surface exposed protein of Moraxella catarrhalis , said protein having an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or an immunogenic or IgD-binding fragment of said protein or variants, is combined with another protein by the use of a recombinant DNA molecule as defined above. In another aspect the present invention relates to a fusion protein or polypeptide, in which an immunogenic or IgD-binding fragment of a surface exposed protein, which fragment can be detected in Moraxella catarrhalis , which has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, is combined with another protein by the use of a recombinant DNA molecule as defined above. In still another aspect the present invention relates to a fusion protein or polypeptide in which an immunogenic and adhesive fragment of a surface exposed protein as defined above, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding erythrocytes and epithelial cells, or naturally occurring or artificially modified variants of said fragment, is combined with another protein by the use of a recombinant DNA molecule as defined in above. In yet another aspect the present invention relates to a fusion product, in which a surface exposed protein of Moraxella catarrhalis , said protein having an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, or an immunogenic or IgD-binding fragment of said protein or variants, is covalently or by any other means bound to a protein, carbohydrate or matrix. In a further aspect the present invention relates to a fusion product in which an immunogenic or IgD-binding fragment of a surface exposed protein, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, is covalently or by any other means bound to a protein, carbohydrate or matrix. In still another aspect the present invention relates to a fusion product in which an immunogenic and adhesive fragment of a surface exposed protein as defined above, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding erythrocytes and epithelial cells, or naturally occurring or artificially modified variants of said fragment, is covalently, or by any other means, bound to a protein, carbohydrate or matrix. Preferably, a fusion product in which an immunogenic or IgD-binding fragment, having an amino acid sequence described in SEQ ID NO 10, is covalently, or by any other means, bound to a protein, carbohydrate or matrix. Preferably, a fusion product in which an immunogenic and adhesive fragment, having an amino acid sequence described in SEQ ID NO 8, is covalently, or by any other means, bound to a protein, carbohydrate or matrix. In one aspect the present invention relates to a method of detecting IgD using a surface exposed protein of Moraxella catarrhalis , said protein having an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, or an immunogenic or IgD-binding fragment of said protein or variants, optionally labelled and/or bound to a matrix. In a further aspect the present invention relates to a method of detecting IgD using an immunogenic or IgD-binding fragment of a surface exposed protein, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, optionally labelled and/or bound to a matrix. In another aspect the present invention relates to a method of detecting IgD using an immunogenic or IgD-binding fragment of a surface exposed protein of Moraxella catarrhalis , having an amino acid sequence as described in SEQ ID NO 10, and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, optionally labelled and/or bound to a matrix. In a further aspect the present invention relates to a method of separating IgD using a surface exposed protein of Moraxella catarrhalis , said protein an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, or an immunogenic or IgD-binding fragment of said protein or variants, optionally bound to a matrix. In yet another aspect the present invention relates to method of separating IgD using an immunogenic or IgD-binding fragment of a surface exposed protein, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, optionally bound to a matrix. In another aspect the present invention relates to a method of separating IgD using an immunogenic or IgD-binding fragment of a surface exposed protein of Moraxella catarrhalis , having an amino acid sequence as described in SEQ ID NO 10, and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, optionally labelled and/or bound to a matrix. In one aspect the present invention relates to a method of isolation of a surface exposed protein of Moraxella catarrhalis , said protein having an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, or an immunogenic or IgD-binding fragment of said protein or variants. Said method comprises the steps: a) subjecting a suspension of Moraxella catarrhalis to an extraction process by adding a zwitterionic or non-ionic detergent, optionally in the presence of EDTA; b) applying the extract comprising the IgD-binding protein of Moraxella catarrhalis from step a) to an adsorption column; c) eluating the IgD-binding protein; and d) separating the IgD-binding protein. In another embodiment the concentration of the detergent in step a) of the method is within the range 0.1-5%, preferrably 3%. In yet another aspect the present invention relates to a method for treatment of an autoimmune disease comprising extra corporal circulation of the blood trough a material comprising a surface exposed protein as defined above, or a fragment thereof as defined above, for removal of IgD from the blood. In one aspect the present invention relates to a purified antibody which is specific to an immunogenic portion of a surface exposed protein Moraxella catarrhalis , said protein having an amino acid sequence as described in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or an immunogenic or IgD-binding fragment of said protein or variants. In another aspect the present-invention relates to a purified antibody as described above, which is specific to an immunogenic or IgD-binding fragment as defined above, having a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment. In still another aspect the present invention relates to a purified antibody as described above, which is specific to an immunogenic or adhesive fragment as defined above, having a capacity of binding erythrocytes and epithelial cells.
Medicament for treating tumours and their metastases
The invention relates to a medicament against tumors and their metastases, which preferentially settle in bone tissue, comprising as active ingredient at least one binding molecule which binds to bone sialoprotein or a fragment thereof in serum or plasma.
1. Medicament against tumors and their metastases, which preferentially settle in bone tissue, comprising as active ingredient at least one binding molecule which binds to bone sialoprotein or a fragment thereof in serum or plasma. 2. Medicament according to claim 1, wherein the active ingredient is an antibody or an aptamer or spiegelmer on DNA or RNA basis. 3. Medicament according to claim 2, wherein the active ingredient binds a molecule that corresponds to a bone sialoprotein chemically or naturally modifed in its glycosylation. 4. Medicament according to claim 1, wherein the binding molecule is an antibody or an aptamer which specifically binds bone sialoprotein from tumor cells. 5. Medicament according to claim 1, wherein the binding molecule binds bone sialoprotein, modified in its glycosylation, from bone material the donor of which was not capable of normal gylcosylation of bone proteins. 6. Medicament according to claim 1, comprising as active ingredient an antibody or a plurality of antibodies against human bone sialoprotein (hBSP), wherein the antibodies bind epitopes which are present on human bone sialoprotein from tumor cells, the post-translation glycosylation of which in the region of amino acids 120 to 135 (SWISSPROT: SIAL13 HUMAN; Acc. No. P21815, incl. signal sequence) containing the amino acids TGLAA (SEQ ID NO: 15) is modified or incomplete in comparison with normal bone sialoprotein from bones. 7. Medicament according to claim 1, comprising as active ingredient an antibody and/or an aptamer, generated against a hBSP epitope, comprising the amino acid sequence TGLAA (SEQ ID NO: 15) or YTGLAA (SEQ ID NO: 16) and optionally sugar groups. 8. Medicament according to claim 1, including as active ingredient IgY antibodies from chicken. 9. Medicament according to claim 8, comprising as active ingredient IgY antibodies from chicken, which are human or humanised. 10. Medicament according to claim 1, wherein the binder molecule as a bispecific antibody comprises also an additional paratope that is preferably specific for epitopes of CD3. 11. Medicament according to claim 1, wherein the active ingredient is an immunotoxin which is a conjugate of binder molecule and a residue having cytotoxic activity. 12. Medicament according to claim 11, wherein the conjugate comprises the ricin-A chain or a non-binding fragment of the diphtheria toxin. 13. Medicament according to claim 1, wherein the binder molecule is coupled with a radionucleotide. 14. Medicament according to claim 1, comprising additionally at least one antibody, ligand or inhibitor, selected from the group comprising adhesion molecules, membrane-associated proteases, receptors which mediate chemotaxis, chemokin receptors, apoptosis inducing substances. 15. Medicament according to claim 14, wherein the inhibitors at least partially block BSP and thus modulate its functioning. 16. Medicament according to any preceding claim for the treatment of tumors from the group comprising prostate, breast, lung, kidney and thyroid tumors, tumor diseases of the blood system, of the lymphatic system, of the heart and circulatory system, of the nervous system, of the respiratory tract, of the digestive tract, of the endocrine system, of the skin including adnexa, of the locomotory system and of the urogenital tract, including the kidneys.
<SOH> BACKGROUND OF THE INVENTION <EOH>At the present time many medicaments are in development which are intended to combat tumors and their metastases which spread in bone. Despite all advances in medicaments, bone metastases, however, do not count as being curable or treatable. The are attempts, by means of antibodies against surface antigens of tumor cells, to combat their metastases. Bone metastases are, however, despite this the cause of death in 73% of cases of tumors of the breast, and 68% of cases for tumors of the prostate gland. For tumors of other tissues the following figures apply: cervix 50%, thyroid gland 42%, bladder 40%, lungs 36%, ovaries 9% and colon 6%. Tumor cells which express the so-called bone sialoprotein have the peculiarity that they prefer to settle in bony tissue and build metastases there, particularly in the case of tumors of the prostate gland, breast, lungs, kidney and thyroid and less frequently in the case of malign and semi-malign tumors. The bone sialoprotein (BSP) is a phosphorylated bone glycoprotein having a relative mass of ca. 80 kDa in the SDS-PAGE. The DNA for BSP codes for a peptide sequence of ca. 33 kDa (Fisher (L. W. et al. (1990), J. Biol. Chem. 265, 2347-51; U.S. Pat. No. 5,340,934). BSP is one the few matrix proteins the occurrence of which on mineralising tissue such as bones, dentin and calcifying cartilage is restricted. BSP represents ca. 10 to 15% if the total non-collagenic proteins in the bone matrix. It is as a rule expressed by cells which take part in the formation of dentin, bones and cartilage, for example osteoblasts, developing osteocytes, hypertrophic chondrocytes, odontoblasts and cementoblasts, but also by the trophoblasts in the placenta and some types of cancer cells, e.g. in the case of lungs, breast, prostate, Kidney, thyroid and neuroblastoma primary and secondary tumors, in the case of multiple myeloma and in bone metastases. The degree of expression of BSP by the tumor closely correlates with the severity of the cancer (Waltregny D. et al., Increased expression of bone sialoprotein in bone metastases compared with visceral metastases in human breast and prostate cancers , in J. Bone Miner. Res., 2000, 15(5), 834-43; Bellahcène, A. et al., Bone sialoprotein expression in primary human breast cancer is associated with bone metastases development , in J. Bone Miner. Res., 1996, 11, 665-670; Waltregny, D. et al., Prognostic value of bone sialoprotein expression in clinically localised human prostate cancer , in Journal of the National Cancer Institute, 1998, 90, 1000-1008; Bellahcène, A. et al., Expression of bone sialoprotein in primary breast cancer is associated with poor survival , in Int, J. Cancer, 1996, 69, 350-353). BSP, as an adhesion molecule, is supposed to bring about attachment and dissemination of cells on the tissue matrix, since in vitro it forms crystallisation nuclei for biological apatite and in vivo takes part in mineralisation. The switching off of the BSP gene in knock-out mice leads to no recognisable disruption of the building and functioning of the skeleton. In tumors BSP is attributed with participation in microcalcification (Castronovo, V. et al., Evidence that breast cancer associated microcalcifications are mineralized malignant cells , in Int. J. Oncol., 1998, 12, 305-308) and the colonisation of bones by metastasising tumor cells (Bellahcène, A. et al., Expression of bone sioloprotein in primary breast caner is associated with poor survival , in Int. J. Cancer, 1996, 69, 350-353). The level of concentration of BSP in the serum of patients with primary carcinomas serves for diagnosis of whether these patients have bone metastases or such are likely to arise from the primary tumor (Diploma Thesis of Ms. Ina-Alexandra Meier, Development of a radioimmunoassay for the determination of bone sialoprotein (BSP) [“ Entwicklung eines Radioimmunoassays zur Bestimmung von Bonesialoprotein ( BSP ) ]”, 1996, Darmstadt, Technical University [Fachhochschule], Specialist Field Chemical Technology [FB Chemische Technologie]; Dissertation of Mr. Markus Karmatschek, Isolation of bone sialoprotein from human bones, Structure of a radioimmunoassay for the measurement thereof in serum [“ Isolierung von Bonesialoprotein aus humanem Knochen, Aufbau eines Radioimmunoassays zur dessen Messung im Serum”], 1996; Specialist Field of Biology of the Technical University of Darmstadt [FB Biologie der Technischen Hochschule Darmstadt]; Diel I. J. et al., Elevated bone sialoprotein in primary breast cancer patients is a potent marker for bone metastases ; in Proceedings of ASCO, 1998, 17, Abstract 461; Diel I. J. et al, Serum bone sialoprotein in patients with primary breast cancer is a prognostic marker for subsequent, bone metastasis , in Clin. Cancer Res., 1999, 5, 3914-19; DE 198 13 633; DO 198 21 533; WO 99/50666). However, in body fluids free BSP is bound by complement factor H with high affinity. Further, BSP can bind to various receptors. Thus, there have been produced in rabbits antibodies against various peptide partial structures of BSP (Fisher, L. W. et al., Antisera and cDNA probes to human and certain animal model bone matrix noncollagenous proteins . Acta Orthop Scand Suppl., 1995, 266, 61-655), against recombinant BSP (Stubbs JT 3 rd et al., Characterization of native and recombinant bone sialoprotein: delineation of the mineral - binding and cell adhesion domains and structural analysis of the RGD domain . J. Bone Miner. Res. 1997 12(8), 1210-22), and against BSP isolated from bones, which antibodies failed to recognise any BSP in human serum. The larger factor H molecule of 150 kDa probably masks the smaller BSP (of ca. 65 kDa), so that antibodies cannot bind. Further, factor H is present in excess in the serum (0.5 mg factor H/mL in comparison to BSP with <20 ng/ml Serum in the case of healthy persons and max. 160 ng/ml in the case of tumor patients). It has been asserted that immunological direct determination of BSP in body fluids is impossible, without reducing sample preparation, due to the binding to the factor H and possibly that trophoblasts and BSP producing tumor cells are thereby protected from attack by the immune system, since the factor. H belongs to the complement system and is known to bring about a restriction of the alternative pathway to complement lysis (Fedarko N. S. et al., Factor H binding of bone sialoprotein and osteopontin enables tumor cell elvasion of complement - mediated attack, in J. Biol. Chem., 200, 275, 16666-16672; WO00/062065). Further, BSP can specifically bind to the integrin receptors on the cell surface through its own recognition, sequence (arginine-glycine-aspartate, RGD). In the case of expression of BSP the tumor cells are then supposed to bind the factor H in the blood and in the tissue fluids to their cell surfaces, or concentrate it around them. Such a protection of BSP from the complement system of the blood of the mother is suspected also for the trophoblasts in the placenta (Fedarko N. S. et al. Factor H binding of bone sialoprotein and osteopontin enables tumor cells evasion of complement - mediated attack , in J. Biol. Chem., 200, 275, 16666-16672; WO 00/062065). Further there is also suspected a function of BSP in angiogenesis. Along with the adhesion of osteoclasts and osteoblasts to the bone matrix—through the binding of the RGD recognition sequence in the matrix to the alpha(v)beta(3) integrin receptors on the cell wall—it is also observed that the adhesion, dissemination and orientation of the endothelial cells is probably mediated by BSP. Namely, blood vessel formation around a tumor occurs in parallel with the BSP expression in the tumor cells (Bellahcène A et al., Bone sialoprotein mediates human endothelial cell attachment and migration and promotes angiogenesis , in Circ. Pes. 2000, 86(8), 885-91). These characteristics thus make BSP a starting point for medicaments of all kinds. Thus, the binding of BSP via the RGD sequence to vitronectin or integrin receptors of tumor and epithelial cells can be restricted by antagonists (U.S. Pat. No. 6,069,158; U.S. Pat. No. 6,008,213; U.S. Pat. No. 5,849,865; van der Pluijm et al., Bone Sialoprotein peptides are potent inhibitors of breast cancer cell adhesion to bone in vitro, in Cancer Res. 1996, 36, 1946-1955) EP 1 064 719 A1 teaches a pharmaceutical composition having BSP as active substance for the support of the repair of damaged bone and connective tissue. WO 94/13310 teaches a composition having a BSP binding protein of staphylococcus aureaus as active ingredient. WO 00/36919 discloses regulatory elements for the purposive monitoring and suppression of the expression of BSP in tumor and connective tissue cells, which promote calcification. Thus, generally the substances of the regulation of the cell growth and cell migration are of particular interest from a diagnostic and therapeutic point of view. There are, however, still very many unknown factors which control cancer growth, whereby primary and secondary tumors and colonised organs interact. Here, important steps are the invasion, adhesion, migration and cell division of the tumor cells. Along with matrix metalloproteinases, adhesion molecules and chemotactic factors play a particular role. A medicament for combating and also for healing bone metastases on the basis of antibodies and binding molecules against BSP is not known. It is also not known that the BSP of tumor cells is different from the BSP of normal healthy cells.
<SOH> SUMMARY OF THE INVENTION <EOH>The subject of the invention is a medicament for therapy of tumors and their metastases, which preferentially settle in bony tissues, including as active ingredient at least one binding molecule which binds to bone sialoprotein or a fragment thereof in serum or plasma. The active ingredient is preferably an antibody or an aptamer or spiegelmer (Noxxon, Berlin, Germany), on a DNA or RNA basis and further binds a molecule which corresponds to a bone sialoprotein modified chemically or naturally in its glycosylation. The binding molecule may be arm antibody or an aptamer which specifically binds bone sialoprotein from tumor cells, or also the binding structure of natural BSP receptor or the factor H molecule. The binding molecule binds to or it can be produced against bone sialoprotein from bone material, which is modified in its glycosylation, the donor of which was not capable of normal glycosylation of bone proteins. In a particularly preferred embodiment, the medicament contains as active ingredient an antibody or a plurality of antibodies against human bone sialoprotein (hBSP), whereby the antibodies bind epitopes which are present on human sialoprotein from tumor cells, the post-translational glycosylation of which is modified or incomplete, in comparison with normal bone sialoprotein from bones, in the region of the amino acids 120 to 135 (SWISSPROT: SIAL_HUMAN, Acc. No. P21815, incl. signal sequence) containing the amino acids TGLAA. The medicament in accordance with the invention may also contain as active ingredient an antibody and/or an aptamer produced against a hBSP epitope, including the amino acid sequence TGLAA or YTGLAA and optionally sugar groups and a carrier molecule. The active ingredient is preferably a chicken IgY antibody. The chicken IgY antibody may also be a corresponding human or humanised antibody. Further preferred are medicaments whereby the binding molecule contains as a bispecific antibody also an additional paratope which is preferably specific for epitopes of CD3. The active ingredient may also be an immunotoxin that is a conjugate of binding molecule and a residue having Cytotoxic activity. The immunitoxin may or example be a conjugate which contains the ricin-A-chain or a non-binding fragment of the diphtheria toxin. The binding molecule may, further, be coupled with a radionucleotide so that the medicament can also be put to use for immune scintigraphy or for the localisation and observation of development of bone metastases. The medicament in accordance with the invention may also contain at least one antibody, ligand or inhibitor, from the group comprising adhesion molecules, membrane associated proteases, receptors which mediate chemotaxis, chemokine receptors, apoptosis inducing substances. The inhibitors can be so selected that they at least partially block BSP and modulate its function. In terms of its range of applications, the medicament in accordance with the invention is thus particularly suitable for the treatment of tumors from the group comprising prostate, breast, lung, kidney and thyroid tumors, tumor diseases of the blood system, of the lymphatic system, and of the heart and circulatory system, the nervous system, the respiratory tract, the digestive tract, the endocrinic system, the skin including adnexa, the locomotory system and of the urogenital tract. There will now be described further features and advantages of the invention with reference to the examples and the accompanying drawings.
Optical fiber coating removing device
An object of the invention is to provide an optical fiber coating removing apparatus which can remove coating from an optical fiber preform without damaging the optical fiber surface. The apparatus is excellent in workability because positioning or the like for coating removal easily. The invention provides an optical fiber coating removing apparatus for holding coating 1b of an end portion of an optical fiber 1 between-a pair of coating removing blades and removing the coating 1b from the optical fiber 1 with the pair of the blades. In each blade 11, 11′, a blade pressing plate 12, 12′, a semicircular blade 13, 13′ having a semicircular eating blade portion 13a, and a flat blade 14, 14′ having a straight-line eating blade portion 14a having a triangular section are integrally laminated onto a blade bearing plate 15, 15′ in that order. The coating 1b is removed without damaging the surface of the optical fiber.
1. An optical fiber coating removing apparatus for nipping coating of an end portion of an optical fiber with a pair of coating removing blades and removing said coating with said pair of coating removing apparatus, wherein each of said coating removing blades includes a semicircular blade having a semicircular eating blade portion and a flat blade having a straight-line eating blade portion having a triangular shape in section. 2. An optical fiber coating removing apparatus according to claim 1, wherein a blade pressing plate and a blade bearing plate are provided in each of said coating removing blades, and said blade pressing plate, said semicircular blade and said flat blade are fixedly laminated onto said blade bearing plate in that order. 3. An optical fiber coating removing apparatus according to claim 2, wherein a V-shaped notch for positioning said optical fiber is formed in said blade bearing plate. 4. An optical fiber coating removing apparatus according to claim 1, wherein heating means for heating said end portion of said optical fiber is provided in a body portion having said coating removing blades attached thereto. 5. An optical fiber coating removing apparatus according to claim 2, wherein heating means for heating said end portion of said optical fiber is provided in a body portion having said coating removing blades attached thereto. 6. An optical fiber coating removing apparatus according to claim 1, wherein an optical fiber holder capable of holding said optical fiber and a holder support table for supporting said optical fiber holder are provided. 7. An optical fiber coating removing apparatus according to claim 2, wherein an optical fiber holder capable of holding said optical fiber and a holder support table for supporting said optical fiber holder are provided. 8. An optical fiber coating removing apparatus according to claim 6, wherein a lifting member is provided for lifting up a front end portion of said optical fiber protruding from said optical fiber holder so as to prevent said front end portion of said optical fiber from colliding with said coating removing blades when said optical fiber holder is moved to a predetermined position, and for releasing said front end portion of said optical fiber from lifting up when said optical fiber holder has been moved to said predetermined position. 9. An optical fiber coating removing apparatus according to claim 7, wherein a lifting member is provided for lifting up a front end portion of said optical fiber protruding from said optical fiber holder so as to prevent said front end portion of said optical fiber from colliding with said coating removing blades when said optical fiber holder is moved to a predetermined position, and for releasing said front end portion of said optical fiber from lifting up when said optical fiber holder has been moved to said predetermined position.
<SOH> BACKGROUND ART <EOH>When optical fibers are connected to each other, or when an optical fiber is connected to an optical component, first the coating in an end portion of each optical fiber is removed to expose the glass portion thereof. After that, the surface of the glass portion of the optical fiber exposed after the coating removal is cleaned by wiping off residual parts of the coating or the like with apiece of gauze or the like impregnated with a solvent. After that, a part of this glass portion is cut off with a fiber cutter (creeper) so that the glass portion has a predetermined exposed length. Thus, a mirror-finished cut surface is obtained. Such a series of steps for terminal working in the end portion of the optical fiber are carried out individually with tools special to those steps respectively, or carried out with an automated apparatus. Removal of coating from an optical fiber is carried out, for example, by nipping the coating of a single-core optical fiber with a pair of coating removing blades, making the blades eat into the coating, and pulling the coating from the optical fiber with the coating removing blades. FIG. 8 is a view showing examples of removal of coating from a conventional optical fiber. FIG. 8 (A) shows an example of removal of coating with flat blades, and FIG. 8 (D) shows an example of removal of coating with semicircular blades. As shown in FIG. 8 (A), when coating 1 b of an optical fiber 1 is removed with a pair of flat blades 2 , the upper and lower flat blades 2 and 2 are made to eat into the coating 1 b shallowly enough not to come into direct contact with the surface of a glass portion 1 a of the optical fiber 1 . In this state, when the optical fiber 1 is pulled in the left direction shown by the arrow in the drawing, the coating 1 b is ripped down at the place where the blades eat. Then, the coating 1 b is removed while forming a swelling on the right of the blades. When the outer diameter of the glass portion 1 a is 0.125 mm and the outer diameter of the coating 1 b is not larger than 0.3 mm, the distance between the pair of flat blades 2 will be about 0.2 mm, and the distance between the glass portion la and each flat blade 2 will be about 0.04 mm. When the thickness of the coating 1 b of the optical fiber 1 is small, pulling out the coating 1 b may make the optical fiber 1 shake up and down. When the coating 1 b is pulled out in the state where the optical fiber 1 shakes up and down, the pair of flat blades 2 and 2 further eat into the coating 1 b so that the surface of the glass portion 1 a of the optical fiber 1 is brought into contact with the blades. Thus, the surface of the glass portion 1 a is apt to be damaged. When the surface of the glass portion 1 a is damaged, the strength in connecting optical fibers to each other by fusion splicing is lowered so that the connection may be broken easily. It is therefore known that a resin layer is added to a coating portion to be removed so that the thickness of the coating is increased, and the coating 1 b is removed together with the added resin layer (see Japanese Patent Laid-Open No. 94925/1994). However, it requires extra labor to form the coating resin layer additionally for coating removal. In addition, when the upper and lower flat blades 2 and 2 are made to eat into the coating 1 b, the sectional shape of the coating 1 b becomes an elliptic shape as shown in FIG. 8 (B). When the coating 1 b is pulled out in this state, the end portion from which the coating 1 b has been removed is expanded like an elliptic trumpet as shown in FIG. 8 (C). Thus, there is also a problem that failure in shape is apt to occur in a molding step in which glass portions 1 b are connected to each other by fusion splicing and the fusion splicing portion thereof is covered with resin or the like. As shown in FIG. 8 (D), when coating is removed with a pair of semicircular blades 3 and 3 , first, the semicircular blade portions are made to eat into the coating 1 b shallowly enough not to come into direct contact with the glass portion 1 a. The semicircular blades 3 are steadier than the flat blades 2 when the optical fiber is pulled out. Thus, it is difficult for the optical fiber surface to abut against the blades. However, when the optical fiber 1 is pulled out in the left direction shown by the arrow in the drawing, the coating 1 b is ripped down at the place where the blades eat into the coating 1 b, coating dust 1 c is collectively concentrated on the right side of the blades. When the coating dust is concentrated, the pulling resistance increases so that it becomes difficult to pull out the optical fiber 1 . When the optical fiber 1 is pulled forcedly, the optical fiber 1 will be broken. In addition, when the adhesive force between the coating 1 b and the glass portion 1 a is great, the coating dust is crushed to powder to increase the residual part of the coating 1 b adhering to the glass portion 1 b. Thus, the workability to remove the coating dust (residual part) deteriorates. In order to facilitate the removal of coating from an optical fiber, there is also known a technique in which a heater is disposed in a coating removing apparatus and heating by the heater is used to weaken the adhesive force between the optical fiber and the coating. However, the aforementioned problem in coating removing blades themselves has not been solved. It is an object of the present invention to provide an optical fiber coating removing apparatus which can carry out coating removal easily without damaging the surface of a glass portion of an optical fiber when the coating of the optical fiber is removed therefrom. Further, it is another object of the present invention to provide an optical fiber coating removing apparatus which is excellent in workability because it can carry out positioning or the like for removing the coating of the optical fiber easily.
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 shows an example of coating removing blades for use in an optical fiber coating removing apparatus according to the present invention, FIG. 1 (A) being an exploded perspective view, FIG. 1 (B) being a sectional view, FIG. 1 (C) being a sectional view taken on line a-a in FIG. 1 (B); FIG. 2 is a side view of the optical fiber coating removing apparatus according to the present invention, showing the state where an optical fiber has been set in the coating removing apparatus and moved forward slightly; FIG. 3 is a side view of the optical fiber coating removing apparatus according to the present invention, showing the state where removal of coating from the optical fiber has been started; FIG. 4 is a side view of the optical fiber coating removing apparatus according to the present invention, showing the state where the removal of coating from the optical fiber is in operation; FIG. 5 is a perspective view showing examples of optical fiber holders for use in the optical fiber coating removing apparatus according to the present invention, FIG. 5 (A) showing the state where a pair of clamp members are provided and an optical fiber has not yet been set, FIG. 5 (B) showing the state where the optical fiber has been set, FIG. 5 (C) showing the state where a single clamp member is provided and an optical fiber has not yet been set, FIG. 5 (D) showing the state where the optical fiber has been set; FIG. 6 is a diagram showing a working process for optical fiber fusion splicing; FIG. 7 is a graph for comparing the strength of fusion splicing after coating removal with the coating removing blades of the optical fiber coating removing apparatus according to the present invention with the strength of fusion splicing after coating removal with conventional coating removing blades; and FIG. 8 is a view for explaining conventional examples of coating removal. detailed-description description="Detailed Description" end="lead"? Incidentally, as for the reference numerals in the drawings, 1 designates an optical fiber; 1 a, a glass portion; 1 b, coating; 11 and 11 ′, coating removing blades; 12 and 12 ′, blade pressing plates; 13 and 13 ′, semicircular blades; 14 and 14 ′, flat blades; 15 , 15 ′, blade bearing plates; 16 , a screw; 17 , a body portion; 18 , a pressing portion; 19 , a mounting screw; 20 , an optical fiber holder; 21 , a holder base; 22 , a clamp member; 23 , a hinge shaft; 24 , an extension portion; 25 , a V-groove; 26 , an elastic member; 27 and 28 , magnets; 30 , a base table; 31 , a holder support table; 32 , a holder clamp; 33 , a rail; 34 , a rear portion stopper; 35 , a lifting member mounting table; 36 , a lifting member; 37 , a heater (heating means); and 38 , a protrusion.
Guide system
A guide system for use by a user who performs an operation in a defined threedimensional region is disclosed, the system including a data processing apparatus for generating images of the subject of the operation in co-registration with the subject, a display for displaying the images to the user, a probe having a position which is visible to the user, and a tracking unit for tracking the location of the probe by the system and transmitting that location to the data processing apparatus, the data processing apparatus being arranged, upon the user moving the probe to a selection region outside and surrounding the defined region, to generate one or more virtual buttons, each of the buttons being associated with a corresponding instruction to the system, the data processing apparatus being arranged to register a selection by the user of any of the virtual buttons, the selection including positioning of the probe in relation to the apparent position of that virtual button, and to modify the computer-generated image based on the selection.
1. A guide system for use by a user who performs an operation in a defined three-dimensional region, the system including a data processing apparatus for generating images of the subject of the operation in co-registration with the subject, a display for displaying the images to the user, a probe having a position which is visible to the user, and a tracking unit for tracking the location of the probe by the system and transmitting that location to the data processing apparatus, the data processing apparatus being arranged, upon the user moving the probe to a selection region outside and surrounding the defined region, to generate one or more virtual buttons, each of the buttons being associated with a corresponding instruction to the system, the data processing apparatus being arranged to register a selection by the user of any of the virtual buttons, the selection including positioning of the probe in relation to the apparent position of that virtual button, and to modify the computer-generated image based on the selection. 2. A system according to claim 1 wherein the data processing apparatus is arranged to generate images of the subject of the operation overlaid on the subject. 3. A system according to claim 1 in which the positioning of the probe includes aligning a longitudinal axis of the probe with the button. 4. A system according to claim 3 in which the computing apparatus is arranged, while the virtual buttons are displayed, to include in the image a line extending from the probe along its longitudinal axis. 5. A system according to any preceding claim in which the display is adapted to be mounted on the head of a user, the user being able to view the subject of the operation through the display, so as to see the computer-generated image superimposed on a true image of the subject of the image, the tracking unit monitoring the position of the display and transmitting the monitored position of the display to the processing apparatus, which is arranged to modify the computer-generated image according to the position of the display to maintain the computer-generated image and the real image stereoscopically in register. 6. A system according to any preceding claim in which the display is adapted to be mounted on a microscope, the user being able to view the microscope image through the display, so as to see the computer-generated image superimposed on the microscope image, the tracking unit monitoring the position of the microscope and transmitting the monitored position of the microscope to the processing apparatus, which is arranged to modify the computer-generated image according to the position of the microscope to maintain the computer-generated image and the real image stereoscopically in register. 7. A method for use by a user who performs an operation in a defined three-dimensional region with guidance from an image guided system, for modifying the image displayed to the user by the image guided system, the system including a data processing apparatus for generating images of the subject of the operation in co-registration with the subject, a display for displaying the images to the user, a probe having a position which is visible to the user, and a tracking unit for tracking the location of the probe by the system and transmitting that location to the data processing apparatus, the method including: the user moving the probe to a selection region outside and surrounding the defined region, the data processing apparatus registering the position of the probe within the selection region, and thereupon generating within the image one or more virtual buttons, each of the buttons being associated with a corresponding instruction to the system, the user selecting one of the buttons, the selection including positioning of the probe in relation to the apparent position of that virtual button, and the data processing apparatus registering the selection, and modifying the computer-generated image based on the corresponding instruction. 8. A method according to claim 7 wherein the data processing generates images of the subject of the operation overlaid on the subject. 9. A method according to claim 7 in which, while the data processing apparatus displays the virtual buttons, it further displays a line extending from the probe along a longitudinal axis thereof, the positioning of the probe includes aligning the longitudinal axis of the probe with the button. 10. A method according to claim 7 in which the display is mounted on the head of a user, the user being able to view the subject of the operation through the display, so as to see the computer-generated image superimposed on a true image of the subject of the image, the tracking unit monitoring the position of the display and transmitting the monitored position of the display to the processing apparatus, which modifies the computer-generated image according to the position of the display to maintain the computer-generated image and the real image stereoscopically in register. 11. A method according to claim 7 in which the display is mounted on a microscope, the user being able to view the microscope image through the display, so as to see the computer-generated image superimposed on the microscope image, the tracking unit monitoring the position of the microscope and transmitting the monitored position of the microscope to the processing apparatus, which modifies the computer-generated image according to the position of the microscope to maintain the computer-generated image and the real image stereoscopically in register.
<SOH> BACKGROUND OF THE INVENTION <EOH>Image guidance systems have been widely adopted in neurosurgery and have been proven to increase the accuracy and reduce the invasiveness of a wide range of surgical procedures. Currently, image guided surgical systems (“Navigation Systems”) are based on a series of images constructed from data gathered before the operation (for example by MRI or CT) which are registered in relation to the patient in the physical world by means of an optical tracking system. To do this, detecting markers are placed on the skin of the patient and they are correlated with their counterparts visible on the imaging data. During the surgical operation the images are displayed on a screen in 3 orthogonal planes through the image volume, while the surgeon holds a probe that is tracked by the tracking system. When the probe is introduced into the surgical field, the position of the probe tip is represented as an icon drawn on the images. By linking the preoperative imaging data with the actual surgical space, navigation systems provide the surgeon with valuable information about the exact localisation of a tool in relation to the surrounding structures and help to relate the intra-operative status to the pre-operative planning. Despite these strengths, the current navigation systems suffer from various shortcomings. Firstly, the surgeon needs to look at the computer monitor and away from the surgical scene during the navigation procedure. This tends to interrupt the surgical workflow and in practice often results in the operation being a two-people job, with the surgeon looking at the surgical scene through the microscope and his assistant looking at the monitor and prompting him. Secondly, the interaction with the images during the surgery (e.g. switching between CT and MRI, changing the screen windows, activating markers or segmented structures from the planning phase, colour and contrast adjustments) requires the operation of a keyboard, a mouse or a touch screen, which is distracting for the surgeon and troublesome since the equipment needs to be packed with sterile drape. Although probe-type control devices have been proposed (see Hinckley K, Pausch R, Goble C J, Kassel N,F: A Survey of Design Issues in Spatial Input, Proceedings of ACM UIST94 Symposium on User Interface Software & Technology, pp. 213-222; and Mackinlay J, Card S. Robertson G: Rapid Controlled Movement Through a Virtual 3D Workspace, Comp. Grap., 24 (4), 1990, 171-176), all have shortcomings in use. Thirdly, a common problem to all current navigation systems which present imaging data as 2D orthogonal slices is the fact that the surgeon has to relate the spatial orientation of the image series including their mentally reconstructed 3D information to the orientation of the patient's head, which is covered during the operation. A system that uses see-through augmentation by combining the naked eye view of the patient with the computer-generated images is currently under investigation (see Blackwell M, O'Toole RV, Morgan F, Gregor L: Performance and Accuracy experiments with 3D and 2D Image overlay systems. Proceedings of MRCAS 95, Baltimore, USA, 1995, pp 312-317; and DiGioia, Anthony M., Branislav Jaramaz, Robert V. O'Toole, David A. Simon, and Takeo Kanade. Medical Robotics And Computer Assisted Surgery In Orthopaedics. In Interactive Technology and the New Paradigm for Healthcare, ed. K. Morgan, R. M. Satava, H. B. Sieberg, R. Mattheus, and J. P. Christensen. 88-9.0. IOS Press, 1995). In this system, an inverted image on an upside-down monitor is overlaid over the surgical scene with a half-silvered mirror to combine the images. The user wears a head tracking system while looking onto the mirror and the patient beneath. However, the authors report significant inaccuracies between the virtual and the real object. Other systems currently under research or development combine computer-generated images with the video of the surgical scene obtained through cameras placed at fixed positions in the operation theatre or a head mounted display of the user. The combined signal is then channelled into the HMD (“Head Mounted Display”) of a user. The three examples of such projects are disclosed at in Fuchs H, Mark A, Livingston, Ramesh Raskar, D'nardo Colucci, Kurtis Keller, Andrei State, Jessica R. Crawford, Paul Rademacher, Samuel H. Drake, and Anthony A. Meyer, MD. Augmented Reality Visualization for Laparoscopic Surgery. Proceedings of First International Conference on Medical Image Computing and Computer-Assisted Intervention (MICCAI '98), 11-13 October 1998, Massachusetts Institute of Technology, Cambridge, Mass., USA; Fuchs H, State A, Pisano E D, Garrett W F, Gentaro Hirota, Mark A. Livingston, Mary C. Whitton, Pizer S M. (Towards) Performing Ultrasound-Guided Needle Biopsies from within a Head-Mounted Display. Proceedings of Visualization in Biomedical Computing 1996, (Hamburg, Germany, Sep. 22-25, 1996), pgs. 591-600; and State, Andrei, Mark A. Livingston, Gentaro Hirota, William F. Garrett, Mary C. Whitton, Henry Fuchs, and Etta D. Pisano (MD). Technologies for Augmented-Reality Systems: realizing Ultrasound-Guided Needle Biopsies. Proceedings of SIGGRAPH 96 (New Orleans, La., Aug. 4-9, 1996), in Computer Graphics Proceedings, Annual Conference Series 1996, ACM SIGGRAPH, pgs. 439-446. Another technique (disclosed in Edwards P J, Hawkes D J, Hill D L G, Jewell D, Spink R, Strong A, Gleeson M: Augmented reality in the stereo microscope for Otolaryngology and neurosurgical Guidance. Proceedings of MRCAS 95, Baltimore, USA, 1995, pp 8-15) uses an operating microscope as a device for overlaid display of 3D graphics. By “image injection” of stereoscopic structures into the optical channels of the microscope the surgeon sees the superimposed image over the surgical scene. This technique overlays simple meshes with a relatively low resolution onto the surgical scene, without providing any interactive capabilities. The authors report difficulties regarding the stereoscopic perception of the overlaid data in relation to the real view. Although meant for guidance of the user, these techniques are all limited in application and usability.
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention aims to address at least one of the above problems, and to propose new and useful navigation systems and methods and devices for controlling them. The present invention is particularly concerned with a system which can be used during a surgical operation. However, the applicability of the invention is not limited to surgical operations, and the systems and methods discussed below may find a use in the context of any delicate operation, and indeed during a planning stage as well as an intra-operative stage. The present invention is motivated by noting that during the navigation procedure in a surgical operating room it is critical to be able easily and quickly to interact with a surgical navigation system, for example to alter the format of the computer-generated images. In addition, it would be advantageous to be able to simulate certain surgical procedures directly at the surgical site by using the computer-generated images. In general terms, the present invention proposes a probe to be held by a user who performs an operation (e.g. a surgical operation) within a defined region while employing an image-based guide system having a display for displaying computer-generated images (3D and/or 2D slices) of the subject of the operation. The probe has a position which is tracked by the system and which is visible to the user (for example, because the system allows the user to see the probe directly, or alternatively because the computer-generated images include an icon representing its position). By moving the probe, the user is able to enter information into the system to control it, such as to cause changes in the physical shape of the subject in the image presented by the computer. The invention proposes a guide system for use by a user who performs an operation in a defined three-dimensional region, the system including a data processing apparatus for generating images of the subject of the operation in co-registration with the subject, a display for displaying the images to the user, a probe having a position which is visible to the user, and a tracking unit for tracking the location of the probe by the system and transmitting that location to the data processing apparatus, the data processing apparatus being arranged, upon the user moving the probe to a selection region outside and surrounding the defined region, to generate one or more virtual buttons, each of the buttons being associated with a corresponding instruction to the system, the data processing apparatus being arranged to register a selection by the user of any of the virtual buttons, the selection including positioning of the probe in relation to the apparent position of that virtual button, and to modify the computer-generated image based on the selection. The defined region is referred to as a “bounding box”, so that the user is able to display the virtual regions by moving the probe out of the bounding box. This means that the amount of movement which a user has to perform to cause the virtual buttons to be displayed is low. To indicate one of the virtual buttons, the user may move the probe to a location within that virtual button, but more preferably he may align a length direction of the probe in the direction of the desired virtual button (e.g. towards the centre of that button with a given tolerance). This latter possibility means that the probe only needs to be moved slightly to indicate one of the buttons. When this alignment has been made to indicate one of the buttons, the user may issue a separate confirmation command (preferably using a separate data input device which is not operated by the user's hand, such as a foot-operated switch) to confirm the selection. To help the user direct the probe accurately, a line may be displayed to the user extending from the probe along the length direction. The line may be a predefined length, or have a length controlled by the user (e.g. so that the user does not just indicate a virtual button by its direction but also by its distance from the probe). Most preferably, the computer-generated images are overlaid on the real image of the subject and are preferably displayed in a semitransparent head-mounted stereo display (HMD), to be worn by a surgeon, so that he or she sees the computer-generated images overlying the real view of the subject of the operation obtained through the semi-transparent display (e.g. semi-transparent eye-pieces). The HDM is tracked, and the computer generates images based on this tracking, so that as the surgeon moves, the real and computer-generated images remain in register. The system can be used in two modes. Firstly, during macroscopic surgery the user looks through the display in semi-transparent mode and sees stereoscopic computer graphics overlaid over the surgical field. This will enable the surgeon see “beyond the normal line of sight” before an incision is made, e.g. visualising the position of a tumour, the skull base or other target structures. Secondly, for microscopic surgery the same stereo display can be attached to (e.g. on top of the binocular of) a stereoscopic microscope, the position of which is tracked (as an alternative to tracking movements of the user). The computer graphics in the display may be linked to the magnification and focus parameters of the tracked microscope and therefore reflect a “virtual” view into the surgical field The 3D data presented in the display may be computer-generated by a computational neurosurgical planning package called VizDexter, which was previously published under the name VIVIAN and was developed by Volume Interactions of Singapore. VizDexter allows the employment of multimodal (CT and MRI fused) images in the Virtual Reality environment of the “Dextroscope” (for example, as disclosed in Kockro R A, Serra L, Yeo T T, Chumpon C, Sitoh Y Y, Chua G G, Ng Hern, Lee E, Lee Y H, Nowinski W L: Planning Simulation of Neurosurgery in a Virtual Reality Environment. Neurosurgery Journal 46[1], 118-137. 2000.9, and in Serra L, Kockro R A, Chua G G, Ng H, Lee E, Lee Y H, Chan C, Nowinski W: Multimodal Volume-based Tumor Neurosurgery Planning in the Virtual Workbench, Proceedings of the First International Conference on Medical Image Computing and Computer-Assisted Intervention (MICCAI), Massachusetts, Institute of Technology, Cambridge Mass., USA, Oct. 11-13, 1998, pp.1007-1016. The disclosure of these publications is incorporated herein in its entirety by reference). Although the invention has been expressed above in terms of a system, it may alternatively be expressed as a method carried out by the user of the system.
Distiller for liquids, liquids distillation method and equipment for treating sewage, which equipment includes said distiller
The distiller includes an exchanger (6) of tubes (7) which fill with the liquid to be treated, means (14) of heating, means (23) for compressing the steam from evaporation of the liquid, means (24) for feeding the compressed steam around the tubes (7) and means (27, 28) for inducing turbulence into the liquid, preferably by making the compressed steam bubble inside it. The method includes heating the liquid and supplying it to the tubes of the exchanger, compressing the resulting steam and bringing it into contact with the exterior of the tubes. The equipment uses a distiller such as that described. The invention permits treatment of sewage and other liquids by distillation, with minimum energy consumption and in a small space.
1. Distiller (1) for liquids, which includes a heat exchanger (6) with a housing which contains a plurality of tubes (7), into which is fed the liquid to be treated, means (9, 10) of supplying the liquid to the tubes, means (14) of heating the liquid, means (23) for compressing the steam from evaporation of the liquid, means (24) for feeding the compressed steam into the housing around the tubes (7) and means (26, 12) for removing on the one hand the condensate and on the other the concentrated residue, wherein the liquid-feed means (9, 10) keep the tubes (7) full of liquid up to an intermediate predetermined level, in that said distiller (1) further includes means (27, 28) for inducing turbulence in the liquid which is in the tubes (7) and in that it comprises a level device (29) with level sensors (30). 2. Distiller as claimed in claim 1, wherein the means for inducing turbulence in the liquid include a duct (28) for injecting some of the steam coming from the compressor into the liquid. 3. Distiller as claimed in claim 2, wherein said duct (28) for injecting some of the steam into the liquid runs in the bottom part of the distiller (1) and consists in a tube (28) with a plurality of lateral steam outlet orifices. 4. Distiller as claimed in claim 1, wherein it also includes a substantially horizontal plate (15) situated inside the housing, above the tubes (7), with a diameter less than that of the housing. 5. Distiller as claimed in claim 1, wherein it also includes a filter (17) of perforated plates (18, 19), situated above the tubes (7). 6. Distiller as claimed in claim 5, wherein said filter (17) is made up of a horizontal plate (19) with a perforated central zone (20) and at least one perforated annular plate (18), fixed onto the bottom part of said horizontal plate (19), around its perforated central zone (20). 7. Distiller as claimed in claim 1, wherein the housing has a lower tank (8), connected to the tubes (7) and surrounded by the means (14) of heating the liquid. 8. Distiller as claimed in claim 7, wherein said lower tank (8) has a duct (12) with a valve (13) for extracting the concentrated residues. 9. Distiller as claimed in claim 1, wherein said means (23) for compressing the steam consist in a side-channel blower. 10. Distiller as claimed in claim 1, wherein it includes a condensate outlet (26) calibrated to a predetermined diameter. 11. Distiller as claimed in claim 1, wherein its housing is made of up an intermediate section (6) with the tubes (7) of the heat exchanger, an upper section (5) with a steam outlet (21) and a lower section (8) with an inlet (9) for the liquid to be treated and with a concentrated residue outlet (12), these three sections (5, 6, 8) being attached to each other by means of detachable fixing means. 12. Liquids distillation method which includes the following operations: heating the liquid to be treated and delivering it inside the tubes (7) of a heat exchanger (6); compressing the steam resulting from evaporation of the liquid and bringing it into contact with the exterior wall of the exchanger (6) tubes (7); and removing the condensate and the concentrated residue; wherein the liquid inside the tubes (7) of the exchanger (6) is maintained up to certain level; and turbulence is induced in the liquid in the tubes (7). 13. Method as claimed in claim 12, wherein said turbulence in the liquid is induced by injecting some of the compressed steam into the liquid to make it bubble. 14. Method as claimed in claim 11, wherein it includes a stage of preheating the liquid to be treated, with recuperation of the heat of the condensate obtained from the steam. 15. Equipment for treating sewage, wherein it includes a distiller (1) as claimed in claim 1. 16. Equipment as claimed in claim 15, wherein it further includes a heat recuperator (40) for preheating the water to be treated, in which the condensate extracted from the distiller (1) gives up heat to the water to be treated, before the water is supplied to the distiller (1). 17. Equipment as claimed in claim 15, wherein it includes a residues storage tank (50) divided into two parts (51, 52) by a filtering divider (53), with one (51) of the parts of the tank connected to an inlet duct for the sewage to be treated and an inlet duct (12) for the concentrate residue from the distiller (1), while the other part (52) of the tank is connected to a suction duct (54) for water to be treated. 18. Equipment as claimed in claim 15, wherein it also includes means for drying (70) the concentrated residues. 19. Equipment as claimed in claim 18, wherein said means of drying (70) include a compartment (71) with means of heating (72) and provided with an inlet (67) for the concentrated residues, an outlet (74) for the dry residues, a gases and smoke outlet (82) and a duct (75) for feeding in air. 20. Equipment as claimed in claims 15, wherein it also includes means (63) of mechanical separation of the solids contained in suspension in the sewage before they are fed to the distiller. 21. Equipment as claimed in claim 20, wherein said means of mechanical separation of the solids include a screw conveyor (63) whose housing has a perforated lower part (65) for the outlet of liquid through it. 22. Equipment as claimed in claim 15, wherein it includes a catalyst for elimination of ammonia, situated at the outlet from the steam compression means (23) or in a smoke outlet duct.
<SOH> BACKGROUND OF THE INVENTION <EOH>On people-transport equipment which has toilet facilities, such as railways, the sewage generated has to be disposed of. One possibility is to store the waste in a tank, from which it is removed at intervals for treatment thereof in conventional fixed installations; this system nevertheless requires periodic maintenance and substantial storage space if the tank is not emptied very often. Some recent systems are based on treatment of the sewage on board the transport equipment itself, though a number of major problems are raised in implementing such solutions. On the one hand, the sewage has a very high level of contamination, since practically all of it is from toilets which are evacuated by means of vacuum systems with very low consumption of water at each flush (of the order of 0.7 litres per flush); as a result, the conventional treatment systems used at treatment plants and other similar facilities are not useful for this application. Furthermore, transport equipment has major limitations both in terms of physical space for housing the facilities and supplies of power for consumption. Finally, it must be borne in mind that solids or liquids with certain levels of contamination cannot simply be disposed of into the environment. Some systems developed for treatment of the sewage on the means of transport itself consist in biological treatments combined with physical treatments. Biological treatments have several disadvantages: on the one hand, the bacteria require maintenance, have a limited life and are delicate when subjected to quite simple aggressions, such as bleach, colognes and other similar products which can be poured into the toilet bowl on a railway train; and on the other hand, the solids obtained from the biological treatment and the subsequent physical treatment must be stored for later treatment at an outside plant, for due to their high levels of contamination they cannot simply be expelled onto the track. There is therefore still a need for emptying of tanks and treatment at fixed facilities. Another disadvantage of this type of systems is that the disinfection treatment following the biological treatment, which is carried out by heating the liquid, involves high energy consumption. Another method might consist in evaporation by direct heating of the liquid to be distilled, though this again has the disadvantage of high energy consumption. There also exist facilities for the desalination of water and treatment of industrial waste waters, all such facilities being large and with high energy consumption, using a distillation system in which the latent heat contained in the evaporated steam is recuperated and used to produce boiling in a heat exchanger system, by means of mechanical compression of the steam In these systems, the water to be treated is recirculated using pumps and is sprayed onto the walls of the tubes of an exchanger, while the previously compressed steam is directed onto the outside of the tubes; by giving up heat to the water through the walls of the tubes, the steam condenses. Spraying of the water is necessary in order to achieve a good thermal exchange coefficient. Although in some respects this treatment system could be suitable for sewage on transport equipment, the necessarily small dimensions of the overall installation make good operation thereof impossible. For example, the recirculation pumps would suffer phenomena of cavitation due to the low head of water available at the suction indraught point, and the spraying nozzles would become blocked continuously due to their small size and the high quantities of solids in suspension.
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>For a better understanding of all that has been set out, some drawings are attached which show schematically and solely by way of non-restrictive example a practical case of embodiment. In said drawings: FIG. 1 is a schematic diagram showing an embodiment of the distiller of the invention, installed in equipment for treating sewage; FIG. 2 is a cross-section of the distiller of FIG. 1 ; FIG. 3 is a schematic diagram of equipment for treating sewage, with a heat recuperator for preheating of the liquid to be treated; FIG. 4 is schematic diagram of equipment similar to that of FIG. 3 , with a storage tank; and FIG. 3 is a schematic diagram of equipment for treating sewage provided with an initial separator of solids and liquids and a device for drying the residue. detailed-description description="Detailed Description" end="lead"?
Use of at least one 10-hydroxy-2-decenoic acid derivative in compositions for treating the cuteous signs of aging
The present invention relates to a method of inhibiting degradation of skin or mucous membranes by inhibiting action of collagenases comprising applying to the skin or mucous membranes a composition comprising at least one of 10-hydroxy-2-decenoic acid derivative. The invention also relates to a process for treating signs of aging.
1. A method of inhibiting the expression, translation or secretion of proteases of the extracellular matrix in skin or mucous membranes comprising: applying to said skin or mucous membranes a composition comprising at least one 10-hydroxy-2-decenoic acid derivative chosen from: 2-dimethylaminoethyl 10-hydroxy-2-decenoate of formula: 2,3-dihydroxypropyl 10-hydroxy-2-decenoate of formula: 2-hydroxypropyl 1,3-bis(10-hydroxy-2-decenoate) of formula: propyl 1,2,3-tris(10-hydroxy-2-decenoate) of formula: 3-[(2-methoxy)ethoxymethoxy]propyl 1,2-bis (10-hydroxy-2-decenoate) of formula: . 2. The method of claim 1, said composition inhibits the expression, translation or secretion of metalloproteases. 3. The method as in claim 1 or 2, wherein said composition inhibits the expression, translation or secretion of type-1 matrix metalloprotease. 4. The method as in claim 1 or 2, wherein said composition treats wrinkles and fine lines. 5. The method as in claim 1 or 2, wherein said composition treats withered skin. 6. The method as in claim 1 or 2, wherein said composition treats thinned skin. 7. The method as in claim 1 or 2, wherein said composition treats a lack of elasticity and/or tone of said skin. 8. The method as in claim 1 or 2, wherein said composition inhibits internal degradation of said skin caused by exposure to ultraviolet radiation. 9. The method of claim 8, wherein said composition inhibits collagen degradation. 10. A method of treating skin complaints associated with the menopause comprising applying to said skin a composition comprising at least one 10-hydroxy-2-decenoic acid derivative as described in claim 1. 11. The method as in claim 1 or 2, wherein said composition comprises between about 0.001% and about 10% of said at least one 10-hydroxy-2-decenoic acid derivative based on the total weight of the composition. 12. The method of claim 11, wherein said composition comprises between about 0.01% and about 1% of said at least one 10-hydroxy-2-decenoic acid derivative based on the total weight of the composition. 13. (cancelled) 14. A process for treating signs of aging of skin, hair or mucous membrane comprising: applying to said skin or ingesting a composition comprising at least one 10-hydroxy-2-decenoic acid derivative as described in claim 1 to said skin, hair or mucous membrane. 15. A process for treating the wrinkles and fine lines, withered skin, thinned skin, the lack of elasticity and/or tone of the skin or any internal change in the skin which is not systematically reflected by a changed external appearance, comprising: applying to said skin or ingesting a composition comprising at least one 10-hydroxy-2-decenoic acid derivative as described in claim 1.
<SOH> BACKGROUND OF THE INVENTION <EOH>Human skin consists of two compartments, namely a surface compartment, the epidermis, and a deep compartment, the dermis. Natural human epidermis is composed mainly of three types of cells: keratinocytes, which form the great majority, melanocytes and Langerhans cells. Each of these cell types contributes, by virtue of its intrinsic functions, toward the essential role played in the body by the skin. The dermis gives the epidermis a solid support. It is also the epidermis' nourishing factor. It consists mainly of fibroblasts and of an extracellular matrix which is itself composed mainly of collagen, elastin and a substance known as ground substance, these components being synthesized by the fibroblasts. Leukocytes, mastocytes and tissue macrophages are also found therein. It also contains blood vessels and nerve fibers. In normal skin, that is to say non-pathological and unscarred skin, the fibroblasts are in the quiescent state, i.e. non-proliferative, metabolically relatively inactive, and immobile. These collagen fibers give the dermis its firmness. Collagen fibers consist of fibrils sealed together, thus forming more than 10 different types of structures. The firmness of the dermis is mainly due to the entanglement of the collagen fibers packed together in all directions. The collagen fibers contribute toward the elasticity and tonicity of the skin and/or mucous membranes. The collagen fibers are under constant renewal, but this renewal decreases with age, leading to thinning of the dermis. This thinning of the dermis is also due to pathological causes such as, for example, the hypersecretion of corticoid hormones, certain pathologies or vitamin deficiencies (which is the case for vitamin C in scurvy). It is also accepted that extrinsic factors such as ultraviolet rays, tobacco or certain treatments (glucocorticoids, vitamin D and derivatives, for example) also have an effect on the skin and its collagen content. However, various factors lead to the degradation of collagen, with all the consequences which may be envisaged on the structure and/or firmness of the skin and/or mucous membranes. Although very strong, collagen fibers are sensitive to certain enzymes known as collagenases. A degradation of collagen fibers leads to the development of flaccid and wrinkled skin, which people, preferring the appearance of smooth and taut skin, have always sought to combat. Collagenases form part of a family of enzymes known as metalloproteases (MMPs) which are themselves members of a family of proteolytic enzymes (endoproteases or endopeptidases) which contain a zinc atom coordinated to 3 cysteine residues and one methionine residue in their active site and which degrade the macromolecular components of the extracellular matrix and of the basal membranes at neutral pH (collagen, elastin, etc.). These enzymes, which are very widespread in the living world, are present, but poorly expressed, in normal physiological situations such as the growth of organs and the renewal of tissues. However, their overexpression in man and their activation are linked to many processes, sometimes pathological processes, which involve the destruction and remodeling of the matrix. This results in either an uncontrolled resorption of extracellular matrix or, conversely, the installation of a state of fibrosis. The metalloprotease family consists of several well-defined groups based on their resemblances in terms of structure and substrate specificity (see Woessner J. F., Faseb Journal, vol. 5, 1991, 2145). Among these groups, mention may be made of the collagenases for degrading fibrillar collagens (MMP-1 or interstitial collagenase, MMP-8 or neutrophil collagehase and MMP-13 or collagenase 3), gelatinases which degrade type IV collagen or any form of denatured collagen (MMP-2 or gelatinase A (72 kDa), MMP-9 or gelatinase B (92 kDa)), stromelysins (MMP-3) whose broad spectrum of activity addresses the proteins of the extracellular matrix such as glycoproteins (fibronectin, laminin), proteoglycans, etc., or alternatively membrane metalloproteases. Prolonged exposure to ultraviolet rays, particularly to ultraviolet rays of A and/or B type, has the effect of stimulating the expression of collagenases, particularly of MMP-1. This is one of the components of photo-induced aging of the skin. Moreover, at the menopause the main changes concerning the dermis are a decrease in collagen content and in the thickness of the dermis. In menopausal women, this results in thinning of the skin and/or mucous membranes. Women then experience a sensation of “dry skin” or of taut skin and there is an increase in surface wrinkles and fine lines. The skin has a rough aspect when touched. Finally, the skin is less supple. The importance of collagen in the structure of tissues, particularly the skin and/or mucous membranes, and the importance of combating its degradation in order thus to combat aging, whether this is chronobiological or photo-induced aging, and the consequences thereof, the thinning of the dermis and/or the degradation of collagen fibers which result in the development of flaccid and wrinkled skin, may thus be appreciated on reading the account hereinabove.
<SOH> SUMMARY OF THE INVENTION <EOH>One of the aims of the present invention is thus to be able to provide a novel product which has an inhibitory effect on collagenases and, if possible, no appreciable side effects. Surprisingly and unexpectedly, the Applicant has now discovered that certain particular 10-hydroxy-2-decenoic acid derivatives have inhibitory activity on the action of collagenases, via inhibition of the production and/or synthesis of collagenases. The expression “inhibition of the production” should be understood as meaning inhibition of the signaling pathways leading to the expression of collagenase messenger RNA and/or inhibition of the translation of the collagenase messenger RNA into proteins and/or inhibition of the secretion of the collagenases outside the collagenase-producing cell. In the publications of the prior art, 10-hydroxy-2-decenoic acid derivatives are known for their property of activating the immune system. To the Applicant's knowledge, it has never been described in the prior art that 10-hydroxy-2-decenoic acid derivatives have inhibitory activity on the action of collagenases. Consequently, the Applicant is proposing the use of certain particular 10-hydroxy-2-decenoic acid derivatives to inhibit the activity of collagenases and thus to combat collagen degradation, thus making it possible to treat the signs of aging of the skin associated with collagen degradation.
Information processing device, method, recording medium, and program
The present invention relates to an information processing apparatus and method, a recording medium, and a program in which usage status information necessary to use content can be managed on a service-by-service basis. The usage status information is stored in a management area for a service node in association with a content ID (CID) and a usage-right ID for management. Accordingly, since the usage status information is managed on a service-by-service basis, even if a piece of usage status information is broken or tampered with, the effects thereof can be blocked within one management area. In other words, the content whose usage status information is stored in a management area in which usage status information which is tampered with is stored cannot be used; however, the content whose usage status information is stored in other management areas can be used without difficulty. The present invention is applicable to a personal computer.
1. An information processing apparatus which uses content based on at least one service provided by at least one server, said information processing apparatus comprising: managing means for managing usage information of the content on a service-by-service basis in association with each service; and executing means for executing a process of using the content based on the usage information. 2. An information processing apparatus according to claim 1, wherein the managing means calculates an ICV of the usage information associated with the service for storage on a service-by-service basis, and the using means calculates an ICV of the usage information managed in association with the service associated with the usage information of the content to be used, and executes the process of using the content when the obtained ICV is identical to the ICV calculated and stored by the managing means. 3. An information processing apparatus according to claim 1, wherein the usage information comprises usage status information indicating usage status of the content or a usage right necessary to use the content. 4. An information processing method for an information processing apparatus which uses content based on at least one service provided from at least one server, said information processing method comprising: a managing step of managing usage information of the content on a service-by-service basis in association with each service; and an executing step of executing a process to use the content based on the usage information. 5. A recording medium having a computer-readable program for an information processing apparatus recorded therein, the information processing apparatus using content based on at least one service provided from at least one server, the program including: a management control step of controlling management of usage information of the content on a service-by-service basis in association with each service; and an execution control step of controlling execution of a process of using the content based on the usage information. 6. A program for an information processing apparatus which uses content based on at least one service provided from at least one server, said program causing a computer to execute: a management control step of controlling management of usage information of the content on a service-by-service basis in association with each service; and an execution control step of controlling execution of a process of using the content based on the usage information.
<SOH> BACKGROUND ART <EOH>In the related art, clients which use content (for example, play back content) based on one or more services provided from one or more license servers collectively manage information indicating, for example, content usage status (for example, the number of playbacks, etc.), which is necessary to use the content (such information is hereinafter referred to as usage status information). For example, an ICV (Integrity Check Value) of the entire usage status information owned by the clients is calculated and stored, thereby managing such information. However, such collective management of usage status information has a problem in that, for example, when a piece of usage status information is broken or tampered with, the remaining pieces of usage status information cannot be decoded, so that all the services cannot be used.
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a block diagram ,of a content providing system according to the present invention. FIG. 2 is a diagram showing the key structure. FIG. 3 is a diagram of category nodes. FIG. 4 is a diagram showing a specific example of the correspondence between nodes and devices. FIG. 5 is a block diagram of a client shown in FIG. 1 . FIG. 6 is a diagram showing the functional structure of the client shown in FIG. 1 . FIG. 7 is a flowchart showing a service registration process of the client shown in FIG. 1 . FIG. 8 is a view showing a management area reserved in a storage unit shown in FIG. 5 . FIG. 9 is a flowchart showing a content downloading process of the client shown in FIG. 1 . FIG. 10 is a flowchart showing a content providing process of a content server shown in FIG. 1 . FIG. 11 is a diagram showing the data structure of content data. FIG. 12 is a flowchart showing a content using process of the client shown in FIG. 1 . FIG. 13 is a flowchart showing the details of a usage right obtaining process of step S 43 shown in FIG. 12 . FIG. 14 is a diagram showing the structure of a usage right. FIG. 15 is a flowchart showing the details of a usage right updating process of step S 45 shown in FIG. 12 . FIG. 16 is a flowchart showing the details of a usage status updating process of step S 47 shown in FIG. 12 . FIG. 17 is a view showing usage status information stored in a management area. FIG. 18 is a flowchart showing a usage right providing process of a license server shown in FIG. 1 . FIG. 19 is a flowchart showing a usage right updating process of the license server shown in FIG. 1 . detailed-description description="Detailed Description" end="lead"?
Armchair adjusting device
The invention concerns a device for adjusting the position of a support element such as an armchair seat in particular a hairdresser's reclining chair comprising at least an actuator capable of modifying the position of the support element, and a three-way distributor (8) comprising a supply channel (10), a working channel (11) and a return channel (12), the supply channel being connected to a public water distribution network, the working channel being connected to the waste water network, the distributor being capable of controlling the displacement of the actuator by introducing therein water under pressure coming from the public water distribution network or by returning water into the waste water network.
1. A device for adjusting a position of a means of support comprising: at least one actuator capable of modifying the position of the support element, and a three way directional control valve comprising a supply line, a use line and a return line, the supply line being connected to a water main, the use line being connected to the actuator, and the return line being connected to a sewer, the directional control valve being capable of controlling movement of the actuator by introducing into the actuator water under pressure from the water main or by returning the water to the sewer. 2. The device as claimed in claim 1, wherein the actuator is a jack connected to the use line, the jack including a piston, the piston being connected to the support element by an axial rod is moved by the water under pressure from the water distribution main. 3. The device as claimed in claim 1, wherein the actuator comprises a structure in a form of a deformable parallelogram and a waterproof extensible bellows connected to the use line enclosed by a mobile upper plate and a fixed lower plate, between a contracted position and an extended position and a mechanism for guiding the movement of the bellows. 4. The device as claimed in claim 3, wherein two links hinged to each other keep the upper and lower plates parallel to each other as the bellows extends. 5. The device as claimed in claim 3, wherein the upper plate and the lower plate are connected by four uprights, the uprights guiding horizontal and vertical movement of the upper plate as the bellows extends. 6. The device as claimed in claim 1, wherein the means of support comprises an adjustable armchair, including at least one of a seat base, a backrest, and a footrest. 7. The device as claimed in claim 2, wherein a piston is connected to an axial rod on which the seat base is arranged, the adjustment of the position of the piston being effected by an action of the directional control valve to introduce water under pressure into a cylinder or by releasing the water present therein into the sewer. 8. The device as claimed in claim 6, wherein an underside of the seat base comprises: a return spring tending to move the armchair forward, means enabling the armchair to slide relative to a plate connected to an axial rod and at least one inelastic cable connecting the armchair to a fixed point, an upward movement of the plate resulting in a rearward movement of the seat base. 9. The armchair as claimed in claim 8, wherein the underside of the seat base has at least three wheels guided in rails formed in the plate. 10. The device as claimed in claim 9, wherein the plate has a pulley on a rear face to guide the inelastic cable. 11. The device as claimed in claim 6, wherein the footrest is hinged to the armchair and comprises a board on which slides a support element, a jack being able to pivot the footrest between a retracted position in which the support element is superposed on the plate and an extended position in which the support element is in a continuation of the board. 12. The device as claimed in claim 11, wherein the support element is, at an extremity adjacent to the hinge between the support element and the armchair, connected to a fixed point on the armchair by a cable running through a sheath connected to the board at a furthest extremity thereof from the hinge between the support element and the armchair. 13. The device as claimed in claim 12, wherein the support element is returned by a spring.
System and method for call routing in an ip telephony network
A method of processing a call request to a callee in a network telephony system is provided which includes mapping a hostname portion of a callee address to a canonical form of the hostname and determining a canonical form of a username portion of a callee address. The canonical form of the user identity of the callee is then used as an index to a user database to retrieve a callee database record. The callee database record is then used to determine call routing based on the retrieved callee database record, such as user location, preferences and policy data stored in the record. The method is generally performed by a signaling server in the network, such as a SIP proxy server. The signaling server can also provide security features such as caller authentication. A scalable signaling and routing architecture is also provided.
1. A method of processing a call request to a callee in a network telephony system comprising: mapping a hostname portion of a callee address to a canonical form of the hostname; determining a canonical form of a username portion of the callee address; applying the canonical form of the hostname and username as an index to a user database to retrieve a callee database record; and determining routing of the call request based on the retrieved callee database record. 2. The method of processing a call request to a callee as defined by claim 1, wherein the hostname mapping operation includes comparing the hostname portion against a range of addresses for an associated domain. 3. The method of processing a call request to a callee as defined by claim 1, wherein the step of determining a canonical form of a username portion of a callee address includes determining whether the username portion corresponds to a known user alias. 4. The method of processing a call request to a callee as defined by claim 1, wherein the step of determining a canonical form of a username portion of a callee address includes determining whether the username portion corresponds to a known e-mail alias. 5. The method of processing a call request to a callee as defined by claim 1, wherein the step of determining a canonical form of a username portion of a callee address includes determining whether the username portion can be associated with a first name, last name combination stored in a user registration file. 6. The method of processing a call request to a callee as defined by claim 5, wherein the user registration file includes a password file. 7. The method of processing a call request to a callee as defined by claim 1, wherein the step of determining a canonical form of a username portion of a callee address includes determining whether the username portion corresponds to a telephone number recognized in a dial plan translation table. 8. The method of processing a call request to a callee as defined by claim 1, wherein the step of determining a canonical form of a username portion of a callee address includes determining whether the username portion corresponds to one of a known user alias; a known e-mail alias, a first name-last name combination from a user registration table, and a telephone number recognized in a dial plan translation table. 9. The method of processing a call request to a callee as defined by claim 1, wherein the callee database record includes callee contact data and callee preference data for routing incoming call requests. 10. The method of processing a call request to a callee as defined by claim 1, further comprising the step of authenticating a caller prior to routing of the call request. 11. The method of processing a call request to a callee as defined by claim 10, wherein for an unknown caller, the step of authenticating a caller comprises: rejecting the initial call request; transmitting an authentication message to an email address corresponding to a user identity of the caller; and if the authentication message is received by the caller, receiving the authentication message from the caller in a subsequent call request from that caller. 12. A computer based process for a signaling server for routing call requests in a network telephony system comprising: mapping a hostname portion of a callee address to a canonical form of the hostname; determining a canonical form of a username portion of a callee address; applying the canonical form of the hostname and username as an index to a user database to retrieve a callee database record; and determining routing of the call request based on the retrieved callee database record. 13. The computer based process for a signaling server for routing call requests in a network telephony system as defined by claim 12, wherein the step of determining a canonical form of a username portion of a callee address includes evaluating a plurality of database tables to determine whether the username portion corresponds to one of a known user alias; a known e-mail alias, a first name-last name combination from a user registration table, and a telephone number recognized in a dial plan translation table. 14. The computer based process for a signaling server for routing call requests in a network telephony system as defined by claim 12, wherein the hostname mapping operation includes comparing the hostname portion against a range of addresses for an associated domain. 15. The computer based process for a signaling server for routing call requests in a network telephony system as defined by claim 12, wherein the step of determining a canonical form of a username portion of a callee address includes determining whether the username portion corresponds to a known user alias. 16. The computer based process for a signaling server for routing call requests in a network telephony system as defined by claim 12, wherein the step of determining a canonical form of a username portion of a callee address includes determining whether the username portion corresponds to a known e-mail alias. 17. The computer based process for a signaling server for routing call requests in a network telephony system as defined by claim 12, wherein the step of determining a canonical form of a username portion of a callee address includes determining whether the username portion can be associated with a first name, last name combination stored in a user registration file. 18. The computer based process for a signaling server for routing call requests in a network telephony system as defined by claim 17, wherein the user registration file includes a password file. 19. The computer based process for a signaling server for routing call requests in a network telephony system as defined by claim 12, further comprising the step of authenticating a caller prior to routing of the call request. 20. The computer based process for a signaling server for routing call requests in a network telephony system as defined by claim 13, wherein for an unknown caller, the step of authenticating a caller comprises: rejecting the initial call request; transmitting an authentication message to an email address corresponding to a user identity of the caller; and if the authentication message is received by the caller, receiving the authentication message from the caller in a subsequent call request from that caller. 21. A scalable telephony network for routing call requests in an IP telephony network comprising: at least one first stage signaling server, the at least one first stage signaling server receiving call requests from callers; at least two second stage signaling servers, each of the at least two second stage signaling servers maintaining a database of a subset of users of the network based on a predetermined property of the user identity, the at least two second stage signaling servers being provided with a portion of the call requests from the at least one first stage signaling server in accordance with the predetermined property of the user identity of the callee.
<SOH> BACKGROUND OF THE INVENTION <EOH>The Internet has evolved into an essential communication tool for millions of users in the business, technical and educational fields. In this regard, a growing use of the Internet relates to Internet protocol (IP) telephony which provides a number of advantages over conventional circuit-switched network telephony systems that are controlled by a separate signaling network. The session initiation protocol (SIP) is gaining in popularity as a standard signaling protocol for use in Internet telephony. As this popularity grows, it will be increasingly desirable to provide a system architecture and method for providing improved services in SIP based systems. Among these services are improved call routing within a network. Such improved call routing functionality can serve to replace or otherwise reduce the system reliance on traditional multi-line Public Branch Exchange (PBX) systems. In addition to call routing functionality, the conventional PBX will generally also provide a measure of security within a telephony system, such as controlling access to the toll lines to authorized users. Therefore it is desirable to provide a network architecture and operating methods which allow a SIP based telephony network to provide security controls on outgoing calls to the toll lines. There is a potentially limitless range of SIP user identifiers that can be used. It is also desirable for such a system to provide authentication of users for incoming calls as an additional security feature.
<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to provide improved systems and methods for call routing in a SIP compliant telephony system. A method of processing a call request to a callee in a network telephony system in accordance with the present invention includes mapping a hostname portion of a callee address to a canonical form of the hostname; determining a canonical form of a username portion of the callee address; applying the canonical form of the hostname and username as an index to a user database to retrieve a callee database record; and determining routing of the call request based on the retrieved callee database record. Preferably, the hostname mapping operation is performed by comparing the hostname portion of the callee's address to a range of addresses for the associated hostname. The operation of determining the canonical form of the username portion preferably includes determining whether the username corresponds to a known alias, a known e-mail address or can be resolved using a name mapping operation. Such operations can be used individually or in combination. In addition, a dial plan translation operation can be used to determine call routing for addresses which are in the form of a telephone number. Preferably, the method is a computer based method which is performed in a signaling server of a telephony network. In addition to call routing, the present methods can also include caller authentication. Such caller authentication can include the steps of rejecting an initial call request from an unknown caller. An email message is then sent to the address corresponding to the identity of the caller which includes an authentication message. The caller then reinitiates a call request using the received authentication message to verify the caller's identity. The email message can also include a link to facilitate the subsequent call request. A scalable telephony network for routing call requests in an IP telephony network is also provided. The scalable telephony network includes at least one first stage signaling server which receives call requests from callers. At least two second stage signaling servers are provided, each of which maintain a database of a subset of users of the network based on a predetermined property of the user identity. The second stage signaling servers are provided with a portion of the call requests from the at least one first stage signaling server in accordance with the predetermined property of the user identity of the callee. For example, calls can be routed by the first stage servers to the particular second stage servers based on the hash of the callee's identifier.
Method for correcting data of several opto-electronic sensors
The invention relates to a method for providing corrected data in order to generate a model of a monitoring area which is located in the respective visual range of at least two opto-electronic sensors for determining the position of detected objects, based on the amounts of raw data elements corresponding to object points in said monitoring area, said amounts of raw data elements being respectively detected by one of the sensors when the visual range is sensed and associated therewith, and the amounts of raw data elements from various sensors which are temporally linked to each other forming a group. The raw data elements corresponding to said amounts respectively comprise at least the coordinates of object points detected by the associated sensor in relation to the position of the detecting sensor. In order to form corrected amounts of corrected data elements corresponding to the amounts of raw data elements of a group for each group quantity, the coordinates contained in the raw data elements are transformed into a common coordinate system, taking into account the relative position of the respective sensors.
1-20. (Cancelled) 21. A method for the provision of corrected data for the production of a model of a monitored zone (26), which lies in respective fields of view (22, 24) of at least two optoelectronic sensors (10, 12), in particular laser scanners, for the determination of the position of detected objects (28) and contains object points, the corrected data being provided on the basis of sets of raw data elements corresponding to object points in the monitored zone, wherein the sets of raw data elements are respectively detected by one of the sensors (10, 12) by scanning its field of view (22, 24) and are associated with it; wherein the sets of raw data elements of different sensors (10, 12) with a specific time association relative to one another form a group; and wherein the raw data elements of the sets include at least the coordinates of object points detected by the associated sensor (10, 12) in each case with respect to the position of the detecting sensor (10, 12), wherein, for the formation of corrected sets of corrected data elements corresponding to the sets of raw data elements of the group, the coordinates contained in the raw data elements are transformed for each set of the group into a common coordinate system, while taking account of the relative position of the respective sensors (10, 12). 22. A method in accordance with claim 21, characterized in that, on the formation of the corrected sets, the coordinates of the raw data elements are corrected for each set, in each case in accordance with the actual movement of the sensor (10, 12) detecting it, or with a movement approximated thereto, and in accordance with the difference between a time defined as the point in time of the detection of the respective raw data elements and a reference point in time. 23. A method in accordance with claim 22, characterized in that a time defined as the point in time of the detection of the set is used as the point in time of the detection of the raw data elements for all raw data elements of a set. 24. A method in accordance with claim 22, characterized in that, on the fixing of the time defined as the point in time of the detection of a raw data element, the time interval of the detection of the individual raw data elements of the set with respect to one another are taken into account. 25. A method in accordance with claim 21, characterized in that, for each set of raw data elements of the group, an object recognition and/or object tracking is carried out on the basis of the raw data elements of the set before the formation of the corresponding corrected set, with raw data elements being associated with each recognized object and movement data calculated in the object tracking being associated with each of these raw data elements; and in that, in the formation of the corrected sets, results of the object detection and/or object tracking on the basis of the individual sets are used for the correction. 26. A method in accordance with claim 25, characterized in that, in the formation of the corrected sets, the coordinates of the raw data elements are corrected for each set in accordance with the movement data associated with them and in accordance with the difference between a time defined as the point in time of the detection of the raw data elements and a reference point in time of the group or set. 27. A method in accordance with claim 22, characterized in that the reference point in time lies between the earliest time (defined as the detection time) of a raw data element of one of the sets of the group and the timewise last time (defined as the detection time) of a raw data element of one of the sets of the group. 28. A method in accordance with claim 25, characterized in that, before or on the formation of the sets of corrected data elements, virtual object groups are formed which are associated in each case with an object (28) and which have virtual objects which were prepared on the basis in each case of one set of raw data and are recognized as corresponding to an object (28); in that, for each virtual object group associated with an object (28), the associated raw data elements in each set are sought; in that, for each virtual object group associated with an object (28), a reference point in time associated with it is fixed; and in that, on the formation of the sets of corrected data elements, for each virtual object the coordinates of the raw data elements associated with it in all sets of the group are corrected in accordance with the movement of the respective sensor (10, 12) detecting these raw data elements and/or in accordance with the movement data associated with these raw data elements and in accordance with the difference between a time defined as the point in time of the detection of the raw data element and the reference point in time for the virtual object. 29. A method in accordance with claim 28, characterized in that the reference points in time respectively associated in each case with the virtual object groups lie between the earliest time (defined as the point in time of the detection) of a raw data element associated with a virtual object of the respective virtual object group and the timewise last time (defined as the point in time of the detection) of a raw data element associated with a virtual object of the respective virtual object group. 30. A method in accordance with claim 25, characterized in that the object recognition and/or object tracking is/are carried out on the basis of the raw data elements of a set in an evaluation unit of the sensor (10, 12) detecting the raw data of the set and the other steps are carried out in another data processing device (20). 31. A method in accordance with claim 28, characterized in that the corrected data elements for an object group also contain the reference point in time associated with this object group. 32. A method in accordance with claim 21, characterized in that it is carried out iteratively for respective sequential scans of the sensors (10, 12), wherein, after reading in of at least one new set of raw data elements which are associated with one of the sensors and which were detected in a later scan than the raw data elements of the set of raw data elements previously contained in the group of sets of raw data elements and associated with the sensor, a new group is formed which contains the sets of raw data elements of the previous group associated with other sensors and contains the new set of raw data elements; and in that is it subsequently also carried out for the new group of sets of raw data elements, with data already obtained in the preceding iteration preferably being used. 33. A method in accordance with claim 21, characterized in that the model is only produced for a monitored zone (26) in which the fields of view (22, 24) of the sensors (10, 12) overlap. 34. A method for the production of a model of a monitored zone (26) which lies in respective fields of view (22, 24) of at least two optoelectronic sensors (10, 12), in particular laser scanners, for the determination of the position of detected objects (28) and contains object points, in which the method for the provision of corrected data is carried out in accordance with claim 21; and an object recognition and/or object tracking takes place on the basis of all corrected data elements. 35. A method in accordance with claim 34, characterized in that an object recognition and/or object tracking is/are carried out on the basis of the corrected data elements separately for each virtual object group, with the associated reference points in time preferably being taken into account. 36. A method in accordance with claim 21, characterized in that it is completely carried out in an evaluation device of one or more sensors (10, 12). 37. A computer program with program code means to carry out claim 21, when the program is carried out on a computer. 38. A computer program product with program code means which are stored on a machine-legible data carrier to carry out the method in accordance with claim 21, when the computer program product is carried out on a computer. 39. An apparatus for the production of a model of a monitored zone (26) comprising at least two optoelectronic sensors (10, 12), in particular laser scanners, whose fields of view (22, 24) include the monitored zone; a data processing device (20) designed to carry out the method in accordance with claim 21; and data links (16, 18) for the transfer of the raw data elements detected by the sensors (10) to the data processing device (20). 40. An apparatus for the production of a model of a monitored zone (26) comprising at least two optoelectronic sensors (10, 12), in particular laser scanners, whose fields of view (22, 24) include the monitored zone (26) and which each have an evaluation device for the object recognition and/or object tracking and for the output of corresponding virtual object data, a data processing device (20) designed to carry out the method in accordance with claim 21, and data links (16, 18) for the transmission of the raw data elements detected by the sensors (10, 12) and virtual object data to the data processing device (20).
Spool for a waste storage device
A spool (10) for storing film for a waste storage device includes a core (12) and a flared funnel portion (14). Film is stored on the core (12) and can be retained in place by shrink-wrapping (34) as a result of which the core can be formed without an outer wall or base, reducing material and moulding costs.
1. A spool for storing tubular film comprising a flared end and an opposing loading end arranged to receive the film loaded in a direction towards the flared end. 2. A spool as claimed in claim 1 in which the flared end includes a funnel. 3. A spool as claimed in claim 1 in which the flared end includes cut-out portions. 4. A spool as claimed in claim 1 further comprising tubular film loaded thereon. 5. A spool as claimed in claim 4 further comprising a shrink-wrapping around the spool and loaded film. 6. A spool as claimed in claim 1 further including a spool housing comprising an outer wall arranged to receive the spool and a base arranged to support the spool. 7. A waste storage device comprising a waste storage opening, a waste storage space and a spool therebetween and arranged to dispense film for storage of waste, the spool including a flared end and a loading end. 8. A method of loading tubular film onto a spool having a flared end and a loading end comprising the steps of mounting the spool with the loading end disposed towards a film dispenser, and loading the film from the film dispenser onto the spool. 9. A method as claimed in claim 8 further comprising the step of compressing the loaded film onto the spool. 10. A method as claimed in claim 8 further comprising the step of loading a compression ring against the loaded film. 11. A method as claimed in claim 8 to further comprising the step of shrink-wrapping the loaded film and spool.
Methods for cloning mammals using remodeling factors
Methods and compositions are provided for remodeling nuclear donor material used in nuclear transfer procedures. By exposing donor chromatin to one or more exogenous remodeling factors, the limited ability of mammalian oocytes to remodel the chromatin of differentiated cells, including fetal and live-born somatic cells, can be increased, resulting in dramatically improved cloning efficiencies.
1. A method for preparing a mammalian embryo by nuclear transfer, comprising: (a) transferring a mammalian cell, or the nucleus thereof, into an enucleated mammalian NT oocyte; (b) introducing into the mammalian NT oocyte one or more remodeling factors prior to, subsequent to, or simultaneous with said transferring step (a); and (c) activating said mammalian NT oocyte to provide said embryo. 2. A method for cloning a mammal by nuclear transfer, comprising: (a) preparing an embryo by the method of claim 1; and (b) transferring the embryo or a re-cloned embryo thereof into the uterus of a host mammal so as to produce a fetus that undergoes full development and parturition. 3. The method of claim 1 or 2, wherein the remodeling factors are obtained from cells selected from the group consisting of Xenopus oocytes, Xenopus eggs, and activated Xenopus eggs. 4. The method of claim 1 or 2, wherein the mammalian NT oocyte is a bovine egg, and the mammalian cell is a bovine cell. 5. The method of claim 1 or 2, wherein the mammalian NT oocyte is a porcine egg, and the mammalian cell is a porcine cell. 6. The method of claim 1 or 2, wherein the mammalian NT oocyte is an ovine egg, and the mammalian cell is an ovine cell. 7. The method of claim 1, wherein the step of introducing said one or more remodeling factors into the mammalian NT oocyte occurs subsequent to said transferring step (a). 8. The method of claim 1, wherein said transferring step (a) comprises fusing the mammalian cell and the egg. 9. The method of claim 1 or 2, wherein the one or more remodeling factors are introduced into the egg by microinjection. 10. The method of claim 1 or 2, wherein one of said one or more remodeling factor(s) is nucleoplasmin. 11. The method of claim 1 or 2, wherein one of said one or more remodeling factor(s) is a cyclin A-dependent kinase. 12. The method of claim 1 or 2, wherein said one or more remodeling factor(s) comprise cyclin A-dependent kinase and nucleoplasmin. 13. The method of claim 1 or 2, wherein the mammalian cell is selected from the group consisting of: an embryonic cell, a fetal cell, a fetal fibroblast cell, an adult cell, a somatic cell, a primordial germ cell, a genital ridge cell, a fibroblast cell, a cumulus cell, an amniotic cell, an embryonic germ cell, an embryonic stem cell, an ovarian follicular cell, a hepatic cell, an epidermal cell, an epithelial cell, a hematopoietic cell, keratinocyte, a renal cell, a lymphocyte, a melanocyte, a muscle cell, a myeloid cell, a neuronal cell, an osteoblast, a mysenchymal cell, a mesodermal cell, an adherent cell, a cell isolated from an asynchronous population of cells, a cell isolated from a synchronous population of cells where the synchronous population is not arrested in the G0 stage of the cell cycle, a cell isolated from a confluent culture, a transgenic embryonic cell, a transgenic fetal cell, a transgenic adult cell, a transgenic somatic cell, a transgenic primordial germ cell, a transgenic fibroblast cell, a transgenic cumulus cell, or a transgenic amniotic cell. 14. A method for preparing a mammalian embryo by nuclear transfer, comprising: (a) transferring a mammalian cell, or the nucleus thereof, into an enucleated mammalian NT oocyte; (b) introducing into the mammalian NT oocyte a cytoplasmic extract obtained from one or more cells selected from the group consisting of Xenopus oocytes, Xenopus eggs, and activated Xenopus eggs, prior to, subsequent to, or simultaneous with said transferring step (a); and (c) activating said mammalian NT oocyte to provide said embryo. 15. A method for cloning a mammal, comprising: (a) preparing an embryo by the method of claim 14; and (b) transferring the embryo or a re-cloned embryo thereof into the uterus of a host mammal so as to produce a fetus that undergoes full development and parturition. 16. The method of claim 14 or 15, wherein the mammalian NT oocyte is a bovine egg, and the mammalian cell is a bovine cell. 17. The method of claim 14 or 15, wherein the mammalian NT oocyte is a porcine egg, and the mammalian cell is a porcine cell. 18. The method of claim 14 or 15, wherein the mammalian NT oocyte is an ovine egg, and the mammalian cell is an ovine cell. 19. The method of claim 14 or 15, wherein the mammalian cell is selected from the group consisting of: an embryonic cell, a fetal cell, a fetal fibroblast cell, an adult cell, a somatic cell, a primordial germ cell, a genital ridge cell, a fibroblast cell, a cumulus cell, an amniotic cell, an embryonic germ cell, an embryonic stem cell, an ovarian follicular cell, a hepatic cell, an epidermal cell, an epithelial cell, a hematopoietic cell, keratinocyte, a renal cell, a lymphocyte, a melanocyte, a muscle cell, a myeloid cell, a neuronal cell, an osteoblast, a mysenchymal cell, a mesodermal cell, an adherent cell, a cell isolated from an asynchronous population of cells, a cell isolated from a synchronous population of cells where the synchronous population is not arrested in the G0 stage of the cell cycle, a cell isolated from a confluent culture, a transgenic embryonic cell, a transgenic fetal cell, a transgenic adult cell, a transgenic somatic cell, a transgenic primordial germ cell, a transgenic fibroblast cell, a transgenic cumulus cell, or a transgenic amniotic cell. 20. A method for preparing a mammalian embryo by nuclear transfer, comprising: (a) contacting a mammalian cell, or a nucleus thereof, with one or more remodeling factors; (b) transferring the mammalian cell, or the nucleus thereof, into an enucleated mammalian NT oocyte; and (c) activating said egg to provide said embryo. 21. A method for cloning a mammal by nuclear transfer, comprising: (a) preparing an embryo by the method of claim 20; and (b) transferring the embryo or a re-cloned embryo thereof into the uterus of a host mammal so as to produce a fetus that undergoes full development and parturition. 22. The method of claim 20 or 21, wherein the remodeling factors are obtained from cells selected from the group consisting of Xenopus oocytes, Xenopus eggs, and activated Xenopus eggs. 23. The method of claim 20 or 21, wherein the plasma membrane of the mammalian cell is permeabilized. 24. The method of claim 20 or 21, wherein the nuclear membrane of the mammalian cell nucleus is permeabilized. 25. The method of claim 23, wherein the plasma membrane of the mammalian cell is permeabilized by exposure to streptolysin-O and/or digitonin prior to contacting the mammalian cell with one or more remodeling factors. 26. The method of claim 20 or 21, wherein the remodeling factors are nucleoplasmin and/or protein kinases. 27. The method of claim 26 wherein the protein kinase is Cdc2, Cdk2, or a combination thereof. 28. The method of claim 20 or 21, wherein the mammalian NT oocyte is a bovine egg, and the mammalian cell is a bovine cell. 29. The method of claim 20 or 21, wherein the mammalian NT oocyte is a porcine egg, and the mammalian cell is a porcine cell. 30. The method of claim 20 or 21, wherein the mammalian NT oocyte is an ovine egg, and the mammalian cell is an ovine cell. 31. The method of claim 20 or 21, wherein the mammalian cell is selected from the group consisting: an embryonic cell, a fetal cell, a fetal fibroblast cell, an adult cell, a somatic cell, a primordial germ cell, a genital ridge cell, a fibroblast cell, a cumulus cell, an amniotic cell, an embryonic germ cell, an embryonic stem cell, an ovarian follicular cell, a hepatic cell, an epidermal cell, an epithelial cell, a hematopoietic cell, keratinocyte, a renal cell, a lymphocyte, a melanocyte, a muscle cell, a myeloid cell, a neuronal cell, an osteoblast, a mysenchymal cell, a mesodermal cell, an adherent cell, a cell isolated from an asynchronous population of cells, a cell isolated from a synchronous population of cells where the synchronous population is not arrested in the G0 stage of the cell cycle, a transgenic embryonic cell, a transgenic fetal cell, a transgenic adult cell, a transgenic somatic cell, a transgenic primordial germ cell, a transgenic fibroblast cell, a transgenic cumulus cell, or a transgenic amniotic cell. 32. A method for preparing a mammalian embryo by nuclear transfer, comprising: (a) contacting a mammalian cell, or a nucleus thereof, with a cytoplasmic extract obtained from one or more cells selected from the group consisting of Xenopus oocytes, Xenopus eggs, and activated Xenopus eggs; (b) transferring the mammalian cell, or the nucleus thereof, into an enucleated mammalian NT oocyte; and (c) activating said mammalian NT oocyte to provide said embryo. 33. A method for cloning a mammal, comprising: (a) preparing an embryo by the method of claim 32; and (b) transferring the embryo or a re-cloned embryo thereof into the uterus of a host mammal so as to produce a fetus that undergoes full development and parturition. 34. The method of claim 32 or 33, wherein the plasma membrane of the mammalian cell is permeabilized by exposure to streptolysin-O and/or digitonin. 35. The method of claim 32 or 33, wherein the nuclear membrane of the mammalian cell nucleus is permeabilized. 36. The method of claim 35, wherein the nuclear membrane of the mammalian cell nucleus is permeabilized by homogenization. 37. The method of claim 32 or 33, wherein the mammalian cell is selected from the group consisting of: an embryonic cell, a fetal cell, a fetal fibroblast cell, an adult cell, a somatic cell, a primordial germ cell, a genital ridge cell, a fibroblast cell, a cumulus cell, an amniotic cell, an embryonic germ cell, an embryonic stem cell, an ovarian follicular cell, a hepatic cell, an epidermal cell, an epithelial cell, a hematopoietic cell, keratinocyte, a renal cell, a lymphocyte, a melanocyte, a muscle cell, a myeloid cell, a neuronal cell, an osteoblast, a mysenchymal cell, a mesodermal cell, an adherent cell, a cell isolated from an asynchronous population of cells, a cell isolated from a synchronous population of cells where the synchronous population is not arrested in the G0 stage of the cell cycle, a transgenic embryonic cell, a transgenic fetal cell, a transgenic adult cell, a transgenic somatic cell, a transgenic primordial germ cell, a transgenic fibroblast cell, a transgenic cumulus cell, or a transgenic amniotic cell.
<SOH> BACKGROUND OF THE INVENTION <EOH>The following discussion of the background of the invention is provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention. Over the past two decades, researchers have been developing methods for cloning mammalian animals, with notable recent success. The reported methods typically include the steps of (1) isolating a cell, often an embryonic cell, but more recently fetal and adult cells as well; (2) inserting the cell or nucleus isolated from the cell into an enucleated recipient cell (e.g., an NT oocyte as defined herein, the nucleus of which was previously extracted), (3) activating the oocyte, and (4) allowing the embryo to mature in vivo. See, e.g., U.S. Pat. No. 4,664,097, “Nuclear Transplantation in the Mammalian Embryo by Microsurgery and Cell Fusion,” issued May 12, 1987, McGrath & Solter; U.S. Pat. No. 4,994,384 (Prather et al.); U.S. Pat. No. 5,057,420 Massey et al.); U.S. Pat. No. 6,107,543; U.S. Pat. No. 6,011,197; Proc. Nat'l. Acad. Sci. USA 96: 14984-14989 (1999); Nature Genetics 22: 127-128 (1999); Cell & Dev. Biol. 10: 253-258 (1999); Nature Biotechnology 17: 456-461 (1999); Science 289: 1188-1190 (2000); Nature Biotechnol. 18: 1055-1059 (2000); and Nature 407: 86-90 (2000); each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings. Successful development of any cloned embryo is believed to involve the “reprogramming” of the somatic nucleus by the egg cytoplasm. Reprogramming involves reversing the genetic programming of the differentiated somatic cell to create a totipotent nucleus. Chromatin structure partly determines the cell's epigenetic memory, which regulates the pattern of gene expression in its descendants. Thus, the regulated change in structure or “remodeling” of somatic chromatin by the egg may reverse this pattern of expression, and as such, facilitate development. Successful cloning demonstrates that the unfertilized egg has the potential to direct the complete reprogramming of the somatic nucleus. However, the relative inefficiency of the process also suggests that important activities can be limiting in nuclear transfer events (Gurdon and Colman, Nature, 402(6763): p. 743-6, 1999). While specific features of donor nuclei certainly contribute to this inefficiency, in theory, virtually all somatic nuclei may have the potential to become totipotent for development if they are correctly and completely reprogrammed. Thus, it may be that it is primarily the limited reprogramming capacity of the egg that is responsible for most cloning failures. This limitation is undoubtedly due to a number of factors, as the egg has evolved to program a sperm nucleus at fertilization and not to reprogram a somatic nucleus following nuclear transfer. However, while many factors may be necessary for complete reprogramming, it is possible that reprogramming can be achieved with only a few factors. See, e.g., Kikyo and Wolffe, J. Cell Sci. 113: 11-20, 2000. If so, then supplementing the egg with these critical factors, or treating somatic nuclei with these factors prior to nuclear transfer, may result in improved development and increased cloning success. Thus, despite the recent progress in cloning mammalian animals, there remains a great need in the art for methods and materials that increase cloning efficiencies.
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides methods for cloning mammals by nuclear transfer. As described herein, exposing an oocyte and/or a somatic cell or nucleus to remodeling factors prior to their use in nuclear transfer procedures can increase the efficiencies of cellular reprogramming. Moreover, by careful selection of such remodeling factors, it can be possible to achieve these increased efficiencies utilizing only one or a small number of remodeling factors. Preferred remodeling factors include, but are not limited to, nucleoplasmin, cyclin A-dependent kinase(s), protein kinases, or a combination of these. The present invention therefore provides, in a first aspect, methods and compositions for preparing a mammalian embryo by nuclear transfer. The methods may comprise transferring a mammalian cell, or the nucleus thereof, into an enucleated mammalian oocyte, introducing into the mammalian oocyte one or more remodeling factors prior to, subsequent to, or simultaneous with the transferring step, and activating the mammalian oocyte to provide an embryo. For purposes of clarity, the mammalian oocyte that is to receive or has received the nuclear donor cell or nucleus is referred to hereinafter as an “NT oocyte.” This is to distinguish such oocytes from those that are used as the source of reprogramming factors and extracts. This designation is purely for convenience, and does not denote that the NT oocyte has received a donor cell or nucleus at the time to which it is referred. In certain embodiments, the methods may comprise preparing an embryo by the methods of the present invention, and transferring the embryo, or a re-cloned embryo thereof, into the uterus of a host mammal so as to produce a fetus that undergoes full development and parturition. “Re-cloning” is described hereinafter. The term “mammalian” as used herein refers to any animal of the class Mammalia. Preferably, a mammal is a placental, a monotreme and a marsupial. Most preferably, a mammal is a canid, felid, murid, leporid, ursid, mustelid, ungulate, ovid, suid, equid, bovid, caprid, cervid, and a human or non-human primate. These terms are defined hereinafter. In preferred embodiments, the mammal may be a bovine, the mammalian NT oocyte may be a bovine oocyte, and/or the mammalian cell may be a bovine cell; the mammal may be a porcine, the mammalian NT oocyte may be a porcine oocyte, and/or the mammalian cells may be porcine cells; and the mammal may be an ovine, the mammalian NT oocyte may be an ovine oocyte, and/or the mammalian cells may be ovine cells. The one or more remodeling factors may be obtained from cells, such as oocytes and eggs, at any stage of maturation and/or development. Thus, the remodeling factors of the instant invention may be obtained before and/or after activation of the source cells. In addition, remodeling factors may also be obtained from cells from multiple maturation and/or developmental stages and pooled. While any species may serve as the source of these remodeling factors, amphibian oocytes and eggs, and particularly Xenopus oocytes, Xenopus eggs, and activated Xenopus eggs, are a particularly rich source of these remodeling factors. The term “oocyte” as used herein with reference to amphibian cells refers to a female germ cell arrested in G2/prophase of meiosis I. The term “egg” as used herein with reference to amphibian cells refers to a c0 female germ cell arrested in metaphase of meiosis II. The term “activated egg” as used herein with reference to amphibian cells refers to a female germ cell that is beyond the “egg” stage due to release from metaphase arrest and progression into interphase. Each of the previous three definitions are known to the skilled artisan. See, e.g., Leno, Methods in Cell Biology 53: 497-515, 1998. Extracts of such cells may be used without fractionation, as these extracts contain the remodeling factors; but in certain embodiments the remodeling factors may be purified factors such as nucleoplasmin, cyclin A-dependent kinase, ATP-dependent chromatin remodeling complexes, or a combination thereof. Remodeling factors may also be obtained by recombinant methods. For example, insect cells may be transformed to produce Xenopus nucleoplasmin, which may be used in the methods described herein. Similarly, mRNA obtained from, for example, Xenopus cells may be translated in vitro to produce Xenopus remodeling factors. Purification in this context does not indicate absolute purity; only that the relative amount of a preferred compound has been enriched. The term “remodeling factor” as used herein refers to any substance that alters the structure and/or composition of chromatin, known as “chromatin restructuring.” Remodeling factors include, but are not limited to, ATP-dependent remodeling factors (e.g., SWI/SNF, ISWI, and ISWI homologs from yeast and Xenopus ; see, e.g., Genes & Development 15: 619-26, 2001; and cyclin-dependent kinases; see, e.g., Hua et al., J. Cell. Biol. 137: 183-192, 1997; Findeisen et al., Eur. J. Biochem. 264: 415-26, 1999); non-ATP-dependent remodeling factors (e.g., nucleoplasmin and polyanionic molecules such as polyglutamic acid (Philpot and Leno, Cell 69: 759-67, 1992; Dean, Dev. Biol. 99: 210-216, 1983); and chromatin components that can replace their counterparts that are preexisting in chromatin (e.g., histone H1 oo or H1 embryonic may replace histone H1 somatic ). Whole cell extracts, whether unpurified or purified, that precipitate chromatin restructuring can also be referred to as remodeling factors. Preferably, the one or more remodeling factors may be introduced into a cell, such as an NT oocyte or a nuclear donor cell, by microinjection (for example using a piezo drill), by delivery in liposomes (e.g., BioPORTER, Gene Therapy Systems, San Diego, Calif.), by transient permeabilization of the recipient cell (e.g. by streptolysin 0 or digitonin treatment), by electroporation, or by any other methods for introducing materials into cells that are known to the artisan. The skilled artisan will recognize that the use of an NT oocyte in these procedures provides a reservoir for exposing nuclear donor material to levels of remodeling factors sufficient to cause successful remodeling of the donor chromatin. Thus, any small chamber may be used as a replacement for the NT oocyte. For example, an enucleated cell of any type (e.g., an enucleated zygote, an enucleated blastomere, etc.) may receive the nuclear donor material and remodeling factor(s). Alternatively, any chamber of approximately the size of a cell (a liposome, chromatin encapsulated by an artificial membrane, etc.) may also be used as a reprogramming chamber. Thus, while the specification discusses the use of NT oocytes, other such reprogramming chambers are within the scope of the invention. In particular, any cultured cell may be considered an appropriate reprogramming chamber; that is, the nucleus may be exposed within the cell to reprogramming factors, and then that cell itself may be treated as one would a nuclear transfer-derived embryo (e.g., to transfer to a recipient animal for development into a fetus or live-born animal, or as a source of cultured cells such as stem cells or stem cell-like cells). Remodeling factor(s) can be introduced into the nuclear transfer procedure at various points. For example, remodeling factor(s) may be introduced into an NT oocyte prior to, subsequent to, or simultaneously with the transfer of nuclear donor material into the NT oocyte. Similarly, remodeling factors can be introduced into an NT oocyte before or after enucleation of the NT oocyte, before, during, or after maturation of an NT oocyte, or before, during, or after activation of the NT oocyte. In preferred embodiments, remodeling factors are introduced between 20 hours before activation and the time of activation, more preferably between 10 hours before activation and the time of activation. Remodeling factor(s) can also be introduced into the nuclear transfer procedure following the generation of a nuclear transfer-derived embryo. For example, remodeling factor(s) may be introduced into a developing embryo in culture. The mammalian cell used as a source of nuclear donor material may be any mammalian cell, but is preferably an embryonic cell, a fetal cell, a fetal fibroblast cell, an adult cell, a somatic cell, a primordial germ cell, a genital ridge cell, a fibroblast cell, a cumulus cell, an amniotic cell, an embryonic germ cell, an embryonic stem cell, an ovarian follicular cell, a hepatic cell, an epidermal cell, an epithelial cell, a hematopoietic cell, keratinocyte, a renal cell, a lymphocyte, a melanocyte, a muscle cell, a myeloid cell, a neuronal cell, an osteoblast, a mysenchymal cell, a mesodermal cell, an adherent cell, a cell isolated from an asynchronous population of cells, a cell isolated from a synchronous population of cells where the synchronous population is not arrested in the G0 stage of the cell cycle, a transgenic embryonic cell, a transgenic fetal cell, a transgenic adult cell, a transgenic somatic cell, a transgenic primordial germ cell, a transgenic fibroblast cell, a transgenic cumulus cell or a transgenic amniotic cell. In particularly preferred embodiments, a nuclear donor cell is a transgenic cell. The term “transgenic” as used herein in reference to cells refers to a cell whose genome has been altered using recombinant DNA techniques. In preferred embodiments, a transgenic cell comprises one or more exogenous DNA sequences in its genome. In other preferred embodiments, a transgenic cell comprises a genome in which one or more endogenous genes have been deleted, duplicated, activated, or modified. In particularly preferred embodiments, a transgenic cell comprises a genome having both one or more exogenous DNA sequences, and one or more endogenous genes that have been deleted, duplicated, activated, or modified. In another aspect, the methods of the present invention for preparing a mammalian embryo by nuclear transfer may comprise transferring a mammalian cell, or the nucleus thereof, into an enucleated mammalian NT oocyte, introducing into the mammalian NT oocyte a cytoplasmic extract obtained from one or more cells, preferably amphibian cells (e.g., Xenopus oocytes, Xenopus eggs, and activated Xenopus eggs), prior to, subsequent to, or simultaneous with the transferring step, and activating the mammalian NT oocyte to provide the embryo. In certain embodiments, the methods may comprise preparing an embryo according to the present invention, and transferring the embryo or a re-cloned embryo thereof into the uterus of a host mammal so as to produce a fetus that undergoes full development and parturition. In yet another aspect, the present invention provides methods for preparing a mammalian embryo by nuclear transfer comprising contacting a mammalian cell, or a nucleus thereof, with one or more remodeling factors, transferring the mammalian cell, or the nucleus thereof, into an enucleated mammalian egg, and activating the egg to provide the embryo. In various embodiments, the plasma membrane of the mammalian cell may be permeabilized and/or the nuclear membrane of the mammalian cell nucleus may be permeabilized by methods known to the skilled artisan, in order to permit the remodeling factor(s) to access the interior of the cell and/or nucleus. For example, in preferred embodiments, the plasma membrane of the mammalian cell may be permeabilized by exposure to streptolysin-O and/or digitonin prior to contacting the mammalian cell with the remodeling factors, and/or the nuclear membrane of the mammalian cell nucleus may be permeabilized by homogenization. In addition to methods in which remodeling factors are introduced into mammalian cell nuclei by permeabilization of the nuclear membrane, in certain embodiments remodeling factors may also be introduced into a mammalian cell nucleus through the use of nuclear localization signals, or by using remodeling factors that are sufficiently small to diffuse through the nuclear pore complexes present in the nuclear membrane. In another aspect the present invention provides methods for preparing a mammalian embryo by nuclear transfer comprising contacting a mammalian cell, or a nucleus thereof, with a cytoplasmic extract obtained from one or more cells such as Xenopus oocytes, Xenopus eggs, and activated Xenopus eggs, transferring the mammalian cell, or the nucleus thereof, into an enucleated mammalian NT oocyte, and activating the mammalian NT oocyte to provide the embryo. The term “nuclear transfer” as used herein refers to introducing a full complement of nuclear DNA from one cell to an enucleated cell. Nuclear transfer methods are well known to a person of ordinary skill in the art. See, e.g., U.S. Pat. No. 4,664,097, “Nuclear Transplantation in the Mammalian Embryo by Microsurgery and Cell Fusion,” issued May 12, 1987, McGrath & Solter; U.S. Pat. No. 4,994,384 (Prather et al.); U.S. Pat. No. 5,057,420 (Massey et al.); U.S. Pat. No. 6,107,543; U.S. Pat. No. 6,011,197; Proc. Nat'l. Acad. Sci. USA 96: 14984-14989 (1999); Nature Genetics 22: 127-128 (1999); Cell & Dev. Diol 10: 253-258 (1999); Nature Biotechnology 17: 456-461 (1999); Science 289: 1188-1190(2000); Nature Biotechnol. 18: 1055-1059 (2000); and Nature 407: 86-90 (2000); each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings. Exemplary embodiments define a nuclear transfer technique that provide for efficient production of totipotent mammalian embryos. The term “enucleated oocyte” as used herein refers to an oocyte which has had part of its contents removed. As discussed above, such an oocyte is also referred to herein as an “NT oocyte,” to distinguish these oocytes from cells that are the source of remodeling factors. Typically a needle can be placed into an oocyte and the nucleus can be aspirated into the inner space of the needle. The needle can be removed from the oocyte without rupturing the plasma membrane. This enucleation technique is well known to a person of ordinary skill in the art. See, U.S. Pat. No. 4,994,384; U.S. Pat. No. 5,057,420; and Willadsen, 1986 , Nature 320: 63-65. An enucleated oocyte can be prepared from a young or an aged oocyte. Definitions of “young oocyte” and aged oocyte” are provided herein. Nuclear transfer may be accomplished by combining one nuclear donor and more than one enucleated oocyte. In addition, nuclear transfer may be accomplished by combining one nuclear donor, one or more enucleated oocytes, and the cytoplasm of one or more enucleated oocytes. The term “injection” as used herein in reference to nuclear transfer methods, refers to the perforation of the NT oocyte with a needle, an insertion of the nuclear donor in the needle into the NT oocyte. In preferred embodiments, the nuclear donor may be injected into the cytoplasm of the NT oocyte or in the perivitelline space of the NT oocyte. This direct injection approach is well known to a person of ordinary skill in the art, as indicated by the publications already incorporated herein in reference to nuclear transfer. For the direct injection approach to nuclear transfer, the whole totipotent mammalian cell may be injected into the NT oocyte, or alternatively, a nucleus isolated from the totipotent mammalian cell may be injected into the NT oocyte. Such an isolated nucleus may be surrounded by nuclear membrane only, or the isolated nucleus may be surrounded by nuclear membrane and plasma membrane in any proportion. The NT oocyte may be pre-treated to enhance the strength of its plasma membrane, such as by incubating the NT oocyte in sucrose prior to injection of the nuclear donor. For the purposes of the present invention, the term “embryo” or “embryonic” as used herein refers to a developing cell mass that has not implanted into the uterine membrane of a maternal host. Hence, the term “embryo” as used herein can refer to a fertilized oocyte, a cybrid (defined herein), a pre-blastocyst stage developing cell mass, a blastocyst stage embryo, a morula stage embryo, and/or any other developing cell mass that is at a stage of development prior to implantation into the uterine membrane of a maternal host. Embryos of the invention may not display a genital ridge. Hence, an “embryonic cell” is isolated from and/or has arisen from an embryo. The term “fetus” as used herein refers to a developing cell mass that has implanted into the uterine membrane of a maternal host. A fetus can include such defining features as a genital ridge, for example. A genital ridge is a feature easily identified by a person of ordinary skill in the art, and is a recognizable feature in fetuses of most animal species. The term “fetal cell” as used herein can refer to any cell isolated from and/or has arisen from a fetus or derived from a fetus. The term “non-fetal cell” is a cell that is not derived or isolated from a fetus. The term “activation” refers to any materials and methods useful for stimulating a cell to divide before, during, and after a nuclear transfer step. An embryo obtained by a nuclear transfer procedure, that is, a combination of an NT oocyte and a nuclear donor cell or cell nucleus, may require stimulation in order to divide after a nuclear transfer has occurred. The invention pertains to any activation materials and methods known to a person of ordinary skill in the art. Although electrical pulses are sometimes sufficient for stimulating activation of nuclear transfer-derived embryos, other means are sometimes useful or necessary for proper activation. Chemical materials and methods useful for activating embryos are described below in other preferred embodiments of the invention. Examples of non-electrical means for activation include agents such as ethanol; inositol trisphosphate (IP 3 ); Ca ++ ionophores (e.g., ionomycin) and protein kinase inhibitors (e.g., 6-dimethylaminopurine (DMAP)); temperature change; protein synthesis inhibitors (e.g., cyclohexamide); phorbol esters such as phorbol 12-myristate 13-acetate (PMA); mechanical techniques; and thapsigargin. The invention includes any activation techniques known in the art. See, e.g., U.S. Pat. No. 5,496,720 and U.S. Pat. No. 6,011,197, entitled “Parthenogenic Oocyte Activation,” incorporated by reference herein in their entirety, including all figures, tables, and drawings. The term “totipotent” as used herein in reference to embryos refers to embryos that can develop into a live born animal. The term “cloned” as used herein refers to a cell, embryonic cell, fetal cell, and/or animal cell having a nuclear DNA sequence that is substantially similar or identical to the nuclear DNA sequence of another cell, embryonic cell, fetal cell, and/or animal cell. The terms “substantially similar” and “identical” are described herein. The cloned embryo can arise from one nuclear transfer, or alternatively, the cloned embryo can arise from a cloning process that includes at least one re-cloning step. The term “substantially similar” as used herein in reference to nuclear DNA sequences refers to two nuclear DNA sequences that are nearly identical. The two sequences may differ by copy error differences that normally occur during the replication of a nuclear DNA. Substantially similar DNA sequences are preferably greater than 97% identical, more preferably greater than 98% identical, and most preferably greater than 99% identical. The term “identity” is used herein in reference to nuclear DNA sequences can refer to the same usage of the term in reference to amino acid sequences, which is described previously herein. The term “maturation” as used herein refers to process in which an oocyte is incubated in a medium in vitro. Oocytes can be incubated with multiple media well known to a person of ordinary skill in the art. See, e.g., Saito et al., 1992 , Roux's Arch. Dev. Biol. 201: 134-141 for bovine organisms and Wells et al., 1997 , Biol. Repr. 57: 385-393 for ovine organisms, both of which are incorporated herein by reference in their entireties including all figures, tables, and drawings. Maturation media can comprise multiple types of components, including microtubule and/or microfilament inhibitors (e.g., cytochalasin B). Other examples of components that can be incorporated into maturation media are discussed in WO 97/07668, entitled “Unactivated Oocytes as Cytoplast Recipients for Nuclear Transfer,” Campbell & Wilmut, published on Mar. 6, 1997, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings. The time of maturation can be determined from the time that an oocyte is placed in a maturation medium and the time that the oocyte is then utilized in a nuclear transfer procedure. The term “cybrid” as used herein refers to a construction where an entire nuclear donor is translocated into the cytoplasm of a recipient oocyte. See, e.g., In Vitro Cell. Dev. Biol. 26: 97-101 (1990). The term “canid” as used herein refers to any animal of the family Canidae. Preferably, a canid is a wolf, a jackal, a fox, and a domestic dog. The term “felid” as used herein refers to any animal of the family Felidae. Preferably, a felid is a lion, a tiger, a leopard, a cheetah, a cougar, and a domestic cat. The term “murid” as used herein refers to any animal of the family Muridae. Preferably, a murid is a mouse and a rat. The term “leporid” as used herein refers to any animal of the family Leporidae. Preferably, a leporid is a rabbit. The term “ursid” as used herein refers to any animal of the family Ursidae. Preferably, a ursid is a bear. The term “mustelid” as used herein refers to any animal of the family Mustelidae. Preferably, a mustelid is a weasel, a ferret, an otter, a mink, and a skunk. The term “primate” as used herein refers to any animal of the Primate order. Preferably, a primate is an ape, a monkey, a chimpanzee, and a lemur. The term “ungulate” as used herein refers to any animal of the polyphyletic group formerly known as the taxon Ungulata. Preferably, an ungulate is a camel, a hippopotamus, a horse, a tapir, and an elephant. Most preferably, an ungulate is a sheep, a cow, a goat, and a pig. The term “ovid” as used herein refers to any animal of the family Ovidae. Preferably, an ovid is a sheep. The term “suid” as used herein refers to any animal of the family Suidae. Preferably, a suid is a pig or a boar. The term “equid” as used herein refers to any animal of the family Equidae. Preferably, an equid is a zebra or an ass. Most preferably, an equid is a horse. The term “caprid” as used herein refers to any animal of the family Caprinae. Preferably, a caprid is a goat. The term “cervid” as used herein refers to any animal of the family Cervidae. Preferably, a cervid is a deer. The term “bovine” as used herein refers to a family of ruminants belonging to the genus Bos or any closely related genera of the family Bovidae. The family Bovidae includes true antelopes, oxen, sheep, and goats, for example. Preferred bovine animals are the cow and ox. Especially preferred bovine species are Bos taurus, Bos indicus . and Bos buffaloes . Other preferred bovine species are Bos primigenius and Bos longifrons. The term “totipotent” as used herein in reference to cells refers to a cell that gives rise to all of the cells in a developing cell mass, such as an embryo, fetus, and animal. In preferred embodiments, the term “totipotent” also refers to a cell that gives rise to all of the cells in an animal. A totipotent cell can give rise to all of the cells of a developing cell mass when it is utilized in a procedure for creating an embryo from one or more nuclear transfer steps. An animal may be an animal that functions ex utero. An animal can exist, for example, as a live born animal. Totipotent cells may also be used to generate incomplete animals such as those useful for organ harvesting, e.g., having genetic modifications to eliminate growth of a head such as by manipulation of a homeotic gene. The term “totipotent” as used herein is to be distinguished from the term “pluripotent.” The latter term refers to a cell that differentiates into a sub-population of cells within a developing cell mass, but is a cell that may not give rise to all of the cells in that developing cell mass. Thus, the term “pluripotent” can refer to a cell that cannot give rise to all of the cells in a live born animal. The term “totipotent” as used herein is also to be distinguished from the term “chimer” or “chimera.” The latter term refers to a developing cell mass that comprises a sub-group of cells harboring nuclear DNA with a significantly different nucleotide base sequence than the nuclear DNA of other cells in that cell mass. The developing cell mass can, for example, exist as an embryo, fetus, and/or animal. The term “confluence” as used herein refers to a group of cells where a large percentage of the cells are physically contacted with at least one other cell in that group. Confluence may also be defined as a group of cells that grow to a maximum cell density in the conditions provided. For example, if a group of cells can proliferate in a monolayer and they are placed in a culture vessel in a suitable growth medium, they are confluent when the monolayer has spread across a significant surface area of the culture vessel. The surface area covered by the cells preferably represents about 50% of the total surface area, more preferably represents about 70% of the total surface area, and most preferably represents about 90% of the total surface area. Nuclear donor cells can be obtained from confluent cultures. In preferred embodiments, (1) the nuclear donor cell is selected from the group consisting of non-embryonic cell, a non-fetal cell, a differentiated cell, a somatic cell, an embryonic cell, a fetal cell, an embryonic stem cell, a primordial germ cell, a genital ridge cell, an amniotic cell, a fetal fibroblast cell, an ovarian follicular cell, a cumulus cell, an hepatic cell, an endocrine cell, an endothelial cell, an epidermal cell, an epithelial cell, a fibroblast cell, a hematopoletic cell, a keratinocyte, a renal cell, a lymphocyte, a melanocyte, a mussel cell, a myeloid cell, a neuronal cell, an osetoblast, a mesenchymal cell, a mesodermal cell, an adherent cell, a cell isolated from an asynchronous population of cells, and a cell isolated from a synchronized population of cells where the synchronous population is not arrested in the G 0 state of the cell cycle. The term “primordial germ cell” as used herein refers to a diploid somatic cell capable of becoming a germ cell. Primordial germ cells can be isolated from the genital ridge of a developing cell mass. The genital ridge is a section of a developing cell mass that is well-known to a person of ordinary skill in the art. See, e.g., Strelchenko, 1996 , Theriogenology 45: 130-141 and Lavoir 1994 , J. Reprod. Dev. 37: 413-424. The terms “embryonic germ cell” and “EG cell” as used herein refers to a cultured cell that has a distinct flattened morphology and can grow within monolayers in culture. An EG cell may be distinct from a fibroblast cell. This EG cell morphology is to be contrasted with cells that have a spherical morphology and form multicellular clumps on feeder layers. Embryonic germ cells may not require the presence of feeder layers or presence of growth factors in cell culture conditions. Embryonic germ cells may also grow with decreased doubling rates when these cells approach confluence on culture plates. Embryonic germ cells of the invention may be totipotent. Embryonic germ cells may be established from a cell culture of nearly any type of precursor cell. Examples of precursor cells are discussed herein, and a preferred precursor cell for establishing an embryonic germ cell culture is a genital ridge cell from a fetus. Genital ridge cells are preferably isolated from procine fetuses where the fetus is between 20 days and parturition, between 30 days and 100 days, more preferably between 35 days and 70 days and between 40 days and 60 days, and most preferably about a 55 day fetus. An age of a fetus can be determined as described above. The term “about” with respect to fetuses can refer to plus or minus five days. As described herein, EG cells may be physically isolated from a primary culture of cells, and these isolated EG cells may be utilized to establish a cell culture that eventually forms a homogenous or nearly homogenous line of EG cells. The term “embryonic stem cell” as used herein refers to pluripotent cells isolated from an embryo that are maintained in in vitro cell culture. Embryonic stem cells may be cultured with or without feeder cells. Embryonic stem cells can be established from embryonic cells isolated from embryos at any state of development, including blastocyst stage embryos and pre-blastocyst stage embryos. Embryonic stem cells are well known to a person of ordinary skill in the art. See, e.g., WO 97/37009, entitled “Cultured Inner Cell Mass Cell-Lines Derived from Ungulate Embryos,” Stice and Golueke, published Oct. 9, 1997, and Yang & Anderson, 1992 , Theriogenology 38: 315-335, both of which are incorporated herein by reference in their entireties, including all figures, tables, and drawings. The term “ovarian follicular cell” as used herein refers to a cultured or non-cultured cell obtained from an ovarian follicle, other than an oocyte. Follicular cells may be isolated from ovarian follicles at any stage of development, including primordial follicles, primary follicles, secondary follicles, growing follicles, vesicular follicles, maturing follicles, mature follicles, and graafian follicles. Furthermore, follicular cells may be isolated when an oocyte in an ovarian follicle is immature (i.e., an oocyte that has not progressed to metaphase II) or when an oocyte in an ovarian follicle is mature (i.e., an oocyte that has progressed to metaphase II or a later stage of development). Preferred follicular cells include, but are not limited to, pregranulosa cells, granulosa cells, theca cells, columnar cells, stroma cells, theca interna cells, theca externa cells, mural granulosa cells, luteal cells, and corona radiata cells. Particularly preferred follicular cells are cumulus cells. Various types of follicular cells are known and can be readily distinguished by those skilled in the art. See, e.g., Laboratory Production of Cattle Embryos, 1994, Ian Gordon, CAB International; Anatomy and Physiology of Farm Animals (5th ed.), 1992, R. D. Frandson and T. L. Spurgeon, Lea & Febiger, each of which is incorporated herein by reference in its entirety including all figures, drawings, and tables. Individual types of follicular cells may be cultured separately, or a mixture of types may be cultured together. The term “amniotic cell” as used herein refers to any cultured or non-cultured cell isolated from amniotic fluid. Examples of methods for isolating and culturing amniotic cells are discussed in Bellow et al., 1996 , Theriogenology 45: 225; Garcia & Salaheddine, 1997 , Theriogenology 47: 1003-1008; Liebo & Rail. 1990 , Theriogenology 33: 531-552; and Vos et al., 1990 , Vet. Rec. 127: 502-504, each of which is incorporated herein by reference in its entirety, including all figures tables and drawings. Particularly preferred are cultured amniotic cells that do not display a fibroblast-like morphology. The skilled artisan will understand that amniotic cells may be both maternal cells and fetal cells. Thus, preferred amniotic cells also include fetal fibroblast cells. The terms “fibroblast,” fibroblast-like,” “fetal,” and “fetal fibroblast” are defined hereafter. The terms “fibroblast-like” and “fibroblast” as used herein refer to cultured cells that have a distinct flattened morphology and that are able to grow within monolayers in culture. The term “fetal fibroblast cell” as used herein refers to any differentiated fetal cell having a fibroblast appearance. While fibroblasts characteristically have a flattened appearance when cultured on culture media plates, fetal fibroblast cells can also have a spindle-like morphology. Fetal fibroblasts may require density limitation for growth, may generate type I collagen, and may have a finite life span in culture of approximately fifty generations. Preferably, fetal fibroblast cells rigidly maintain a diploid chromosomal content. For a description of fibroblast cells, see, e.g. Culture of Animal Cells: a manual of basic techniques (3 rd edition), 1994, R. I. Freshney (ed), Wiley-Liss, Inc., incorporated herein by reference in its entirety, including all figures, tables, and drawings. The terms “morphology” and “cell morphology” as used herein refer to form, structure, and physical characteristics of cells. For example, one cell morphology is significant levels of alkaline phosphatase, and this cell morphology can be identified by determining whether a cell stains appreciably for alkaline phosphatase. Another example of a cell morphology is whether a cell is flat or round in appearance when cultured on a surface or in the presence of a layer of feeder cells. Many other cell morphologies are known to a person of ordinary skill in the art and are cell morphologies are readily identifiable using materials and methods well known to those skilled in the art. See, e.g., Culture of Animal Cells: a manual of basic techniques (3 rd edition), 1994, R. I. Freshney (ed.). Wiley-Liss, Inc. The term “cumulus cell” as used herein refers to any cultured or non-cultured cell isolated from cells and/or tissue surrounding an oocyte. Persons skilled in the art can readily identify cumulus cells. Examples of methods for isolating and/or culturing cumulus cells are discussed in Damiani et al., 1996 , Mol. Reprod. Dev. 45: 521-534; Long et al., 1994 , J. Reprod. Fert. 102: 361-369; and Wakayama et al., 1998 , Nature 394: 369-373, each of which is incorporated herein by reference in its entireties, including all figures, tables, and drawings. Cumulus cells may be isolated from ovarian follicles at any stage of development, including primordial follicles, primary follicles, secondary follicles, growing follicles, vesicular follicles, maturing follicles, mature follicles, and graafian follicles. Cumulus cells may be isolated from oocytes in a number of manners well known to a person of ordinary skill in the art. For example, cumulus cells can be separated from oocytes by pipeting the cumulus cell/oocyte complex through a small bore pipette, by exposure to hyaluronidase, or by mechanically disrupting (e.g. vortexing) the cumulus cell/oocyte complex. Additionally, exposure to Ca ++ /Mg ++ free media can remove cumulus from mature and/or immature oocytes. Also, cumulus cell cultures can be established by placing mature and/or immature oocytes in cell culture media Once cumulus cells are removed from media containing increased LH/FSH concentrations, they can to attach to the culture plate. The term “hepatic cell” as used herein refers to any cultured or non-cultured cell isolated from a liver. Particularly preferred hepatic cells include, but are not limited to, an hepatic parenchymal cell, a Küpffer cell, an Ito cell, an hepatocyte, a fat-storing cell, a pit cell, and an hepatic endothelial cell. Persons skilled in the art can readily identify the various types of hepatic cells. See, e.g., Regulation of Hepatic Metabolism, 1986, Thurman et al. (eds.), Plenum Press, which is incorporated herein by reference in its entirety including all figures, drawings, and tables. The term “asynchronous population” as used herein refers to cells that are not arrested at any one stage of the cell cycle. Many cells can progress through the cell cycle and do not arrest at any one stage, while some cells can become arrested at one stage of the cell cycle for a period of time. Some known stages of the cell cycle are G 0 , G 1 , S, G 2 , and M. An asynchronous population of cells is not manipulated to synchronize into any one or predominantly into any one of these phases. Cells can be arrested in the G 0 stage of the cell cycle, for example, by utilizing multiple techniques known in the art, such as by serum deprivation. Examples of methods for arresting non-immortalized cells in one part of the cell cycle are discussed in WO 97/07669, entitled “Quiescent Cell Populations for Nuclear Transfer,” hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings. The terms “synchronous population” and “synchronizing” as used herein refer to a fraction of cells in a population that are arrested (i.e., the cells are not dividing) in a discreet stage of the cell cycle. Synchronizing a population of cells, by techniques such as serum deprivation, may render the cells quiescent. The term “quiescent” is defined below. Preferably, about 50% of the cells in a population of cells are arrested in one stage of the cell cycle, more preferably about 70% of the cells in a population of cells are arrested in one stage of the cell cycle, and most preferably about 90% of the cells in a population of cells are arrested in one stage of the cell cycle. Cell cycle stage can be distinguished by relative cell size as well as by a variety of cell markers well known to a person of ordinary skill in the art. For example, cells can be distinguished by such markers by using flow cytometry techniques well known to a person of ordinary skill in the art. Alternatively, cells can be distinguished by size utilizing techniques well known to a person of ordinary skill in the art, such as by the utilization of a light microscope and a micrometer, for example. In yet another aspect, the present invention relates to cells and cell lines derived from the embryos and/or the reprogrammed cells described herein; and to uses thereof in cellular and tissue therapies.
Fluid bedding
Bedding comprises a sealed bag body 3 having an elastic body 1 with a plurality of continuous pores and gas 2 sealed therein and having looseness in the upper part thereof, and a water-sealed bag body 6 disposed on the upper part of the bag body 3 having an elastic body 4 with a plurality of continuous pores and liquid 5 sealed therein and having looseness on the upper and lower parts thereof, whereby bedding capable of providing comfortable sleep can be realized by uniformly supporting a human body, with the buoyancy of the liquid while reducing the amount of the liquid required therein.
1. Bedding comprising: a sealed bag body 3 containing an elastic body 1 with a plurality of continuous pores and gas 2 sealed therein and having looseness in an upper part thereof; and a water-sealed bag body 6, disposed on the upper part of the bag body 3, containing an elastic body 4 with a plurality of continuous pores and liquid 5 sealed therein and having looseness on upper and lower parts thereof. 2. Bedding comprising: an elastic body 1 having a plurality of continuous pores; and a water-sealed bag body 6, disposed on an upper part of the elastic body 1, containing an elastic body 4 with a plurality of continuous pores and liquid 5 sealed therein and having looseness on upper and lower parts thereof. 3. Bedding according to claim 1, characterized in having paths 7 which are independent from each other or continuous with each other on surfaces or in interior portion(s) of said elastic body 1 and/or said elastic body 4. 4. Bedding characterized in forming a piece of bedding through connecting together a plural bedding, each bedding being made up in small size according to claim 1. 5. Bedding according to claim 2, characterized in having paths 7 which are independent from each other or continuous with each other on surfaces or in interior portion(s) of said elastic body 1 and/or said elastic body 4. 6. Bedding characterized in forming a piece of bedding through connecting together a plural bedding, each bedding being made up in small size according to claim 2. 7. Bedding characterized in forming a piece of bedding through connecting together a plural bedding, each bedding being made up in small size according to claim 3. 8. Bedding characterized in forming a piece of bedding through connecting together a plural bedding, each bedding being made up in small size according to claim 5.
<SOH> BACKGROUND ART <EOH>Conventionally, as bedding which uses fluid therein, bedding so-called an air mattress and an air pillow, which are filled with air therein; and bedding so-called a waterbed and a water pillow, which are filled with water therein are available. Conventional air mattress and air pillow support a human body mainly with tension of an air-sealed bag. However, pressure, which is generated by the tension of a bag body and supports the human body, is extremely uneven spot by spot depending on the shape of the part of the human body, and widely varies depending on the state of contact between the bag body and the human body. Accordingly, the bedding fails to support the human body constantly with uniform pressure and thus comfortable sleep is hardly obtained. Also, as for a waterbed and a water pillow, a method to constantly support the human body uniformly, which mainly uses buoyancy of water, has been invented. However, in the conventional waterbed and water pillow with no frame, in order to maintain the shape as bedding, an equivalent amount of water approximately to the maximum volume of an inner space of the water-sealed bag body has to be filled therein. Since a large ratio of a force that supports the human body is tension working on the bag, comfortable sleep is hardly obtained. In view of the above problems, as a technique to maintain the shape of the bedding while reducing the volume of the fluid-sealed bag body therein, such configuration that an elastic body with continuous air bubbles which has been formed into a desired shape as bedding is inserted in the bag body, and the surface of the continuous member and the inner surface of the bag body are all bonded to each other so as to maintain the shape as bedding, has been proposed. However, in this case, since the bag body is bonded to the elastic body via the surfaces thereof, there is not enough flexibility to deform freely, therefore, when the human body is supported, there has been such problem that a high ratio of the tension of the bag body in the force of supporting the human body is resulted in. Also, as a technique to reduce the ratio of the tension in the force of supporting the human body, an appropriate looseness is provided to the bag in the portions of the water-sealed bag, where the human body comes into contact with, so as not to generate the tension. And to maintain the configuration as bedding, a part of the bag is formed of a solid material, or the periphery of the bag is enclosed with a solid material. However, in this case, although almost entire part of the supporting force of the human body is born by buoyancy, the above technique is applicable only to such a large bed that the solid portion does not come into contact with the human body. And further, to obtain enough buoyancy, liquid of considerable depth has to be sealed inside the bed. Accordingly, a larger weight of the bed is resulted in and thus there resides such problem that the bed can be installed only in a building, which is designed based on a special standard. As described above, no conventional bedding comfortably supports a head or an entire body by means of buoyancy at a constant pressure, nor being light in weight, small in size, and inexpensive in cost.
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is an explanatory view illustrating a structure of fluid bedding; FIG. 2 is a view illustrating a manner of embodying the fluid bedding (Embodiment 1); FIGS. 3 a - 3 c are explanatory views illustrating examples of paths for gas; FIG. 4 is an explanatory view illustrating a manner in which the fluid bedding supports a human body; FIG. 5 is an explanatory view showing a manner of embodying the fluid bedding (Embodiment 2); FIGS. 6 a , 6 b are views showing manners of embodying the fluid bedding (Embodiment 3); and FIGS. 7 a , 7 b are views showing manners of embodying the fluid bedding (Embodiment 4). detailed-description description="Detailed Description" end="lead"?
Antibacterial material
An antibacterial material, a method of producing an antibacterial material and a method of inhibiting infection during the treatment of wounds using an antibacterial material. The antibacterial including a fibrous substrate impregnated with a carrier and a controlled release biocide dispersed through the carrier.
1. A method of inhibiting infection during the treatment of a wound by applying an antibacterial material to an affected skin area on a patient, said antibacterial material including: a fibrous substrate, said fibrous substrate being impregnated with a carrier; and a controlled release biocide dispersed through said carrier. 2. A method according to claim 1, wherein said biocide is mobile within said carrier. 3. A method according to claim 1, wherein said substrate is a substantially cellulose fibre blend. 4. A method according to claim 3, wherein said substrate is paper. 5. A method according to claim 4, wherein said paper has an open weave configuration. 6. A method according to claim 3, wherein the weight of said substrate is in the range of 50 to 200 gsm. 7. A method according to claim 6, wherein the weight of said substrate is in the range of 80 to 150 gsm. 8. A method according to claim 7, wherein the weight of said substrate is in the range of 100 to 120 gsm. 9. A method according to claim 3, wherein said substrate dissolves in water. 10. A method according to claim 1, wherein said biocide is in a latent state capable of subsequent activation by external stimulus. 11. A method according to claim 10, wherein said external stimulus is pressure. 12. A method according to claim 10, wherein said external stimulus is light. 13. A method according to claim 12, wherein said light is ultraviolet light. 14. A method according to claim 10, wherein said biocide is encapsulated. 15. A method according to claim 1, wherein said biocide is inactivated by blood serum. 16. A method according to claim 1, wherein said biocide is non-allergenic to humans. 17. A method according to claim 1, wherein said biocide inhibits the growth of E. coli, Legionella or Staphylococcus. 18. A method according to claim 1, wherein said biocide is triclosan. 19. A method according to claim 18, wherein said triclosan is that known under the trademark CANSAN®TCH. 20. A method according to claim 1, wherein said biocide is present in an amount of 10 to 10,000 ppm. 21. A method according to claim 1, wherein said carrier transports said biocide to the site of action. 22. A method according to claim 1, wherein said carrier is a glycol. 23. A method according to claim 22, wherein said glycol is propylene glycol. 24. A method according to claim 1, wherein 100 ml of said carrier is used per m2 of said substrate. 25. A method according to claim 1, wherein said material further includes a nylon resin. 26. A method according to claim 25, wherein said nylon is a Kymene resin. 27. A method according to claim 1, wherein said wound is an ulcer. 28. A method according to claim 1, wherein said wound is a burn. 29. Use of an antibacterial material including a fibrous substrate impregnated with a carrier, and a controlled release biocide dispersed through said carrier, in the manufacture of a dressing for inhibiting infection during the treatment of wounds. 30. A wound dressing including an antibacterial material including a fibrous substrate impregnated with a carrier, and controlled release biocide dispersed through said carrier. 31. A wound dressing according to claim 30, further including a waterproof backing material or an elastic-backed sticking plaster. 32. An antibacterial material suitable for use as a wound dressing including: a fibrous substrate having a weight of from 50 to 200 gsm, said fibrous substrate being impregnated with a carrier; and a controlled release biocide dispersed through, and mobile within, said carrier. 33. An antibacterial material according to claim 32, wherein said substrate is a substantially cellulose fibre blend. 34. An antibacterial material according to claim 33, wherein said substrate is paper. 35. An antibacterial material according to claim 34, wherein said paper has an open weave configuration. 36. An antibacterial material according to claim 35, wherein the weight of said substrate is in the range of 80 to 150 gsm. 37. An antibacterial material according to claim 36, wherein the weight of said substrate is in the range of 100 to 120 gsm. 38. An antibacterial material according to claim 33, wherein said substrate dissolves in water. 39. An antibacterial material according to claim 32, wherein said biocide is encapsulated. 40. An antibacterial material according claim 32, wherein said biocide is triclosan. 41. An antibacterial material according to claim 40, wherein said triclosan is that known under the trademark CANSAN®TCH. 42. An antibacterial material according to claim 32, wherein said biocide is present in an amount of 10 to 10,000 ppm. 43. An antibacterial material according to claim 32 wherein said carrier is a glycol. 44. An antibacterial material according to claim 43, wherein said glycol is propylene glycol. 45. An antibacterial material according to claim 32, wherein 100 ml of said carrier is used per m2 of said substrate. 46. An antibacterial material according to claim 32, wherein said material further includes a nylon resin. 47. An antibacterial material according to claim 46, wherein said nylon is a Kymene resin. 48. A process for making an antibacterial material according to claim 32, said process including: dispersing a controlled release biocide within a carrier to provide an active carrier mixture; and impregnating a fibrous substrate having a weight of from 50 to 200 gsm with said active carrier mixture.
<SOH> BACKGROUND OF THE INVENTION <EOH>The treatment and dressing of slow healing open wounds has been a long-standing medical problem. Wound healing is an extremely complex biological process and there are many factors to be taken into account when considering the best possible treatment and dressing. Factors such as risk of infection, time of treatment and patient comfort are important considerations. Ulcers are a particularly painful form of slow healing wound and are often difficult to treat due to underlying circulatory problems and the continuous risk of infection. Ulcers are secreting skin wounds which produce exudates such as blood, pus and other wound fluids. These fluids can accumulate in the wound cavity and form a rich pool of nutrients which promotes bacterial growth. This, in turn, delays or prevents the healing process, or in some cases leads to further tissue damage or even the possibility of systemic life threatening infection. It is would be desirable to allow such wounds to heal in a slightly moist environment provided infection can be controlled and excess wound exudates can be removed to prevent blisters forming under the dressing or the dressing becoming embedded in the wound. Examples of conventional dressings used to protect open wounds and promote the healing process include traditional cloth, cotton or lint pads, non-adherent latex dressings and synthetic-algin coatings. However, each of these dressings has drawbacks. Traditional dressings in direct contact with a wound can interfere with the healing process. This is particularly so with chronic ulcerated wounds because of the repeated mechanical impact and interaction of the bandage with the fragile and pressure sensitive tissues within the wound. This problem is amplified by the repeated dressing changes which are often required. In many cases because there is new tissue growth the dressings adhere to the wound. This makes them difficult to remove without disrupting the healing process. Non-adherent latex dressings have a high rate of allergic reaction. Along with some other types of conventional dressings, they are unable to absorb exuded fluids at a rate commensurate with the rate of fluid production by the wound or alternatively, their fluid holding capacity is not significant to allow long periods of time in contact with the wound. The progressive build up of bacteria then delays healing and may also produce unpleasant odours. Algin type dressings are designed to avoid adhering to healing wounds and for easy removal. They present the appearance of being coated with jelly-like substance. While these may avoid adhering to a healing wound, they have a propensity to go mouldy when exposed to bacteria filled fluids for extended periods. There is a need for a wound dressing that can continuously inhibit the growth of bacteria and be easily removed from the wound. Moreover, it would be particularly desirable if the dressing could be left on the wound for a longer period of time than conventional dressings allow, in order to avoid disrupting the wound and thus accelerating the healing process. It is an object of the invention to overcome or ameliorate at least some of the disadvantages of the prior art, or to provide a useful alternative.
Multilayer containers and process for forming multilayer containers
A co-injection process is provided for fabricating improved multi-layer containers, including but not limited to blood collection tubes, evacuated blood collection tubes, culture bottles, centrifuges tubes, and syringe barrels. The container includes a tube (10) having a bottom wall (12) and a side wall (14) with an open end (18). The container can be provided with a stopper having an upper portion (22) and a skirt with an outside portion (30), a well (34) and a cavity (36). The tube and stopper assembly can be used for medical purposes including containing a blood sample within an enclosed interior space (40).
1. A process for fabricating a container having a bottom wall, a top edge, and a sidewall between the bottom wall and top edge, comprising the steps of: providing a first molten polymeric material and a second molten polymeric material, the first and second polymeric materials being non-compatible; and directing the first and second molten polymeric materials through a nozzle section into a mold cavity that comprises a region for integrally forming the bottom wall of the container, wherein the first and second molten polymeric materials co-flow in the mold cavity for at least a portion of the fabrication process, wherein, during the co-flow, the nozzle section directs the first and second molten polymeric materials into the mold cavity as inner and outer skin layers of the first molten polymeric material with a core layer of the second molten polymeric material between the inner and outer skin layers. 2. The process of claim 1, wherein the inner and outer skin layers are directly adjacent the core layer. 3. The process of claim 1, wherein the first and second polymeric materials are selected from the group consisting of ethylene vinyl alcohol copolymer, polyester, copolymers of ethylene vinyl alcohol copolymer and polyester, cyclic olefin copolymers, and polypropylene. 4. The process of claim 3, wherein the first and second polymeric materials are, respectively, polypropylene and ethylene vinyl alcohol copolymer, or cyclic olefin copolymer and ethylene vinyl alcohol copolymer, or polypropylene and polyester, or cyclic olefin copolymer and polyester. 5. The process of claim 4, wherein the first and second polymeric materials are, respectively, polypropylene and ethylene vinyl alcohol copolymer. 6. The process of claim 1, wherein the first polymeric material is a gas barrier material and the second polymeric material is a liquid vapor barrier material, or the first polymeric material is a liquid vapor barrier material and the second polymeric material is a gas barrier material. 7. The process of claim 1, wherein the core layer is encapsulated by the skin layers. 8. The process of claim 1, wherein the core layer exhibits substantially continuous coverage throughout the bottom wall and throughout the side wall. 9. The process of claim 1, wherein the core layer exhibits substantially continuous coverage throughout the bottom wall and in the side wall up to a region of the container that is intended to be contacted by a stopper. 10. A container comprising: a bottom wall, a top edge, and a sidewall between the bottom wall and the top edge, wherein at least the sidewall comprises inner and outer polymeric skin layers with a polymeric core layer located between and directly adjacent the inner and outer polymeric skin layers, the polymeric skin layers being non-compatible with the polymeric core layer. 11. The container of claim 10, wherein the container is free of adhesive or tie layers between the skin layers and core layer. 12. The container of claim 10, wherein the skin and core layers are formed from polymers selected from the group consisting of ethylene vinyl alcohol copolymer, polyester, copolymers of ethylene vinyl alcohol copolymer and polyester, cyclic olefin copolymers, and polypropylene. 13. The container of claim 12, wherein the skin and core layers are, respectively, polypropylene and ethylene vinyl alcohol copolymer, or cyclic olefin copolymer and ethylene vinyl alcohol copolymer, or polypropylene and polyester, or cyclic olefin copolymer and polyester. 14. The container of claim 13, wherein the skin and core layers are, respectively polypropylene and ethylene vinyl alcohol copolymer. 15. The container of claim 10, wherein the skin layer is a gas barrier material and the core layer is a liquid vapor barrier material, or the skin layer is a liquid vapor barrier material and the core layer is a gas barrier material. 16. The container of claim 10, wherein the container is formed by a coinjection process. 17. The container of claim 10, wherein the sidewall and the bottom wall comprise inner and outer polymeric skin layers with a polymeric core layer located between and directly adjacent the inner and outer polymeric skin layers, the polymeric skin layers being non- compatible with the polymeric core layer. 18. The container of claim 10, wherein the core layer exhibits substantially continuous coverage throughout both the bottom wall and throughout the side wall. 19. The container of claim 10, wherein the core layer exhibits substantially continuous coverage throughout the bottom wall and in the side wall up to a region of the container that is designed to be contacted by a stopper. 20. The container of claim 18, wherein the core layer is encapsulated by the skin layers. 21. The container of claim 10, wherein the container is a tube. 22. The container of claim 20, wherein the tube comprises a cap having an elastomeric region. 23. The container of claim 21, wherein the tube is an evacuated blood collection tube. 24. The container of claim 10, wherein the core layer, the skin layers, or both the core layer and the skin layers comprise one or more materials selected from the group consisting of organic or inorganic fillers, dyes, plasticizers, slip agents, processing aids, stabilizers, ultraviolet light barriers, molecular scavenger materials, radiation barrier materials, chargeable dyes, materials that react to temperature or pressure changes, and structural additives. 25. The container of claim 10, wherein the core layer, the skin layers, or both the core layer and the skin layers comprise a nanocomposites material. 26. A process for fabricating.a container having a bottom wall, a top edge, and a sidewall between the bottom wall and top edge, comprising the steps of: providing a first molten polymeric material and a second molten polymeric material; and directing the first and second molten polymeric materials through a nozzle section into a mold cavity that comprises a region for integrally forming the bottom wall of the container, wherein the first and second molten polymeric materials co-flow in the mold cavity for at least a portion of the fabrication process, wherein, during the co-flow, the nozzle section directs the first and second molten polymeric materials into the mold cavity as annular inner and outer skin layers of the first molten polymeric material with an annular core layer of the second molten polymeric material between the inner and outer skin layers, and wherein a cold sprue is located between the nozzle section and the mold cavity, such that the polymeric materials pass through the cold sprue before flowing into the mold cavity. 27. A process for fabricating a container having a bottom wall, a top edge, and a sidewall between the bottom wall and top edge, comprising the steps of: providing a first molten polymeric material, a second molten polymeric material, and a third molten polymeric material, the first and second polymeric materials being non-compatible, and the second and third polymeric materials being non-compatible; and directing the first and second and third molten polymeric materials through a nozzle section into a mold cavity that comprises a region for integrally forming the bottom wall of the container, wherein the first and second and third molten polymeric materials co-flow in the mold cavity for at least a portion of the fabrication process, wherein, during the co-flow, the nozzle section directs the first and second and third molten polymeric materials into the mold cavity as, respectively, an inner skin layer, a core layer, and an outer skin layer, with the core layer located between the inner and outer skin layers. 28. The process of claim 27, wherein the inner and outer skin layers are directly adjacent the core layer. 29. The process of claim 27, wherein the first, second, and third molten polymeric materials are identical or different, and one or more of the polymeric materials comprise one or more additives selected from the group consisting of organic or inorganic fillers, dyes, plasticizers, slip agents, processing aids, stabilizers, ultraviolet light barriers, molecular scavenger materials, radiation barrier materials, chargeable dyes, materials that react to temperature or pressure changes, and structural additives. 30. The process of claim 27, wherein the first and second and third polymeric materials are identical or different, and are selected from the group consisting of ethylene vinyl alcohol copolymer, polyester, copolymers of ethylene vinyl alcohol copolymer and polyester, cyclic olefin copolymers, and polypropylene. 31. The process of claim 27, wherein the first and second and third polymeric materials include at least one gas barrier material and at least one liquid vapor barrier material. 32. The process of claim 27, wherein the core layer is encapsulated by the skin layers. 33. The process of claim 27, wherein one or more of the first, second, and third polymeric materials comprise a nanocomposite material.
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention This invention relates to plastic articles and more particularly relates to medical articles having improved gas and liquid vapor barriers. 2. Description of the Related Art Polypropylene (PP) has long been used in molding and extruding operations for articles such as plastic medical containers and films for the food packaging industry. Polyethylene terephthalate (PET) has more recently been used in molding and extruding operations for these articles. However, PP and PET are somewhat permeable to nitrogen, oxygen, and other gases and vapors. As a result, PP and PET containers are inherently subject to transmission of gases. As the medical industry begins to place increased emphasis on the use of plastic medical products, these permeability problems have become more acute. In particular, evacuated blood collection tubes must meet certain performance standards. Such performance standards generally include the ability to maintain greater than about 90% original draw volume over a one year period, and gas permeability clearly is detrimental to this need. Moreover, materials must also be capable of being sterilized by radiation, and substantially avoid interfering with tests and analysis. Thus, materials for such containers not only must resist gas and liquid vapor permeability problems, but they must also meet several other requirements. Various techniques have therefore been devised in an attempt to reduce gas and vapor permeability of containers fabricated from PP, PET and other resins. Such techniques include addition of inorganic fillers, coating the containers with resins having barrier properties, plasma chemical vapor deposition coating of inorganic materials, and blending, laminating or co-extruding the resins with barrier resins. While such efforts have offered some improvement, the need to consistently meet high performance standards demands further improvement.
<SOH> SUMMARY OF THE INVENTION <EOH>The invention address the problems of the prior art, and provides a process for fabricating improved containers meeting the needs discussed above. Such containers include but are not limited to blood collection tubes, evacuated blood collection tubes, centrifuge tubes, culture bottles, and syringe barrels. In one embodiment, the process of the invention involves providing a first molten polymeric material and a second molten polymeric material, the first and second polymeric materials being non-compatible, and directing the first and second molten polymeric materials through a nozzle section into a mold cavity that comprises a region for integrally forming the bottom wall of the container. The first and second molten polymeric materials co-flow in the mold cavity for at least a portion of the fabrication process. During the co-flow, the nozzle section directs the first and second molten polymeric materials into the mold cavity as inner and outer skin layers of the first molten polymeric material with an core layer of the second molten polymeric material between the inner and outer skin layers. In another embodiment, a tube of the invention comprises a bottom wall, a top edge, and a sidewall between the bottom wall and the top edge. At least the sidewall comprises inner and outer polymeric skin layers with a polymeric core layer located between and directly adjacent the inner and outer polymeric skin layers, with the polymeric skin layers being non- compatible with the polymeric core layer. A tube having non-compatible polymers, yet without the need for adhesive or tie layers between the distinct polymers is thereby achieved.
Attenuated circovirus
An isolated attenuated circovirus having a mutation in viral nucleic acid encoding viral protein 2 (VP2). The attenuated circovirus is particularly suitable for use in conferring immunity in an animal, particularly birds.
1. An isolated attenuated circovirus having a mutation in viral nucleic acid encoding viral protein 2 (VP2). 2. The isolated attenuated circovirus according to claim 2, wherein the circovirus being derived or obtained from Chicken anaemia virus (CAV), a TT virus (TTV) or other similar virus expressing a VP2 protein. 3. The isolated attenuated circovirus according to claim 2, wherein the circovirus being derived or obtained from Chicken anaemia virus (CAV). 4. The isolated attenuated circovirus according to my claim 1, wherein the mutation is present in a region of nucleic acid encoding the signature motif of VP2. 5. The isolated attenuated circovirus according to claim 4, wherein the mutation alters viral PTPase activity, PTPase motifs, acidic alpha helical regions or basic beta sheet regions. 6. The isolated attenuated circovirus according to claim 3, wherein the mutation is present in the region of nucleic residues 80 to 110, 128 to 143, 151 to 158 and, 160 to 170 in CAV VP2. 7. The isolated attenuated circovirus according to claim 6, wherein the sites targeted for mutagenesis within CAV VP2 are selected from the group consisting of 86, 95, 97, 101, 103 and 169. 8. The isolated attenuated circovirus according to claim 6, wherein the mutations are selected from the group consisting of mut C86 R, mut C 95 S, mut C 97 S, mut R 101 G, mut K 102 E, mut H 103 Y, mut R 129 G, mut Q 131 P, mut R/K/K 150/151/152 G/A/A, mut D/E 161/162 G/G, mut L 163 P, mut D 169 G, mut E 186 G, and combinations thereof. 9. The isolated attenuated circovirus according to claim 8 comprising mut D 169 G. 10. The isolated attenuated circovirus according to an claim 1 having a nucleic acid sequence selected from the group consisting of sequence ID no's 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27. 11. A circovirus vaccine composition comprising an attenuated circovirus having a mutation in viral nucleic acid encoding viral protein 2 (VP2) together with an acceptable carrier or diluent. 12. The circovirus vaccine composition according to claim 11, wherein the circovirus being derived or obtained from Chicken anaemia virus (CAV), a TT virus (TTV) or other similar virus expressing a VP2 protein. 13. The circovirus vaccine composition according to claim 12, wherein the circovirus being derived or obtained from Chicken anaemia virus (CAV). 14. The circovirus vaccine composition according to claim 11, wherein the mutation is present in a region of nucleic acid encoding the signature motif of VP2. 15. The circovirus vaccine composition according to claim 14, wherein the mutation alters viral PTPase activity, PTPase motifs, acidic alpha helical regions or basic beta sheet regions. 16. The circovirus vaccine composition according to claim 13, wherein the mutation is present in the region of nucleic residues 80 to 110, 128 to 143, 151 to 158 and 160 to 170 in CAV VP2. 17. The circovirus vaccine composition according to claim 16, wherein the sites targeted for mutagenesis within CAV VP2 are selected from the group consisting of 86, 95, 97, 101, 103 and 169. 18. The circovirus vaccine composition according to claim 16, wherein the mutations are selected from the group consisting of mut C86 R, mut C 95 S, mut C 97 S, mut R 101 G, mut K 102 E, mut H 103 Y, mut R 129 G, mut Q 131 P, mut R/K/K 150/151/152 G/A/A, mut D/E 161/162 G/G, mut L 163 P, mut D 169 G, mut E 186 G, and combinations thereof. 19. The circovirus vaccine composition according to claim 18 comprising mut D 169 G. 20. The circovirus vaccine composition according to any one of the claims 11 to 19 claim 11 comprising a nucleic acid sequence selected from the group consisting of sequence no's 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27. 21. A method for imparting immunity to circovirus infection in an animal comprising administering to the animal an effective amount of circovirus vaccine according to claim 11. 22. The method for imparting immunity according to claim 21 wherein the animal is a bird. 23. The method for imparting immunity according to claim 22 wherein the bird is a chicken. 24. The method for imparting immunity according to claim 21 wherein the vaccine is administered parenterally, intramuscularly, subcutaneously, orally, intranasally, or in ovo route. 25. The method for imparting immunity according to claim 24 wherein the animal is a bird and the route of administration of the vaccine is by mucosal administration, aerosol administration or via drinking water. 26. The method for imparting immunity according to claim 25 wherein the bird is a chicken. 27. The method for imparting immunity according to claim 21 wherein the vaccine is administered in a dosage range from 1 to 100 million TCID50. 28. The method for imparting immunity according to claim 27 wherein the vaccine is administered in a dosage range of about 1000 TCID50. 29. An isolated nucleic acid molecule derived or obtained from a circovirus genome, the nucleic acid molecule including at least a portion of a coding region for viral protein 2 (VP2) having a mutation therein. 30. The isolated nucleic acid molecule according to claim 29, wherein the circovirus being derived or obtained from Chicken anaemia virus (CAV), a TT virus (TTV) or other similar virus expressing a VP2 protein. 31. The isolated nucleic acid molecule according to claim 30, wherein the circovirus being derived or obtained from Chicken anaemia virus (CAV). 32. The isolated nucleic acid molecule according to claim 29, wherein the mutation is present in a region of nucleic acid encoding the signature motif of VP2. 33. The isolated nucleic acid molecule according to claim 32, wherein the mutation alters viral PTPase activity, PTPase motifs, acidic alpha helical regions or basic beta sheet regions. 34. The isolated nucleic acid molecule according to claim 31, wherein the mutation is present in the region of nucleic residues 80 to 110, 128 to 143, 151 to 158 and 160 to 170 in CAV VP2. 35. The isolated nucleic acid molecule according to claim 34, wherein the sites targeted for mutagenesis within CAV VP2 are selected form the group consisting of 86, 95, 97, 101, 103 and 169. 36. The isolated nucleic acid molecule according to claim 34, wherein the mutations are selected from the group consisting of mut C86 R, mut C 95 S, mut C 97 S, mut R 101 G, mut K 102 E, mut H 103 Y, mut R 129 G, mut Q 131 P, mut R/K/K 150/151/152 G/A/A, mut D/E 161/162 G/G, mut L 163 P, mut D 169 G, mut E 186 G, and combinations thereof. 37. The isolated nucleic acid molecule according to claim 36 comprising mut D 169 G. 38. The isolated nucleic acid molecule according to claim 29 comprising a sequence selected from the group consisting of sequence no's 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27. 39. The vaccine composition comprising an isolated nucleic acid molecule according to claim 29 together with an acceptable carrier or diluent. 40. A method of conferring immunity in an animal against a circovirus infection, the method comprising administering to the animal a vaccine composition according to claim 39. 41. The method of conferring immunity according to claim 40 wherein the circovirus infection is a CAV infection. 42. The method of conferring immunity according to claim 41 wherein the animal is a bird. 43. The method of conferring immunity according to claim 42 wherein the bird is a chicken. 44. Use of an isolated attenuated circovirus according to claim 1 in the manufacture of a vaccine for conferring immunity in an animal against a circovirus infection. 45. Use of an isolated nucleic acid molecule according to claim 29 in the manufacture of a vaccine for conferring immunity in an animal against a circovirus infection. 46. A method of producing a circovirus vaccine comprising: (a) inoculating an isolated nucleic acid molecule derived or obtained from a circovirus genome into the yolk sac of an embryonated egg, wherein the nucleic acid molecule includes at least a portion of a coding region for viral protein 2 (VP2) having a mutation therein; (b) allowing circovirus to replicate from the isolated nucleic acid; and (c) harvesting the circovirus from the egg 47. The method according to claim 46, wherein the isolated nucleic acid molecule being derived or obtained from Chicken anaemia virus (CAV), a TT virus (TTV) or other similar virus expressing a VP2 protein. 48. The method according to claim 47, wherein the isolated nucleic acid molecule being derived or obtained from Chicken anaemia virus (CAV). 49. The method according to claim 46, wherein the mutation is present in a region of nucleic acid encoding the signature motif of VP2. 50. The method according to claim 49, wherein the mutation alters viral PTPase activity, PTPase motifs, acidic alpha helical regions or basic beta sheet regions. 51. The method according to claim 48, wherein the mutation is present in the region of nucleic residues 80 to 110, 128 to 143, 151 to 158 and 160 to 170 in CAV VP2. 52. The method according to claim 51, wherein the sites targeted for mutagenesis within CAV VP2 are selected from the group consisting of 86, 95, 97, 101, 103 and 169. 53. The method according to claim 51, wherein the mutations are selected from the group consisting of mut C86 R, mut C 95 S; mut C 97 S, mut R 101 G, mut K 102 E, mut H 103 Y, mut R 129 G, mut Q 131 P, mut R/K/K′150/151/152 G/A/A, mut D/E 161/162 G/G, mut L 163 P, mut D 169 G, mut E 186 G, and combinations thereof. 54. The method according to claim 53 wherein the mutation comprises mut D 169 G. 55. The method according to claim 46 wherein the circovirus has a nucleic acid sequence selected from the group consisting of sequence no's 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27.
<SOH> BACKGROUND ART <EOH>Chicken anaemia virus (CAV) is a member of the Circoviridae family. The Circoviridae include a number of plant and animal viruses that are characterised by the possession of a single stranded, negative-sense, circular DNA genome. There is minimal similarity in the genomic sequence and organisation between CAV and the other characterised animal circoviruses: Psittacine Beak and Feather Disease Virus (PBFDV), Pigeon Circovirus and Porcine Circoviruses (PCV) 1 and 2. TT viruses (TTV) have recently been identified in human hosts and other species as a heterogeneous cluster of single stranded, negative-sense, circular DNA viruses. Sequence analysis of this group of viruses has demonstrated greatest overall homology to CAV and others have recently proposed the classification of the TTV, SANBAN, YONBAN, TLMV (TTV Like Mini Viruses) and CAV viruses as the Paracircoviridae, however, the phylogeny remains an area of active revision. The highest sequence homology to CAV is seen in the non-coding region and between open reading frame (ORF2) of TTV and VP2 of CAV. The high level of sequence conservation between CAV and TTV suggests VP2 may play a critical role in viral infection and pathogenesis. CAV encodes only three proteins, with overlapping ORFs in three frames. ORF3 encodes the major 45-52 kDa capsid protein VP1, ORF2 encodes the 11-13 kDa VP3 that has demonstrated apoptotic activity in transformed cell lines, and ORF1 encodes a 28 kDa non-structural protein VP2 with unknown function. VP2 is expressed at barely detectable levels during infection, but has been shown to be essential for viral infection and replication in cells. The low level of expression of VP2 is consistent with a non-structural, regulatory protein involved in viral replication and infection. CAV pathogenesis is characterised by immunosuppression and pancytopaenia arising from panmyelophthisis and thymocyte depletion. Immunosuppression results in increased rates of morbidity and mortality associated with coinfections and vaccination failure in CAV infected chicks. CAV infection is directly cytotoxic to two distinct T-cell populations of the thymus and spleen. Thymic infection involves immature lymphoblastic precursors, whereas splenic infection is of mature T-lymphocytes that are highly activated. There is a second indirect-component of immunosuppression found in uninfected immune effector cells. Reductions in macrophage and APC effector functions and B cell antigenic responses have been documented. Limited cytokine profiles from infected cells are suggestive of a basis for generalised indirect immunosuppression. There is a reduction in interleukin 2 (IL-2), interferon gamma (IFNγ), lymphocyte stimulation index, IL-1, T-cell growth factor activity and Fc receptor levels in lymphocytes of infected birds. The molecular basis for viral modulation of cytokine profiles and indirect immunosuppression is unknown. Preliminary comparisons of the CAV VP2 sequence to sequences available in the Genbank database suggested similarity to a number of eukaryotic receptor PTPases (R-PTPase alpha). Database searches identified the human placental, rat, mouse and chicken R-PTPase alpha precursors as homologous to the CAV VP2 sequence. Reversible protein phosphorylation is universal in the regulation of cellular processes, including metabolism, gene regulation, cell cycle control, cytoskeletal organisation and cell adhesion. The PTPase family is highly diverse and includes the eukaryotic receptor-like transmembrane proteins and soluble cytosolic proteins, as well as bacterial PTPases, such as the YopH PTPase from pathogenic Yersinia , and a viral PTPase VH1 found in Vaccinia virus, a member of the Poxyiridae. During Vaccinia virus infection the VH1 protein blocks interferon γ signalling thereby evading the immune response to virus infection. The role of the VH1 PTPase in infection, although currently the only viral PTPase with a characterised in vivo function, does highlight the potential for virus encoded PTPases to be involved in mechanisms of immune evasion and virus persistence. Commercial poultry producers require a chicken anaemia virus (CAV) vaccine that will reduce the economic losses incurred through both clinical and subclinical infections. The elimination of subclinical disease in adult birds associated with CAV infection requires overcoming immunosuppression due to infection. CAV infection is of greatest economic significance in broiler flocks. Both clinical and subclinical infections impact on commercial broiler performance and profitability. Whilst clinical infection produces a more marked reduction in performance parameters, subclinical infection is responsible for a greater degree of financial loss as it is of higher incidence. There is a strong need for a vaccine suitable for pullets, broilers and breeders. Such a vaccine may be administered to birds at the point of lay and therefore must be safe in the event of vertical transmission to embryos. The development of a CAV vaccine has international applications. Chicken anaemia virus (CAV) has a worldwide distribution based on serological surveillance, and is endemic in both SPF and commercial chicken flocks, with the exception of Australian SPF flocks. Countries from which CAV isolates have been characterised and their complete genome sequences published include Germany, UK, USA, Japan, Australia, and the Netherlands. All isolates are classified within a single serotype based on cross reactivity in immunofluorescence and neutralisation tests utilising polyclonal antiserum. Genome sequence conservation is a key feature of all CAV isolates. All field isolates demonstrate equivalent pathogenicity in experimental infection and any variation in the morbidity and severity of disease with CAV exposure is attributed to a range of interacting, epidemiological factors. Viral dose is the key determinant of the severity of CAV induced disease in the field. It is expected that live attenuated vaccines developed from any one isolate will be protective in poultry flocks internationally. An attenuated CAV strain should be infectious whilst having reduced pathogenicity. Clinical disease is best characterised in the literature in birds infected at 1 day old. Clinical disease in chicks infected at 1 day of age is characterised by weakness, depression, stunting and anaemia. By 7 days post infection, there is a transient but severe, peracute anaemia due to destruction of erythroblastoid cells and immunodeficiency due to depletion of cortical lymphocytes. Severe bone marrow hypoplasia, thymic and lymphoid atrophy and thrombocytopaenia are apparent at 14-21 days post infection. Petecchial and ecchymotic haemorrhages develop due to a primary coagulopathy. Immunosuppression is a significant feature of CAV induced disease and secondary infections are common. CAV affected birds have an increased incidence of malignant oedema, gangrenous dermatitis, colibacillosis and pulmonary aspergillosis. The recovery phase extends from 14-35 days post infection. Erythrocytopoiesis precedes granulocytopoiesis during recovery. At 16 days post infection there are a high proportion of circulating immature erythrocytes, thrombocytes and granulocytes, and the haematocrit is completely restored by 28 days post infection. The thymus is repopulated by the third wave of migrating lymphocytes at 21 days. CAV affected birds develop a severe anaemia of myelophthisis. The haematocrit is less than 27%, and typically between 9-23% (normally in chicks 7-14 days of age it is 32-37.5%). Cyanosis is evident in the non-feathered integument and on mucosal membranes. There is a leukopaenia attributable to a heterocytopaenia and lymphopaenia. Prolonged clotting times are associated with petecchial and ecchymotic haemorrhages observed over the integument, skeletal muscle, mucosa of the proventriculus and rarely the pericardium. The bone marrow appears yellow to white and watery in texture due to panmyelophthisis and compensatory adipocyte hyperplasia. This is most obvious in the proximal femoral medullary cavity. The thymus undergoes severe atrophy. Affected thymuses have a quantifiable reduction in weight and a diameter of 2-4 mm. They appear red-brown instead of grey due to a reduction in parenchymal lymphocyte populations, hyperplasia of reticular cells and hyperaemia of the tissue. There is a generalised depletion of lymphoid follicular components of all tissues. The bursa of Fabricius undergoes transient, moderate atrophy but is not swollen or oedematous. Bursal atrophy can be mild to unapparent in clinically affected chicks. The liver, kidneys and spleen are diffusely discoloured and swollen at 14 days post infection. Focal, dermal haemorrhagic lesions are most prominent on the wings, but also involve the head, rump, sides of thorax and abdomen, thighs, legs and feet. The lesions progress to large ulcers with a serosanguinous extravasation due to ischaemic necrosis of the overlying dermis. A purulent exudate develops in association with secondary infections. The lesions are prone to complicating abrasive and mutilation injury in the environment of the commercial broiler rearing unit. An experimental model for CAV pathogenesis is required for the assessment of attenuation. Such a model does not need to represent the full spectrum of pathology observed in field infection but must demonstrate attenuation under conditions that produce most severe pathology. Yolk sac inoculation of 7 day embryos with high doses of virus is the most stringent model available. This model best approximates the field situation in which naïve breeder birds at the point of lay are exposed to CAV and transmit virus transovarially. Chicks infected by vertical transmission have the highest rates of morbidity (100%) and mortality (10-70%) and the pathology is of greatest severity. Extensive studies of the pathology of embryos experimentally infected at 7 days by yolk sac inoculation have not been reported in the literature. Chickens of all ages are susceptible to CAV infection, however there is an age-specific resistance to the development of disease in chickens older than 14 days. Embryos and 1 day old chicks have the highest disease susceptibility. Age resistance may relate to the developing capacity of the bird to produce a serum neutralising humoral response. Co-infection with synergistic avian pathogens such as IBDV will eliminate age-related resistance and will result in outbreaks of acute severe disease in older birds. The majority of commercial breeder flocks have been exposed to CAV and have long lasting neutralising humoral immunity. Antibody persists for at least 20 weeks after seroconversion. Serological surveys of breeder flocks typically demonstrate 97.5-100% of birds remain seropositive over an extended period post infection. Maternal antibody is important in protection against clinical disease in chickens up to 2 weeks of age, and persists until 3 weeks of age. The decay of maternal antibody follows a linear relationship and has a half life of approximately 1 week. Low levels of maternal antibody are effective in preventing clinical disease with infection. The majority of hatchlings derived from immune breeder flocks are infected horizontally following the waning of maternal antibody, develop subclinical disease and seroconvert between 8-12 weeks post infection. In an exposed flock, approximately 10% of breeders will be seronegative at any point post exposure. A minor proportion of chickens are infected vertically and excrete high titres of virus acting as the source of horizontal infection for other hatchlings. There may be between 16 and 25% birds sub-clinically affected in the progeny of immune breeder flocks. Vaccination will therefore improve performance even in flocks with endemic CAV and persistent neutralising humoral immunity. The present inventors have developed live attenuated CAV and CAV DNA capable for use in vaccines suitable for the inoculation of pullets, broiler and breeder flocks, based on the identification of the function of the VP2 as a novel protein tyrosine phosphatase and the identification of regions of its sequence required for full function.
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 shows Chou-Fasman plots of two predicted regions of amphipathic α-helix from residues 128 to 143 and amphipathic β-sheet from residues 151 to 158 of VP2. FIG. 2 shows transfection of mut C86 R into MSB1 cells. FIG. 3 shows transfection of mut C 95 S into MSB1 cells. FIG. 4 shows transfection of mut C 97 S into MSB1 cells. FIG. 5 shows transfection of mut R 101 G into MSB1 cells. FIG. 6 shows transfection of mut H103 Y into MSB1 cells. FIG. 7 shows transfection of mut R129 G into MSB1 cells. FIG. 8 shows transfection of mut Q 131 P into MSB1 cells. FIG. 9 shows transfection of mut R/K/K 150/151/152 G/A/A into MSB1 cells. FIG. 10 shows transfection of mut D/E 161/162 G/G into MSB1 cells. FIG. 11 shows transfection of mut L 163 P into MSB1 cells. FIG. 12 shows transfection of mut DI 69 G into MSB1 cells. FIG. 13 shows R-PTPase homologues aligned to the CAV VP2 amino acid sequence using the ECLUSTALW software (WebANGIS) and displayed graphically using the Seqvu software (Garvin Institute). Row 1: chicken protein-tyrosine phosphatase alpha (Z32749), residues 302-306, homology score 30%. Row 2: human R-PTPase alpha (PP18433), residues 301-353, homology score 32%. Row 3: rat R-PTPase alpha (Q03348), residues 295-347, homology score 32%. Row 4: mouse R-PTPase alpha (P18052), residues 328-380, homology score 32%. Row 5: human R-PTPase alpha (17011300A). Row 6: human placental protein-tyrosine phosphatase (CAA38065), residues 292-345, homology score 32%. Row 7: CAV VP2. FIG. 14 shows alignment of CAV VP2 amino acid sequence and SANBAN TTV sequence using the ECLUSTALW software (WebANGIS) and displayed graphically using the Seqvu software (Garvin Institute). The Genbank accession numbers for the TT viruses are shown. FIG. 15 shows an electrophoresis separation of glutathione-S-transferase (GST) fusion proteins on a 12.5% polyacrylamide gel and visualisation with Coomassie blue staining. Lane 1, Broad range molecular weight standards (Biorad); lane 2, 2.6 μg CAV VP2-GST fusion; lane 3, 3.0 μg GST. FIG. 16 shows a western blot probed with a mouse polyclonal antiserum raised against the COOH-terminal region of VP2. Lane 1, Broad range molecular weight standards (Biorad); lane 2, 3.0 μg GST; lane 3, 2.6 μg CAV VP2-GST fusion. FIG. 17 shows a western blot probed with a rabbit polyclonal antiserum raised against GST. Lane 1, molecular weight standards; lane 2, 3.0 μg GST; lane 3, 2.6 μg chicken anaemia virus VP2-GST fusion. FIG. 18 shows a time course study of phosphate release from ENDY(Pi)INASL as catalysed by VP2-GST or a GST control preparation. Reactions were carried out with 15 nmol substrate. Activity [V] was measured in nmol of phosphate released for each timepoint. FIG. 19 shows PTPase activity of VP2-GST and GST control proteins in the PTPase assay. Reactions were carried out with 10 nmol substrate and for 1 min. Initial activity [V o ] was measured in nmol phosphate released for each substrate concentration. The standard error of the mean for each substrate concentration tested was less than 0.101. FIG. 20 shows TLMV VP2 PTPase activity relative to CAV VP2 activity. detailed-description description="Detailed Description" end="lead"?
Apparatus, method and article of manufacture for managing changes on a compute infrastructure
Provided herein is a system and method for detecting unauthorized and accidental changes to a compute infrastructure. In an exemplary embodiment of the present invention, the system comprises: Manager Nodes (e.g., Managers, Managers with Gateways), Gateways, and Managed Nodes (e.g., Managed Nodes with Agents, Agentless Managed Nodes, Managed Software Components, such as application software, and Managed Special Devices). Agents are comprised of multiple Simple or Dynamic Beans that are used to manage list of Attributes. Simple Beans manage fixed lists of Attributes and Dynamic Beans manage variable lists of Attributes. The system provides for specialized reporting of unauthorized or accidental changes to the compute infrastructure by, among other things, enabling the Attributes to be reported as a single attribute and/or as a group of attributes.
1. In a network having a plurality of nodes having related attributes, a computer-implemented method of detecting and reporting unauthorized changes within said network, comprising: providing a baseline node having predefined baseline attributes associated therewith; selecting at least one target node having target attributes associated therewith; comparing said baseline attributes with said target attributes; generating a display comprising drill down details of said comparison results and said baseline and target attributes. 2. The method as in claim 1 further comprising: encapsulating said comparison results and generating a display comprising drill down details of said encapsulated comparison results and said baseline and target attributes; 3. In a network having a plurality of nodes having related attributes, a computer-implemented method of detecting and reporting unauthorized changes within said network, comprising the steps of: providing a set of predefined baseline attributes; selecting a group of target nodes, each group member having target attributes associated therewith; comparing said set of predefined baseline attributes with said target attributes of said group members to detect change; and generating a display comprising drill down details of said comparison results and said baseline and target attributes. 4. The method as in 3 wherein said generating step further comprises: encapsulating said comparison results and generating an interactive display comprising drill down details of said encapsulated comparison results and said baseline and target attributes. 5. The method as in 3 wherein the target node group comprises at least one target node, subgroups of target nodes or a combination thereof. 6. In a network having a plurality of nodes having related attributes, a computer-implemented method of detecting and reporting unauthorized changes within said network, comprising the steps of: providing a group of baseline attributes; selecting a target node having target attributes associated therewith; comparing said group of baseline attributes with said target attributes to detect change; and generating a display comprising drill down details of said comparison results and said baseline and target attributes. 7. The method as in 6 wherein said generating step further comprises the step of: encapsulating said comparison results and generating an interactive display comprising drill down details of said comparison results and said baseline and target attributes. 8. The method as in 6 wherein said target node comprises at least one target node, subgroups of target nodes or a combination thereof. 9. The method as in 6 wherein said baseline attributes group comprises a set of baseline attributes, at least one subgroup of baseline attributes, or a combination thereof. 10. In a network having a plurality of nodes having related attributes, a computer-implemented method of detecting and reporting authorized changes within said network, comprising: providing a baseline attribute having an attribute transformation function; selecting a target node having a target attribute; comparing said attribute transformation function with said target attribute to detect change; and generating a display comprising drill down details of said comparison results and said baseline and target attributes. 11. The method as in 10 wherein said generating step further comprises the step of: encapsulating said comparison results and generating an interactive display comprising drill down details of said comparison results and said baseline and target attributes. 12. The method as in claim 10 further comprising the steps of: providing an attribute group having an aggregation function; and associating said attribute transformation function with said attribute group, wherein said attribute transformation function references said aggregation function thereby combining said aggregation function and said transform function into a single value for comparison and reporting purposes. 13. In a network having a plurality of nodes having associated node attributes, a computer-implemented method of detecting and reporting unauthorized changes within said network, said method comprising: providing a manager node having predefined baseline attributes for use in detecting changes to said node attributes; providing a node having node attributes to be managed by said manager node; providing a database associated with said manager node for storing node attribute change information; said manager node: 1) polling said node attributes to detect differences between said baseline attributes and said node attributes; and 2) updating said database with data relating to reflect said detected differences. 14. The method as in claim 13 further comprising the step of reporting information related to said detected differences. 15. The method as in claim 14 wherein the reporting step further comprises the step of: genera ting an interactive display comprising drill down details of said detected differences. 16. The method as in claim 13 wherein said node has an agent associated therewith, said agent comprises a control bean and a dynamic bean; said manager updates said control bean with said predefined baseline attributes. 17. In a computer-implemented network comprising a plurality of agentless nodes, a method for managing change events occurring within said network and initiated by said plurality of agentless nodes, said method comprising: providing an agentless node; providing a manager node for managing said agentless node, and providing a gateway node situated between said manager node and said agentless node; said gateway node configured to interface with said manager node and said agentless node to provide a bridge therebetween to enable said agentless node to notify said manager node of a change event affecting said agentless node. 18. A method as in claim 17, wherein said manager node also comprises a database for storing said agentless node change event information. 19. A method as in 18, further comprising the step of reporting said agentless node change event information. 20. The method as in claim 19 wherein said reporting step further comprises the step of: encapsulating said agentless node change event information and generating an interactive display comprising drill down details of said agentless node change event information. 21. The method as in 20, wherein said agentless node change event information is encapsulated into a digital check sum. 22. In a computer network having a plurality of nodes comprising one or more attributes having associated attribute tests, a method for scheduling the execution of said attribute tests to manage change events within said network, said method comprising: providing an attribute test having a trigger condition associated therewith; monitoring said network to detect said trigger condition; and automatically executing said attribute test in response to said trigger condition. 23. A computer-readable medium having stored thereon an archive object data structure for use in a computer-implemented network comprising a plurality of nodes, said archive object data structure to store information relating to change events occurring within said network, said archive object data structure comprising: an archive field containing data representing node state information; and a first interface that receives and stores said node state information in said archive field, a second interface that extracts said stored node state information from said archive field, and a comparison behavior that compares incoming node state information with stored node state information to detect change events occurring within said network. 24. In a JAVA JMX network having a plurality of nodes, a method for extending the Java JMX framework to manage non-Java applications without utilizing a JMX adapter, said method comprising: providing a non-Java application to be managed; providing a Java management bean object for managing said non-Java application; said Java management bean object, 1) invoking a non-Java system command to perform a predefined test having predefined parameters associated with said non-Java application; 2) processing and reporting the results of said non-Java system command invocation; wherein said Java management bean object comprises: a first bean field containing data representing said results of said non-Java system command invocation, a second bean field containing predefined benchmark data, a first exposed bean interface to receive incoming non-Java system command invocation results information, a second exposed bean interface to invoke said non-Java system command; a first bean behavior to store said non-Java system command invocation results information in said command results field, a second bean behavior to compare said incoming non-Java system command invocation information with said stored non-Java system command invocation information to detect and report changes therebetween, and a third bean behavior to trigger an alert notification when said non-Java system command invocation information comparison results deviate from said predefined benchmark data. 25. A method as in claim 24 wherein said bean object is simple or dynamic. 26. A method as in claim 24 wherein said invoking step is done interactively or via a schedule. 27. In a JAVA JMX network embodying the Java JMX framework, a method for extending said Java JMX framework without utilizing a JMX adapter to manage a non-Java application executing within said network, said method comprising: providing a system command interpreter for interpreting a non-Java system command invoked by said non-Java application into a Java JMX command; and providing a Java bean object having a pipe coupled to said system command interpreter, said Java bean object for sending said non-Java system command to said system command interpreter via said pipe. 28. In a computer-implemented network having a plurality of nodes, a method for providing a data warehouse to store network information relating to interactions among said nodes, said method comprising the steps of: providing target nodes; providing manager nodes for managing said target nodes; providing a database associated with said target and manager nodes to store information relating to the interaction among said manager nodes and said target nodes; providing archive objects associated with said manager nodes, said archive objects to store information relating to the interaction among said manager nodes and said target nodes; and determining and distributing said manager-target node interaction information between said archive objects and said database. 29. A method as in claim 28, further comprising retrieving and aggregating said archive object's manager-target node interaction information and reporting said aggregated information.
<SOH> BACKGROUND OF THE INVENTION <EOH>Heretofore, compute infrastructure change management techniques involve methodologies that publicize the change before it occurs so that all potential impacts can be understood and appropriate sign-off achieved. However, the foregoing methodologies are often time-consuming and cumbersome. Additionally, organizations that implement a formal change process are often plagued by unauthorized or accidental changes bundled with authorized changes wherein the unauthorized or even accidental changes are not handled. Accordingly, what is needed is a solution that detects unauthorized and accidental changes on a compute infrastructure and further allows such changes to be minimized by exposing variability with unique data visualization techniques thereby allowing that variability to be minimized or eliminated altogether.
<SOH> SUMMARY OF THE INVENTION <EOH>The present solution addresses the aforementioned problems of the prior art by providing for, among other things, an improved apparatus, method and article of manufacture for managing changes on a compute infrastructure, one that simplifies the complexity of that compute infrastructure by providing a means to reduce the variability of configuration settings, audit those settings and thereby reduce change. Therefore, in accordance with one aspect of the present invention and further described in the Reporting and Grouping section, there is provided at least one exemplary approach for grouping of nodes and attributes in order to manage changes on an exemplary compute infrastructure. In accordance with a second aspect of present invention and further described in the Multi-Line Configuration section, there is provided at least one exemplary approach for reporting multiple attributes as a single attribute at a high-level using a value such as a checksum or digital signature to summarize the values of the multiple lines into a single value. A user can then drill-down to the change details. In accordance with a third aspect of the present invention and further described in the Database Updates section, there is provided at least one exemplary approach for using change notification events to keep multiple database tables synchronized with a source copy. In accordance with a fourth aspect of the present invention and further described in the Dynamic and Control Bean Pairs section, there is provided at least one exemplary approach for using dual Beans, one as a Dynamic Bean and a second as a Control Bean, to manage the attributes and configuration of the Dynamic Bean. In accordance with a fifth aspect of the present invention and further described in the Attribute Test section, there is provided at least one exemplary approach for using commands as a means for populating the values associated with attributes, the commands being executed using the Simple or Dynamic Bean. The commands can be internal Java commands, methods or functions, an external system, application utilities or interactive programs. The commands can be executed on any node and the results stored into a relational database schema. In accordance with a sixth aspect of the present invention and further described in the Extending Java/JMX section, there is provided a bridge between a Java program and system or application utility or interactive command, including the use of pipes to connect Java to non-Java application commands, including interactive commands. In accordance with a seventh aspect of the present invention and further described in the Gateways section, there is provided at least one exemplary approach for using Java/JMX to manage an agentless node and how to extend Java/JMX as a tunnel through a Firewall. In accordance with an eighth aspect of the present invention and further described in the New Data Warehouse Architecture section, there is provided at least one exemplary approach for building a corporate data warehouse architecture leveraging an Archive Object. The new data warehouse model does not store data centrally; rather it uses the Archive Object at Managed Nodes or Gateways to store data. This avoids the purchase of a large centralized data warehouse node, and takes advantages of previously untapped resources (CPU, Disk and Memory) on corporate Managed Nodes to perform the data warehouse function. At the time of this invention, most large computers ran at 30% CPU busy with excess disk, memory and network bandwidth resources. In accordance with a ninth aspect of the present invention, change can be detected and nodes can be synchronized to a baseline. The one-to-many node comparison allows multiple nodes to be synchronized to a master baseline or another node. This provides the tools to reduce the complexity of compute infrastructure by reducing variability of product or node configurations. In accordance with a tenth aspect of the present invention, the scope of attributes are defined in a manner that facilitates the easy comparison of results to multiple nodes, so that the results within a scope type can be filtered. This further improves the node comparison reporting, by providing a finder degree of control of the displayed results. In accordance with an eleventh aspect of the present invention, a unique configuration of agent Mbeans is disclosed, one that uses a set of Mbeans (as Control is and Attribute pairs) to manage both agent and agentless connectivity. In accordance with the twelfth aspect of the present invention, the detection of change by the disclosed framework can cause other events to occur, such as the update of a database table with the newly changed data, or the execution of another attribute test, alert or email. These and other aspects, features and advantages of the present invention will become better understood with regard to the following description and accompanying drawings.
Nasal devices
The present invention relates to a nasal delivery device for and a method of delivering a substance, in particular one of a liquid, as a suspension or solution, or a powder containing a medicament, especially systemic or topical pharmaceuticals, or a vaccine to the nasal airway of a subject.