Modified bovine G-CSF polypeptides and uses thereof are provided.

The economic impact of infectious diseases in food animal production is well documented. Infectious diseases reduce profits, increase production costs, and endanger food products, as well as affect the performance, health, and welfare of the animal. Diseases can reduce the yield and quality of milk resulting in great economic loss to dairy farmers and beef producers, particularly when in some cases infectious microbial diseases cause morbidity and mortality of newborn, young (e.g., replacement stock) or adult animals. Two such diseases, mastitis and bovine respiratory disease (BRD), can have devastating effects on food animal production.
Mastitis is defined as an inflammation of the mammary gland. It may affect any mammal, for example cows, ewes, and goats. Bovine mastitis is an infection of the udder of ruminants such as cows, mainly caused by gram positive and gram negative bacteria and especially in cows in intensive milk producing units. The bacterial infection results in the inflammation of the mammary gland (i.e. teats and udder). Animals may become more susceptible to mastitis due to impaired neutrophil microbicidal function during the periparturient period. The disease is particularly troublesome and of considerable economic importance because the pathogen is readily transferred from one animal to another during the milking process. It often develops in the first few weeks around parturition and can recur with each lactation. Some of the main pathogenic microorganisms causing bovine mastitis are Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Streptococcus dysgalactiae, Escherichia coli, Aerobacter aerogenes, Klebsiella pneumoniae, and Pseudomonas aeruginosa. See also Bovine Mastitis, edited by Glenys Bloomfield, V&O Publications 1987, hereby incorporated by reference. These microorganisms invade the udder through the teat canal and produce inflammation of the milk-producing tissue causing the formation of scar tissue which, once formed, may cause a permanent reduction in the cow's milk production. An infection can also alter the composition, quantity, appearance and quality of the milk. Mastitis-causing pathogens fall into two categories, namely, contagious and environmental. Contagious bacteria, such as Streptococcus agalactiae and Staphylococcus aureus, primarily colonize host tissue sites such as mammary glands, teat canals, and teat skin lesions; and are spread from one infected cow to another during the milking process. Environmental bacteria, often streptococci, enterococci, and coliform organisms, are commonly present within the cow's surroundings from sources such as cow feces, soil, plant material, bedding, or water; and infect by casual opportunistic contact with an animal. The distinction between contagious and environmental pathogens, although not exclusive, is of practical importance because different dairy herd maintenance measures are needed for the different groups of microorganisms. In all bovine mastitis cases, whatever the causal microorganism, the route of transmission of the invading pathogen into the inner gland of the udder is through the teat orifice and teat canal. The common sources of harmful microorganisms include unsanitary milking equipment, the milker, other mastitic animals, an unsanitary stable environment, and the animals' own elimination (defecation/urination) processes.
There are a variety of forms or types of bovine mastitis, with varying severity and symptomatology, including the following: (1) Udder infection: The invasion of the udder cavity by microorganisms that multiply within the gland and cause inflammation; (2) Nonclinical or subclinical mastitis; A form of mastitis in which there is no swelling of the gland or observable abnormality of the milk, although there are changes in the milk that can be detected by specific tests. This type of mastitis is by far the most prevalent and causes the greatest overall loss in most herds. It often is referred to as "hidden" mastitis: (3) Clinical mastitis; A form of mastitis in which the abnormal conditions of the udder and secretion are observable. Mild clinical mastitis involves changes in the milk such as flakes, clots, and a watery or unusual appearance. Heat and sensitiveness of the udder are slight or absent, but there may be signs of swelling. Severe clinical mastitis involves a sudden onset with swelling of the infected quarter which is hot, hard and sensitive. The milk appears abnormal and milk production drops. Sometimes, in addition to the local effects in the udder, the cow herself becomes sick. There are signs of fever, rapid pulse, depression, weakness and loss of appetite. The combination of these conditions often is referred to as acute systemic mastitis, because not only the udder, but the whole animal is affected; and (4) Chronic mastitis; A form of mastitis caused by a persistent udder infection that exists most of the time in the nonclinical form but occasionally can develop into an active clinical form. After these "flare-ups" the nonclinical form usually returns temporarily. (See generally Current Concepts of Bovine Mastitis, published by The National Mastitis Council, Inc., 2nd Ed. 1978 at p. 5.)
Mastitis continues to cause large economic losses to the dairy industry. Mastitis affects the profitability of a herd in a number of ways, both directly and indirectly, including: (1) loss of milk production; (2) higher culling rates of infected cows; (3) decreased value of milk; (4) discarded milk following antibiotic treatment; (5) veterinary costs (antibiotics and veterinary visits); and (6) deaths. (Bovine Mastitis, Glenys Bloomfield, supra, at p. 33.)
Another common disease affecting the cattle industry is shipping fever (bovine respiratory disease or BRD). BRD has been referred to by some as a "disease complex" for two reasons: it usually is caused by a variety of pathogens, both viral and bacterial, that interact with one another to produce full-blown disease, and because the behavior of the pathogens can follow a sequential process that, step by step, results in sick animals. Bacterial pathogens are one of the best known causes of the acute syndrome. The bacterial pathogens may invade the bovine respiratory tract after it has been compromised by a viral infection and other factors, such as the stress of weaning, shipping, change of feed and variation in ambient temperature and humidity, may precede and contribute to infection. In many instances this is added to the cattle's exposure to pathogens during shipping when they are commingling with cattle of other origin in trucks, stockyards and auction barns, resulting in the high incidence of the disease in cattle delivered to the feedlot.
Several species of bacteria have been isolated and associated with BRD, and some of the most common are Mannhemia haemolytica, Pasteurella multocida and (or) Histophilus somni. Haemophilus somnus is a virulent pathogen that causes septicemia in cattle and sometimes the resulting manifestations have been referred to as "Haemophilus somnus complex," of which one form is respiratory disease, viruses such as infectious bovine rhinotracheitis (IBR), bovine viral diarrhea (BVD) and bovine respiratory syncytial virus (BRSV) may also be involved in initiating a BRD complex, often opening the door to secondary bacterial infections.
Because it is virtually impossible to eliminate these organisms from the environment, the BRD complex must be approached from the standpoint of preventing these disease-causing agents from taking hold, and detecting and treating clinical cases as quickly and effectively as possible. Respiratory diseases are a major cause of disease loss in beef cattle. It is generally recognized that the ultimate cause of death in most cases of shipping fever is a bacterial (usually Pasteurella) pneumonia. Pasteurella haemolytica, particularly type 1A, is the most common bacterium isolated from cases of respiratory disease in North America. Vaccination against some of the infectious agents involved in shipping fever is sometimes helpful, but vaccines are available and efficacious for only a few of the agents known to be involved in the disease complex.
Antibiotic therapy has been a major component of mastitis and BRD control strategy. U.S. Pat. No. 7,182,948, which is incorporated by reference herein in its entirety, indicates that antimicrobial teat dips containing iodine have been shown to be effective against mammary infections and mastitis-causing bacteria (Pankey, J. W. et al., (1983) J. Dairy Sci. 66 (1), 161 167). These compositions are usually administered to the teat by dipping or spraying the teat prior to milking as well as after removal of the milking cup. To reduce mastitis, commercial teat dips have been developed containing a variety of antimicrobial agents including iodophors, quaternary ammonium compounds, chlorine release compounds (e.g. alkali hypochlorites), oxidizing compounds (e.g. hydrogen peroxide, peracids), protonated carboxylic acids (e.g. heptanoic, octanoic, nonanoic, decanoic, undecanoic acids), acid anionics (e.g. alkylaryl sulfonic acids), chlorine dioxide (from chlorite), and bisbiguanides such as chlorhexidine. These agents, which have varying degrees of effectiveness, limit the transmission of mastitis by reducing pathogen populations on the teat. However, there are problems associated with the use of antimicrobials. The most prevalent are irritation to the teat and teat cracking. To alleviate these problems, emollient additives such as glycerin and lanolin have been included in such compositions. However, even with the use of these emollients skin irritation can still occur.
U.S. Pat. No. 6,790,867, which is incorporated by reference herein in its entirety, indicates that subcutaneous injections of formulations combining a non-steroidal anti-inflammatory drug (NSAID) such as flunixin, with a fluorinated chloramphenicol or thiamphenicol derivative antibiotic such as florfenicol, may be used to treat BRD. U.S. Patent Application Publication No. 20070155799, which is incorporated by reference herein in its entirety, discloses new fenicol compounds that may be used as antibiotic prodrugs and in combination with NSAIDs or other antibiotics.
The NMC (formerly the National Mastitis Council), a not-for-profit organization devoted to reducing mastitis and enhancing milk quality, stresses the importance of proper teat sanitation, but also proper teat care for the prevention of mastitis. The economic harm caused by mastitis has led to much research in its control. Physical stresses as well as environmental conditions have been reported to be large contributors to mastitis infection. See U.S. Patent Publication No: 20020051789, which is incorporated by reference. Since it was documented that sub-clinical mastitis was directly related to poor teat condition (Neijenhuis, P. et al., (2001) J. Dairy Sci. (84) 2664 2672), a number of commercial teat dip solutions incorporating conditioning agents have evolved (National Mastitis Council, Summary of Peer-Reviewed Publications on Efficacy of Premilking and Postmilking Teat Disinfectants Published Since 1980; January 2002). Teat end callosity and roughness have been shown to have a direct relationship with clinical mastitis (Neijenhuis, F. et al., (2001) J. Dairy Sci. (84) 2664 2672). The reduction of chapping and irritation of teats as well as keeping the teat flexible is very important in controlling mammary infections. Glycerin has also been used as a teat conditioner in teat dip solutions. However, studies indicate no significant decrease in mastitis-causing bacteria such as Staphylococcus aureus, Streptococcus agalactiae, or coliforms when the glycerin content is increased from 2% to 10% in a 1% iodine teat dip solution (National Mastitis Council, Summary of Peer-Reviewed Publications on Efficacy of Premilking and Postmilking Teat Disinfectants Published Since 1980; January 2002). Thus, although products such as teat dip solutions are available, there is still an unmet need to modulate the incidence, recurrence, and/or severity of mastitis.
U.S. Pat. No. 5,849,883, which is incorporated by reference herein in its entirety, discloses a number of the antibiotics used in the treatment of mastitis including but not limited to, beta-lactam antibiotics such as penicillins (ampicillin, cloxacillin, hetacillin, nafcillin, penicillin G, (benzyl penicillin), procaine penicillin) and cephalosporins (cefoperazone, cefuroxime, Cefalonium, cefapirin, cefoxazole, cefracetrile); aminoglycoside antibiotics (framycetin, neomycin, novobiocin, streptomycin); macrolide antibiotics (erythromycin); tetracyclines (chlortetracycline, oxytetracycline); and polypeptide antibiotics (Polymyxin B). Antibiotic treatment for mastitis is usually given by means of intramammary infusions, either in lactating cows when clinical mastitis is detected, or at drying off (dry cow therapy). (Bovine Mastitis, supra, at p. 69.) In cases where severe clinical disease is present, antibiotics must be given parenterally since intramammary infusions are ineffective because of blockage of the ducts).
The early hopes that antibiotics would allow complete control of the disease have not been realized. None of the above mentioned antibiotics utilized thus far has been entirely satisfactory. Additionally, it has been found to be very desirable to replace antibiotic treatment with treatment by non-antibiotic chemo-therapeutic drug compounds, for the following reasons: (1) Antibiotics effective in human medicine should not be utilized in veterinary medicine, in order not to build up strain resistance of bacteria appearing in human diseases; (2) Antibiotics should be reserved for such diseases for which no chemo-therapeutic drug compound would be available, as it has been proved that bacterial strains build up resistance to an antibiotic after extended use of such antibiotic; and (3) Staphylococcus aureus, one of the above-noted pathogens, has already built up a resistance against most of the antibiotics utilized in the treatment of bovine mastitis.
One such method for treatment by a non-antibiotic chemo-therapeutic drug compound is described in U.S. Pat. No. 4,610,993, which is incorporated by reference herein, which claims a method for treating animals for bovine mastitis with an effective amount of at least one pyridine-N-oxide disulfide compound. Another method by the same inventors is described in U.S. Pat. No. 4,401,666, which is incorporated by reference herein, which claims a method for treating animals for bovine mastitis with an effective amount of at least one metallic salt of pyridine 2-thione-N-oxide. Despite these several published methods, it remains very important to find cost-effective methods utilizing non-antibiotic compounds which would substantially overcome the drawbacks of antibiotics used thus far and yet would be effective in treating and preventing mastitis.
Another common disease affecting the cattle industry is shipping fever (bovine respiratory disease). Respiratory diseases are a major cause of disease loss in beef cattle. The term "shipping fever" is used to describe the respiratory disease complex observed in cattle 6 months of age or older after shipment either into feedlots or onto pasture. The stresses of weaning, castration, dehorning, fasting, overcrowding, exposure to infectious agents, dietary changes, transportation, environmental temperature extremes, and other stressors combined with viral, bacterial, mycoplasmal, and/or chlamydial infections contribute to the shipping fever complex. Mixing calves from different farms and/or salebarns greatly facilitates exposure to infectious agents. U.S. Pat. No. 6,497,869, which is incorporated by reference herein, describes some of initial infectious agents that may affect cattle. Population mixing may be a more important predisposing factor to shipping fever than stressors, although disease can occur without mixing and stressors usually dramatically worsen respiratory disease. Attempts to reduce stress by weaning, castrating, dehorning, etc. and acclimating cattle to new diets days or weeks prior to shipment are sometimes successful (but may not be cost-effective) in reducing the incidence of shipping fever. Vaccination against some of the infectious agents involved in shipping fever is sometimes helpful, but vaccines are available and efficacious for only a few of the agents known to be involved in the disease complex.
It is generally recognized that the ultimate cause of death in most cases of shipping fever is a bacterial (usually Pasteurella) pneumonia. Pasteurella haemolytica, particularly type 1A, is the most common bacterium isolated from cases of respiratory disease in North America. Attempts to experimentally reproduce bacterial pneumonia in cattle are usually unsuccessful without severe stress and predisposing damage to the respiratory tract. It is generally believed that during times of stress, viruses, mycoplasma, and/or chlamydia most often provide the initial damage to the respiratory tract which predisposes to severe bacterial infection and disease.
A typical clinical respiratory disease outbreak usually begins within hours or days of the cattle's arrival at the feedlot. Recently shipped cattle in the 400 to 500 pound weight range commonly have 10 to 80% morbidity and 1 to 10% mortality, or more, to respiratory tract disease. When the serum of cattle is analyzed for a four-fold antibody rise (seroconversion) and the respiratory tract and its secretions subjected to microbiologic isolations, a myriad of etiologic agents can be identified. Many animals, those sick and those apparently healthy, can be shown to have undergone infection by one or more agents (respiratory tract disease is probably seldom due to only one infectious agent). Although bovine respiratory disease complex is recognized clinically in the feedlot after arrival, the infections giving rise to clinical disease probably start at the salebarns, where cattle are first assembled from different farms. See also Bovine Respiratory Disease, Loan, R. W. Texas A & M University Press, 1984, hereby incorporated by reference.
Administration of a compound that treats or modulates the incidence, recurrence, duration, and/or severity of mastitis or respiratory disease in cattle or other infections in non-human animals, including but not limited to, cattle, poultry, swine, horses, dogs, and cats would be useful in veterinary medicine. Examples of such infections include but are not limited to, neonatal septicemia in horses, pleuropneumaonia in pigs, and pneumonia in non-human animals. Such compounds may restore or modulate neutrophil function in the animal.
The growth hormone (GH) supergene family (Bazan, F. Immunology Today 11: 350-354 (1991); Mott, H. R. and Campbell, I. D. Current Opinion in Structural Biology 5: 114-121 (1995); Silvennoinen, O. and Ihle, J. N. (1996) SIGNALING BY THE HEMATOPOIETIC CYTOKINE RECEPTORS) represents a set of proteins with similar structural characteristics. Each member of this family of proteins comprises a four helical bundle. While there are still more members of the family yet to be identified, some members of the family include the following: growth hormone, prolactin, placental lactogen, erythropoietin (EPO), thrombopoietin (TPO), interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subunit), IL-13, IL-15, oncostatin M, ciliary neurotrophic factor, leukemia inhibitory factor, alpha interferon, beta interferon, gamma interferon, omega interferon, tau interferon, epsilon interferon, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and cardiotrophin-1 (CT-1) ("the GH supergene family"). Members of the GH supergene family have similar secondary and tertiary structures, despite the fact that they generally have limited amino acid or DNA sequence identity. The shared structural features allow new members of the gene family to be readily identified. Four helical bundle polypeptides are described in WO 2005/074650 entitled "Modified Human Four Helical Bundle Polypeptides and Their Uses," which is incorporated by reference in its entirety.
A member of the GH supergene family is Granulocyte Colony Stimulating Factor (G-CSF). Granulocyte colony stimulating factor (G-CSF) is one of several glycoprotein growth factors known as colony stimulating factors (CSFs) because they support the proliferation of haemopoietic progenitor cells. G-CSF stimulates the proliferation of specific bone marrow precursor cells and their differentiation into granulocytes. It is distinguished from other CSFs by its ability to both stimulate neutrophilic granulocyte colony formation in semi-solid agar and to induce terminal differentiation of murine myelomonocytic leukemic cells in vitro. Granulocyte Colony-Stimulating Factor is a potent stimulus for neutrophil proliferation and maturation in vivo (Cohen et al., Proc. Natl. Acad. Sci. 1987; 84: 2484-2488 see also Heidari et al., Vet. Immunol. Immunopathol. 2001; 81:45-57, herein incorporated by reference). G-CSF is also capable of inducing functional activation or "priming" or mature neutrophils in vitro (Weisbart, R. H., Gasson, C. G., and D. W. Golde. Annals of Internal Medicine 1989; 110:297-303). G-CSF has been shown to prime human granulocytes, and enhance superoxide release stimulated by the chemotactic peptide, N-formyl-methionyl-leucyl-phenalalanine (S. Kitagawa, et al., Biochem. Biophys, Res. Commun. 1987; 144:1143-1146, and C. F. Nathan, Blood 1989; 74:301-306), and activate human neutrophil IgA mediated phagocytosis (Weisbart, R. H., et al., Nature 1988; 332: 647-649).
Neutrophils are a critical component of host defense mechanisms against bacterial and fungal infections. G-CSF is capable of inducing an increase in the absolute number of circulating neutrophils and enhances neutrophil function.
The cDNA cloning and expression of recombinant human G-CSF (hG-CSF) has been described, and it has been confirmed that the recombinant hG-CSF exhibits most, if not all, of the biological properties of the native molecule (Souza, L. et al. Science 232, 61-65 (1986)). Sequence analysis of the cDNA and genomic DNA clones has allowed the deduction of the amino acid sequence and reveals that the protein is 204 amino acids long with a signal sequence of 30 amino acids. The mature protein is 174 amino acids long and possesses no potential N-linked glycosylation sites but several possible sites for O-linked glycosylation.
The cloning and expression of cDNA encoding human G-CSF has been described by two groups (Nagata, S. et. al., Nature 319, 415-418 (1986); Souza, L. M. et al., Science 232, 61-65 (1986)). The first report of a G-CSF cDNA clone suggested that the mature protein was 177 amino acids in length. The authors reported that they had also identified a cDNA clone for G-CSF that coded for a protein that lacked a stretch of three amino acids. This shorter from of G-CSF cDNA expresses the expected G-CSF activity. The second report describes a cDNA sequence identical to this short form and makes no mention of other variants. Since these authors confirmed that the short cDNA expresses G-CSF with the expected profile of biological activity, it is probable that this is the important form of G-CSF and that the longer form is either a minor splicing variant or the result of a cloning artifact.
Matsumoto et al., in Infection and Immunity, Vol. 55, No. 11, p. 2715 (1987) discuss the protective effect of human G-CSF on microbial infection in neutropenic mice.
The following patent publications relate to G-CSF: WO 8703689, which is incorporated by reference herein, describes hybridomas producing monoclonal antibodies specific for human G-CSF and their use in the purification of G-CSF; WO 8702060, which is incorporated by reference herein, discloses human G-CSF like polypeptides and methods of producing them; U.S. Pat. No. 4,810,643, which is incorporated by reference herein, discloses human G-CSF like polypeptides, sequences encoding them and methods of their production; and WO 8604605 and WO 8604506, which are incorporated by reference herein, disclose a gene encoding human G-CSF and infection inhibitors containing human G-CSF. Isolation of h-GCSF and production of G-CSF in host cells such as E. coli are described in, e.g., U.S. Pat. Nos. 4,810,643; 4,999,291; 5,580,755; and 6,716,606, which are incorporated by reference herein.
G-CSF is a pharmaceutically active protein which regulates proliferation, differentiation, and functional activation of neutrophilic granulocytes (Metcalf, Blood 67:257 (1986); Yan, et al. Blood 84(3): 795-799 (1994); Bensinger, et al. Blood 81(11): 3158-3163 (1993); Roberts, et al., Expt'l Hematology 22: 1156-1163 (1994); Neben, et al. Blood 81(7): 1960-1967 (1993); Welte et al. PNAS-USA 82: 1526-1530 (1985); Souza et al. Science 232: 61-65 (1986) and Gabrilove, J. Seminars in Hematology 26:2 1-14 (1989)). G-CSF was purified to homogeneity from cell culture supernatants of the human bladder carcinoma cell line 5637 (Welte et al., Proc. Natl. Acad. Sci (1985) 82:1526-30). The sequence of the cDNA coding for native hG-CSF is known from Souza et al., Science (1986) 232:61-65. As a consequence of alternative splicing in the second intron two naturally occurring forms of hG-CSF exist with 204 or 207 amino acids of which the first 30 represent a signal peptide (Lymphokines, IRL Press, Oxford, Washington D.C., Editors D. Male and C. Rickwood). The mature protein was shown to have a molecular weight of about 19 kDa and has 5 cysteine residues which can form intermolecular or intramolecular disulfide bridges. Binding studies have shown that hG-CSF binds to neutrophilic granulocytes. Little to no binding is observed with erythroid, lymphoid eosinophilic cell lines as well as with macrophages.
In humans, endogenous G-CSF is detectable in blood plasma (Jones et al. Bailliere's Clinical Hematology 2:1 83-111 (1989)). hG-CSF is produced by fibroblasts, macrophages, T cells, trophoblasts, endothelial cells and epithelial cells and is the expression product of a single copy gene comprised of four exons and five introns located on chromosome seventeen. Transcription of this locus produces a mRNA species which is differentially processed, resulting in two forms of hG-CSF mRNA, one version coding for a protein of 177 amino acids, the other coding for a protein of 174 amino acids (Nagata et al, EMBO J 5: 575-581 (1986)), and the form comprised of 174 amino acids has been found to have the greatest specific in vivo biological activity, hG-CSF is species cross-reactive, such that when human G-CSF is administered to another mammal such as a mouse, canine or monkey, sustained neutrophil leukocytosis is elicited (Moore et al. PNAS-USA 84: 7134-7138 (1987)).
G-CSF can be obtained and purified from a number of sources. Natural human G-CSF (nhG-CSF) can be isolated from the supernatants of cultured human tumor cell lines. The development of recombinant DNA technology, see, for instance, U.S. Pat. No. 4,810,643 (Souza) incorporated herein by reference, has enabled the production of commercial scale quantities of G-CSF in glycosylated form as a product of eukaryotic host cell expression, and of G-CSF in non-glycosylated form as a product of prokaryotic host cell expression.
G-CSF has been found to be useful in the treatment of indications where an increase in neutrophils will provide benefits. G-CSF can mobilize stem and precursor cells from bone marrow and is used to treat patients whose granulocytes have been depleted by chemotherapy, or as a prelude to bone marrow transplants. For example, for cancer patients, G-CSF is beneficial as a means of selectively stimulating neutrophil production to compensate for hematopoietic deficits resulting from chemotherapy or radiation therapy. Other indications include treatment of various infectious diseases and related conditions, such as sepsis, which is typically caused by a metabolite of bacteria. G-CSF is also useful alone, or in combination with other compounds, such as other cytokines, for growth or expansion of cells in culture, for example, for bone marrow transplants.
The G-CSF receptor (G-CSFR) is a member of the hematopoietic/cytokine/growth factor receptor family, which includes several other growth factor receptors, such as the interleukin (IL)-3, -4 and -6 receptors, the granulocyte macrophage colony-stimulating factor (GM-CSF) receptor, the erythropoietin (EPO) receptor, as well as the prolactin and growth hormone receptors. See, Bazan, Proc. Natl. Acad. Sci USA 87: 6934-6938 (1990). Members of the cytokine receptor family contain four conserved cysteine residues and a tryptophan-serine-X-tryptophan-serine motif positioned just outside the transmembrane region. The conserved sequences are thought to be involved in protein-protein interactions. See, e.g., Chiba et al., Biochim. Biophys. Res. Comm. 184: 485-490 (1992). The G-CSF receptor consists of a single peptide chain with a molecular weight of about 150 kD (Nicola, Immunol. Today 8 (1987), 134).
Glycosylated hG-CSF has been compared with de-glycosylated hG-CSF, prepared by in vitro enzymatic digestion with neuraminidase and endo-α-N-acetylgalactosaminidase, with respect to its stability as a function of pH and temperature (Oh-eda et al., 1990, J. Biol. Chem. 265 (20): 11432-35). The de-glycosylated hG-CSF, dissolved at a concentration of 1 μg/mL in 20 mM phosphate buffer containing 0.2 M NaCl and 0.01% Tween 20 was rapidly inactivated within the pH range of from about pH 7 to about pH 8 after a two-day incubation at 37° C. In contrast, glycosylated hG-CSF retained over 80% of its activity under the same conditions. Furthermore, evaluation of the thermal stability of both forms of hG-CSF, measured by biological assay and calorimetric analysis, indicated that de-glycosylated hG-CSF was less thermally stable than the native form of hG-CSF.
A number of approaches have been taken in order to provide stable, pharmaceutically acceptable G-CSF compositions. One approach to improving the composition stability of G-CSF involves the synthesis of derivatives of the protein. U.S. Pat. No. 5,665,863 discloses the formation of recombinant chimeric proteins comprising G-CSF coupled with albumin, which have new pharmacokinetic properties. U.S. Pat. Nos. 5,824,784 and 5,320,840, disclose the chemical attachment of water-soluble polymers to proteins to improve stability and provide protection against proteolytic degradation, and more specifically, N-terminally modified G-CSF molecules carrying chemically attached polymers, including polyethylene glycol.
Structures of a number of cytokines, including G-CSF (Zink et al., FEBS Lett. 314:435 (1992); Zink et al., Biochemistry 33:8453 (1994); Hill et al., Proc. Natl. Acad. Sci. USA 90:5167 (1993)), GM-CSF (Diederichs, K., et al. Science 154: 1779-1782 (1991); Walter et al., J. Mol. Biol. 224:1075-1085 (1992)), IL-2 (Bazan, J. F. Science 257: 410-411 (1992); McKay, D. B. Science 257: 412 (1992)), IL-4 (Redfield et al., Biochemistry 30: 11029-11035 (1991); Powers et al., Science 256:1673-1677 (1992)), and IL-5 (Milburn et al., Nature 363: 172-176 (1993)) have been determined by X-ray diffraction and NMR studies and show striking conservation with the GH structure, despite a lack of significant primary sequence homology.
An alternative approach to increasing stability of G-CSF in composition involves alteration of the amino acid sequence of the protein. U.S. Pat. No. 5,416,195 discloses genetically engineered analogues of G-CSF having improved composition stability, wherein the cysteine residue normally found at position 17 of the mature polypeptide chain, the aspartic acid residue found at position 27, and at least one of the tandem proline residues found at positions 65 and 66, are all replaced with a serine residue. U.S. Pat. No. 5,773,581 discloses the genetically engineered G-CSF analogues of G-CSF that have been covalently conjugated to a water soluble polymer.
The various forms of human G-CSF, including their preparation and purification, useful in a method for treating or preventing mastitis are described in detail in U.S. Pat. No. 4,810,643, which is hereby incorporated by reference. U.S. Pat. No. 4,810,643 describes and claims novel gene segments, biologically functional recombinant plasmids and viral DNA vectors and prokaryotic and eukaryotic host cells, which contain a G-CSF gene or a genetically engineered variant of a G-CSF gene. The host cells express biologically active G-CSF or a genetically engineered variant of G-CSF. U.S. Pat. No. 5,849,883 and WO 89/10932 describe various studies with human G-CSF in cattle. The studies were performed evaluated respiratory diseases (Pasteurella hemolytica), responses to bacterial challenges (Klebsiella pneumonia), or coliform mastitis (E. coli) in cattle.
U.S. Pat. No. 5,849,883, which is incorporated by reference herein in its entirety, presents the polynucleotide and polypeptide sequence of mature bovine G-CSF (bG-CSF), and describes methods to clone, isolate, and purify the polypeptide and analogs thereof. Mature b-GCSF is 174 amino acids in length (SEQ ID NO: 1) that has 82% homology to hG-CSF. A bG-CSF polypeptide with an initial methionine amino acid residue is shown as SEQ ID NO: 2. The polynucleotide sequence that encodes SEQ ID NO: 1 is shown as SEQ ID NO: 3. The polynucleotide sequence that encodes SEQ ID NO: 2 is shown as SEQ ID NO: 4. Heidari et al. describe the expression, purification, and biological activities of bG-CSF in Veterinary Immunology and Immunopathology (2001) 81:45-57.
Covalent attachment of the hydrophilic polymer poly(ethylene glycol), abbreviated PEG, is a method of increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending the circulation time of many biologically active molecules, including proteins, peptides, and particularly hydrophobic molecules. PEG has been used extensively in pharmaceuticals, on artificial implants, and in other applications where biocompatibility, lack of toxicity, and lack of immunogenicity are of importance. In order to maximize the desired properties of PEG, the total molecular weight and hydration state of the PEG polymer or polymers attached to the biologically active molecule must be sufficiently high to impart the advantageous characteristics typically associated with PEG polymer attachment, such as increased water solubility and circulating half life, while not adversely impacting the bioactivity of the parent molecule.
PEG derivatives are frequently linked to biologically active molecules through reactive chemical functionalities, such as lysine, cysteine and histidine residues, the N-terminus and carbohydrate moieties. Proteins and other molecules often have a limited number of reactive sites available for polymer attachment. Often, the sites most suitable for modification via polymer attachment play a significant role in receptor binding, and are necessary for retention of the biological activity of the molecule. As a result, indiscriminate attachment of polymer chains to such reactive sites on a biologically active molecule often leads to a significant reduction or even total loss of biological activity of the polymer-modified molecule. R. Clark et al., (1996), J. Biol. Chem., 271:21969-21977. To form conjugates having sufficient polymer molecular weight for imparting the desired advantages to a target molecule, prior art approaches have typically involved random attachment of numerous polymer arms to the molecule, thereby increasing the risk of a reduction or even total loss in bioactivity of the parent molecule.
Reactive sites that form the loci for attachment of PEG derivatives to proteins are dictated by the protein's structure. Proteins, including enzymes, are composed of various sequences of alpha-amino acids, which have the general structure H2N—CHR—COOH. The alpha amino moiety (H2N—) of one amino acid joins to the carboxyl moiety (—COOH) of an adjacent amino acid to form amide linkages, which can be represented as —(NH—CHR—CO)n—, where the subscript "n" can equal hundreds or thousands. The fragment represented by R can contain reactive sites for protein biological activity and for attachment of PEG derivatives.
For example, in the case of the amino acid lysine, there exists an —NH2 moiety in the epsilon position as well as in the alpha position. The epsilon —NH2 is free for reaction under conditions of basic pH. Much of the art in the field of protein derivatization with PEG has been directed to developing PEG derivatives for attachment to the epsilon —NH2 moiety of lysine residues present in proteins. "Polyethylene Glycol and Derivatives for Advanced PEGylation", Nektar Molecular Engineering Catalog, 2003, pp. 1-17. These PEG derivatives all have the common limitation, however, that they cannot be installed selectively among the often numerous lysine residues present on the surfaces of proteins. This can be a significant limitation in instances where a lysine residue is important to protein activity, existing in an enzyme active site for example, or in cases where a lysine residue plays a role in mediating the interaction of the protein with other biological molecules, as in the case of receptor binding sites.
A second and equally important complication of existing methods for protein PEGylation is that the PEG derivatives can undergo undesired side reactions with residues other than those desired. Histidine contains a reactive imino moiety, represented structurally as —N(H)—, but many chemically reactive species that react with epsilon —NH2 can also react with —N(H)—. Similarly, the side chain of the amino acid cysteine bears a free sulfhydryl group, represented structurally as —SH. In some instances, the PEG derivatives directed at the epsilon —NH2 group of lysine also react with cysteine, histidine or other residues. This can create complex, heterogeneous mixtures of PEG-derivatized bioactive molecules and risks destroying the activity of the bioactive molecule being targeted. It would be desirable to develop PEG derivatives that permit a chemical functional group to be introduced at a single site within the protein that would then enable the selective coupling of one or more PEG polymers to the bioactive molecule at specific sites on the protein surface that are both well-defined and predictable.
In addition to lysine residues, considerable effort in the art has been directed toward the development of activated PEG reagents that target other amino acid side chains, including cysteine, histidine and the N-terminus. See, e.g., U.S. Pat. No. 6,610,281 which is incorporated by reference herein, and "Polyethylene Glycol and Derivatives for Advanced PEGylation", Nektar Molecular Engineering Catalog, 2003, pp. 1-17. A cysteine residue can be introduced site-selectively into the structure of proteins using site-directed mutagenesis and other techniques known in the art, and the resulting free sulfhydryl moiety can be reacted with PEG derivatives that bear thiol-reactive functional groups. This approach is complicated, however, in that the introduction of a free sulfhydryl group can complicate the expression, folding and stability of the resulting protein. Thus, it would be desirable to have a means to introduce a chemical functional group into bioactive molecules that enables the selective coupling of one or more PEG polymers to the protein while simultaneously being compatible with (i.e., not engaging in undesired side reactions with) sulfhydryls and other chemical functional groups typically found in proteins.
As can be seen from a sampling of the art, many of these derivatives that have been developed for attachment to the side chains of proteins, in particular, the —NH2 moiety on the lysine amino acid side chain and the —SH moiety on the cysteine side chain, have proven problematic in their synthesis and use. Some form unstable linkages with the protein that are subject to hydrolysis and therefore decompose, degrade, or are otherwise unstable in aqueous environments, such as in the bloodstream. Some form more stable linkages, but are subject to hydrolysis before the linkage is formed, which means that the reactive group on the PEG derivative may be inactivated before the protein can be attached. Some are somewhat toxic and are therefore less suitable for use in vivo. Some are too slow to react to be practically useful. Some result in a loss of protein activity by attaching to sites responsible for the protein's activity. Some are not specific in the sites to which they will attach, which can also result in a loss of desirable activity and in a lack of reproducibility of results. In order to overcome the challenges associated with modifying proteins with poly(ethylene glycol) moieties, PEG derivatives have been developed that are more stable (e.g., U.S. Pat. No. 6,602,498, which is incorporated by reference herein) or that react selectively with thiol moieties on molecules and surfaces (e.g., U.S. Pat. No. 6,610,281, which is incorporated by reference herein). There is clearly a need in the art for PEG derivatives that are chemically inert in physiological environments until called upon to react selectively to form stable chemical bonds.
The use of conjugates of hydroxyalkylstarch, and in particular the use of hydroxyethylstarch (HES), covalently linked to a polypeptide have been disclosed in order to potentially alter the polypeptide's immunogenicity and/or allergenicity. HESylation is an alternative technology that has been disclosed in a series of patent applications assigned to Fresenius Kabi AB including U.S. Patent Publication Numbers 20050063943, 20060121073, 20010100163, 20050234230, 20050238723, 20060019877, 20070134197, 20070087961, as well as U.S. Pat. No. 7,285,661, all of which are incorporated herein by reference. HES is a modified natural polymer that has been clinically used as a plasma volume expander and HESylation represents the technology of coupling drug substances with HES derivatives in order to modify drug characteristics, such as pharmacokinetics or water solubility. This also includes the prolongation of protein plasma circulation via an increased stability of the molecule and a reduced renal clearance, resulting in an increased biological activity. In addition, the immunogenicity or allergenicity might be reduced. By varying different parameters, such as the molecular weight of HES, a wide range of HES conjugates can be customized. Nevertheless, hydroxyethyl starch shares a common disadvantage with all other presently available polymers: its polydispersity. The polymer conjugates are a mixture of molecules having molecular weights distributed around an average value. This lack of homogeneity results in a low level of chemical and biochemical characterization and could prevent the pharmaceutically active component to reach its site of action (receptor, enzyme, etc.). In these cases the drug to be active requires its delivery in the original unconjugated form, and thus cleavage of the polymer by metabolic reactions is required for its pharmaceutical efficacy.
Recently, an entirely new technology in the protein sciences has been reported, which promises to overcome many of the limitations associated with site-specific modifications of proteins. Specifically, new components have been added to the protein biosynthetic machinery of the prokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001), Science 292:498-500) and the eukaryote Saccharomyces cerevisiae (S. cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), which has enabled the incorporation of non-genetically encoded amino acids to proteins in vivo. A number of new amino acids with novel chemical, physical or biological properties, including photoaffinity labels and photoisomerizable amino acids, photocrosslinking amino acids (see, e.g., Chin, J. W., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:11020-11024; and, Chin, J. W., et al., (2002) J. Am. Chem. Soc. 124:9026-9027), keto amino acids, heavy atom containing amino acids, and glycosylated amino acids have been incorporated efficiently and with high fidelity into proteins in E. coli and in yeast in response to the amber codon, TAG, using this methodology. See, e.g., J. W. Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), Chem Bio Chem 3(11):1135-1137; J. W. Chin, et al., (2002), PNAS United States of America 99:11020-11024; and, L. Wang, & P. G. Schultz, (2002), Chem. Comm., 1:1-11. All references are incorporated by reference in their entirety. These studies have demonstrated that it is possible to selectively and routinely introduce chemical functional groups, such as ketone groups, alkyne groups and azide moieties, that are not found in proteins, that are chemically inert to all of the functional groups found in the 20 common, genetically-encoded amino acids and that may be used to react efficiently and selectively to form stable covalent linkages.
The ability to incorporate non-genetically encoded amino acids into proteins permits the introduction of chemical functional groups that could provide valuable alternatives to the naturally-occurring functional groups, such as the epsilon —NH2 of lysine, the sulfhydryl —SH of cysteine, the imino group of histidine, etc. Certain chemical functional groups are known to be inert to the functional groups found in the 20 common, genetically-encoded amino acids but react cleanly and efficiently to form stable linkages. Azide and acetylene groups, for example, are known in the art to undergo a Huisgen [3+2] cycloaddition reaction in aqueous conditions in the presence of a catalytic amount of copper. See, e.g., Tornoe, et al., (2002) J. Org. Chem. 67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599. By introducing an azide moiety into a protein structure, for example, one is able to incorporate a functional group that is chemically inert to amines, sulfhydryls, carboxylic acids, hydroxyl groups found in proteins, but that also reacts smoothly and efficiently with an acetylene moiety to form a cycloaddition product. Importantly, in the absence of the acetylene moiety, the azide remains chemically inert and unreactive in the presence of other protein side chains and under physiological conditions.
The present invention addresses, among other things, problems associated with the activity and production of bG-CSF polypeptides, and also addresses the production of a bG-CSF polypeptide with improved biological or pharmacological properties and/or improved therapeutic half-life.

Modified bovine G-CSF polypeptides and their uses


A host may be selected for a peer-to-peer grid based on discovered NAT type. NAT profile information may be collected by each of the peers and shared with the other peers. Each peer receives NAT profile information for the other peers. Each peer may determine from the NAT profile information for the peer and the NAT profile information for the other peers which of the two or more peers to designate as the host.

Presently, during game play amongst several clients of a computer network, clients communicate directly with the server. The central server processes data from every client relaying this data to all other clients so that several clients in the network can engage in game play with each other through a central server. The central server's ability to communicate data amongst several clients is limited by bandwidth, and so communication outside of game play data is limited.
Aside from game play data, clients may want to communicate other information to each other without having to go through a bandwidth-limited central server. Examples of such communication may include voice over Internet protocol (VoIP), bit torrents, video data, file sharing, and data streaming. By allowing clients to communicate amongst each other without the need of a central server, some of the central server's responsibilities may be offloaded to the clients.
A peer-to-peer network is established when a host is determined amongst the numerous clients participating in the P2P network. The host takes the duty of a central server and directs communication amongst clients (hereinafter referred to as peers when referencing non-hosts in a P2P network).
It is within this context the embodiments of the present invention arise.

Method for host selection based on discovered NAT type


When fresh water is produced from salt water or similar by evaporation of untreated or crude liquid in an evaporation device under partial vacuum and by vapor condensation in a condensation device connected with the vapor outlet of the evaporation device a high degree of evaporation and cost effectiveness can be obtained in that the evaporation device and the condensation device in a disconnected condition, are filled with crude or clean liquid, respectively, and are subsequently exposed to a partial vacuum created by volume enlargement under hermetically sealed conditions and that the evaporation device and the condensation device are not flow-connected with each other until they are under partial vacuum.

Such a process and device is known from DE 33 45 937 A1 where the connecting line between an evaporation device and a condensation device cannot be shut off. To produce the desirable partial vacuum, the evaporation device is connected with at least one tank which is filled with untreated liquid, and provided at a height that is at least by the water column producible by the ambient air pressure above a water level, which tank is provided with a downpipe that is immersed in the water level and can be shut off. By opening the downpipe, liquid flows off causing a partial vacuum in the evaporation device if venting thereof is prevented, which partial vacuum is in this case passed on to the condensation device via the open connecting line. The required location of the tank at a high level and the immersing of the bottom end of the downpipe in an existing water level result in a relatively large overall height of the device and high constructional expenditure. Nevertheless, due to the existing connection between the evaporation device and the condensation device, the obtainable partial vacuum is relatively small. In addition, the liquid in which the downpipe is immersed may contain gas bubbles produced by gas emission etc., which may accumulate at the bottom end of the downpipe and then rise in it, which may result in a further deterioration of the obtainable partial vacuum.

Method and device for producing a pure liquid from a crude liquid


A braking device comprises first and second braking device respectively constrained to rotate with the brake case and with a brake shaft. The device comprises a braking piston, resilient device for displacing the piston axially towards a braking position, hydraulic device for displacing the piston axially in the opposite direction towards a brake-release position, and mechanical brake-release device for bringing the piston into its brake-release position and for holding it there. These mechanical brake-release device comprise at least two brake-release systems each comprising a bore having at least one substantially radial first segment, a screw displaceable in the bore, and displacement transfer device comprising at least one intermediate member suitable for transforming the radial displacement of the screw into axial displacement for pushing away the piston.

Patent FR-A-2 655 091 discloses a hydraulic motor whose braking piston includes, on its outside face, an axial tapped region in alignment with the axis of rotation of the motor. To provide mechanical brake release, a screw passing through a central bore of the motor cover situated outside the brake piston can be screwed into said tapped region while its head is directly or indirectly in abutment against the cover, thereby displacing the braking piston in the second direction and holding it in the brake-release position. That system is effective, providing it is possible to gain access to the head of the screw, and that requires sufficient space next to the axial end of the motor adjacent to the cover. This is not always the case, since the motor may be placed in a space of small dimensions, or, for example, it may be axially integrated in the transmission system of a vehicle.


The present invention is a method for handling multiple aspects of the acquisition of vehicles, such as automobiles and trucks, in a unified and streamlined fashion. A system for acquisition of vehicles that integrates various product and service providers with retailers and consumers is presented from various aspects in order to realize efficiencies and opportunities that are impossible in the present mode of vehicle acquisition.

The present invention relates to a method and corresponding software program for handling vehicle acquisitions.
In the traditional mode of vehicle acquisition, many individual transactions must be performed in order to complete a transaction, such as pre-qualification for financing, organization of value-added products, insurance and more. Each of these transactions must be handled one at a time, with qualification and/or applicability evaluated for each by a retailer and each party involved. Financing must be qualified, applied for, negotiated, and finalized. Insurance services must be determined for each transaction. Value-added services, such as warranties, must be determined on a case-by-case basis. Trades, payoffs and purchases are handled separately. In short, each aspect of the vehicle acquisition transaction is handled individually as part of a single acquisition, making the overall experience time consuming, complex and prone to error, both because of retailer inexperience and high turnover in the retail vehicle industry, as well as the typical complications that arise from such an involved transaction.
Further, from a service and product provider's standpoint, the system is fraught with complication as well. Retailers must be trained on many different systems, which not only amount to difficulty from a transactional standpoint, but can have legal ramifications as well. For example, serious repercussions can arise should consumer credit laws not be fully complied with. Additionally, retailers generally do not have the resources or know-how to have working relationships with lenders, and certainly do not have the capability to have such relationships with multiple lenders in order to better match consumers to financial institutions. As a result, the ability to negotiate diminishes. Smaller retailers may also not be savvy enough to be cognizant of, much less knowledgeable in, many emerging developments and/or finer points of the transaction, such as extended warranties, GAP insurance and the like. They may also be unaware or unable to exploit additional fee income opportunities, like VIN etching, credit insurance or special credit insurance.
From a lender's perspective, the system is also less than perfect. Risk management in the used car financing realm is commonly filled with defaults, slow pays and collection issues. Reaching the appropriate customers for a financial institution is a "hit or miss" strategy at best, with lenders pointlessly charging many dollars in application fees which will likely never come to fruition. A large number of applications are also never approved, which translates to wasted time and money on the processing of unqualified applicants.
Similarly, other service and product providers are often unable to tap an appreciable percentage of the market because impediments to the market exist. Cost, time and the simple knowledge of who to target leave these providers generally to deal with only a portion of the franchise retailers, who comprise less than half of the estimated overall market, leaving independents a giant untapped resource. Expanding the product base to the many retailers that may not be implementing products and services such as GAP insurance and extended warranties is thus a goal as well.
Obvious downsides to the current method of retail exist. Having to individually handle each component of these vehicle transactions, coupled with the numerous vendors of the varying services, makes the dispensation of the transaction a long and arduous task, as well as making the single act of acquiring an automobile a multi-faceted process rife with the possibility of error and inefficiency. For example, certain financial institutions only offer financing to customers of certain financial aptitude, making submission of some customers' lending requests a moot issue. Certain value-added services are only available to certain customers, and therefore should not be offered. Certain other value-added services should be offered, and oft are not, effectually missing sales. Compliance and consumer credit are required for certain portions of a transaction, and not others. All these nuances in the transaction, coupled with the relative lack of knowledge (partly as result of high turnover) in the vehicle sales representative business, make mistakes and time commitment a very relevant issue.
The consequence of these issues is delayed acquisition, loss of sales, reduced productivity, consumer dissatisfaction, increased floor plan expenses, increases in lender processing, loss of value-added product sales, possible compliance violations and other lost opportunities for consumers, financiers and retailers.
The present invention of a unified transaction is thus a much-improved mode of handling this acquisition process, primarily because it overcomes the multitude of problems recited above by unifying the transactions into a single process, decreasing complexity and costs while simultaneously increasing productivity, breadth of products and offerings available, the number of retailer and service provider relationships, volume of sales and products sold as well as consumer satisfaction. This overall transactional process can be defined as a "metamarket," or a set of related activities that accomplish a single consumer goal. In this case, the metamarket goal is that of the acquisition of an automobile.
In addition to the inherent efficiencies of a metamarket driven by a metamediary, another benefit of the present invention is the resultant drastic reduction in risk and optimized risk management for retailers and lenders in the vehicle acquisition transaction. In the present invention, the "metamediary" (or third party that routes information to appropriate destinations to present a seamless approach to the consumer) has the option to undertake the risk associated with the transaction, such as repossession, collections, re-purchase and the like. In this way, the retailer and bank can be at least partially absolved of responsibility associated with these aspects of the lending transaction, as the metamediary may simply buy back the loan, and undertake to collect or repossess as necessary. Having a cooperative retailer repurchase the repossessed automobiles would also be an option, which could benefit both parties to such a transaction and be yet another aspect of this invention.
Risk is further mitigated by checking for retailer integrity, performing background checks, which aid in the prevention of fraud perpetuated on lenders. All parties can be checked as well, for credit background, financials and the like on all parties involved. This is imperative because the principals of the business may be "clean," but all parties involved should be checked, such as the finance manager, credit manager and so forth. This helps to encourage lenders and other service providers to deal with the smaller retailers, since lenders are often reluctant to deal with retailers whose integrity they cannot account for. It thus becomes difficult to track down and manage bad dealers. The present system thus establishes proper dealer setup procedures, complete with risk management tools. System alerts, thresholds and notifications can be setup to help mitigate risk as well. This type of system can detect loan "hopping" whereby a customer goes from retailer to retailer in an attempt to get a loan. This type of system can detect this behavior and notify the system operator of same. Effective data management by the metamediary thus enables much better client, consumer, lender and service provider management so that the metamarket as a whole is more organic rather than the current mode of disjointed processes.
Of course, there are good reasons for lenders and service providers to be cautious, since it is not unheard of for retailers to (either intentionally or negligently) setup so-called straw purchases, wherein one party signs a loan to purchase a car for someone else; in essence, the party responsible for the loan is not the party who will be driving or housing the automobile. This type of arrangement leads to banks not being able to locate the automobile if and when a repossession is required; therefore this method helps to eliminate risks such as these, and this is but one example of the problems that can be reduced or eradicated by having the metamediary manage the metamarket.
The target of the invention is all vehicle retailers, including car, marine and RV dealers, both franchise (manufacturer-oriented) and individual (local pre-owned) retailers. Other dealers with a similar business plan could also feasibly benefit from this type of system. Individual retailers are more targeted by the present invention, since they stand to gain more from the present invention, and they generally have less infrastructure set up for the aspects of the vehicle acquisition transactions than do the franchise retailers. They also generally do not have the resources or expertise to deal with multiple banks, or other preferred sources of financing. And since there are estimated to be over twice as many individual retailers (55,000 estimated as of this writing) than franchise retailers (25,000 estimated as of this writing), the present invention stands to benefit many businesses across the country. Being able to offer the three primary services to an independent (financing, floor planning and technology) would be a great boon to the marketplace—one this invention seeks to bring about. Essentially then, the metamediary brings together a multiple-retailer, multiple-service provider, multiple lender marketplace for multiple consumers.
Other inventors have attempted to address the presented problem, such as the inventions disclosed in U.S. Pat. Nos. 5,878,403 and 6,587,841 to DeFrancesco and 6,208,979 to Sinclair. However, these references only address the issue of loan applications, and do not address the entire metamarket as does the present invention. Clearly then, the present invention offers a much more complete and efficient solution that has yet to be addressed.
All of these aspects of the current mode of vehicle retailing lead to an increased need for a revised method of vehicle acquisition with minimized cost and complexity, all of which the present invention addresses.


A single crystal alloy for high AN2 applications has a composition consisting essentially of from 4.0 to 10 wt % chromium, from 1.0 to 2.5 wt % molybdenum, up to 5.0 wt % tungsten, from 3.0 to 8.0 wt % tantalum, from 5.5 to 6.25 wt % aluminum, from 6.0 to 17 wt % cobalt, up to 0.2 wt % hafnium, from 4.0 to 6.0 wt % rhenium, from 1.0 to 3.0 wt % ruthenium, and the balance nickel. Further, these single crystal alloys have a total tungsten and molybdenum content in the range of from 1.0 to 7.5 wt %, preferably from 2.0 to 7.0 wt %, a total refractory element content in the range of from 9.0 to 24.5 wt %, preferably from 13 to 22 wt %, a ratio of rhenium to a total refractory element content in the range of from 0.16 to 0.67, preferably from 0.20 to 0.45, a density in the range of from 0.300 to 0.325 lb/in3, and a specific creep strength in the range from 106×103 to 124×103 inches. These alloys provide (a) increased creep strength for a given density and (b) specific creep strengths as high as or higher than all 2nd generation single crystal alloys with a significant decrease in density.

(1) Field of the Invention
The present invention relates to lower density single crystal alloys that have particular use in turbine engine components.
(2) Prior Art
High rotor speed turbine engine components, such as turbine blades, require materials with as low density as possible while maintaining reasonable levels of high temperature creep-rupture strength. Alloy design philosophy has previously been to achieve maximum creep capability without undue regard to alloy density. New engine designs require that extremely high levels of performance be achieved, which can only be met at very high AN2 conditions where A=Area; N=Rotor speed. This in turn necessitates a new look at alloy design philosophy. For advanced high rotor speed designs, turbine blade weight (density) is critical to minimize the blade pull on the disk and thus minimize the overall disk size. Current second generation single crystal alloys with densities ranging from 0.312 to 0.323 lb/in3 are widely deployed in production, while third and fourth generation single crystal alloys with increasing strength capability have correspondingly higher densities ranging from 0.324 to 0.331 lb/in3. If reduced alloy density can be achieved for a given level of creep capability, significant savings in engine weight and increased engine performance would result.

Moderate density, low density, and extremely low density single crystal alloys for high AN


The invention provides an agent for preventing or treating arthritis, a cartilage protecting agent, a joint destruction inhibitor and a synovial membrane growth inhibitor comprising an anti-FGF-8 neutralizing antibody as an active ingredient, as well as a diagnostic agent of arthritis comprising an anti-FGF-8 antibody as an active ingredient and a method for judging arthritis using the antibody.

The number of persons complaining of arthropathy has been surely increased in the aging society. It is very important to perform early diagnosis or screening of diseases such as osteoarthritis and rheumatoid arthritis as typical articular diseases or exact prognostic analysis of patients, and the treatment thereof leads to the improve in quality of life of many aged persons. However, satisfactory diagnostic and therapeutic methods have not yet been established.
The articular cartilage is a tissue that comprises a small number of chondrocytes covering the movable surface of the joint and a large number of extracellular matrix. Blood vessels or nerves are not distributed therein, and nutrients are supplied mainly from a synovial fluid produced from the synovial membrane covering the inner surface of the joint. Further, it is not only avascular but also exhibits strong resistance to invasion of blood vessels from the peripheral tissues rich in vasoganglion. Chondrocytes intricately control both of synthesis and degradation of extracellular matrix to play a major role in maintaining homeostasis of extracellular matrix. Chemical factors such as cytokines and growth factors and dynamic factors such as weight loading act on chondrocytes and change the balance of both the synthesis and the degradation of extracellular matrix to influence metabolism of extracellular matrix.
Osteoarthritis is caused by the aging or mechanical stresses to thereby induce disruption of the articular cartilage surface accompanied by growth of new cartilages around joints, deformation of joints and failure of adaptability and to lead to inflammation of synovial membranes of joints. The osteoarthritis is a monoarthritis disease with delayed denaturation of the articular cartilage, and characteristics thereof are often pains and functional loss (Manek M. J. and Lane N. E., Am. Fam. Physician, 61, 1795-1804, 2000).
In rheumatoid arthritis, inflammatory cells invade synovial membranes because of immunological abnormality or infectious diseases, and the growth of synovial fibroblasts progresses according to angiogenesis to form an inflammatory synovial granulation tissue called pannus. When the pannus is formed, destruction of bones or cartilages proceeds to cause irreversible disorder in joints. During the destruction of bones or cartilages, various extracellular matrix present in large quantities, such as collagen and proteoglycan are degraded.
In articular diseases such as osteoarthritis and rheumatoid arthritis, the synovitis and the destruction of extracellular matrix lead to the functional loss of articular cartilages.
Osteoarthritis and rheumatoid arthritis are quite different diseases, but have many common points in the articular cartilage destruction mechanism. Many types of matrix metalloproteases are produced and secreted in the articular synovial fluid and articular portions such as a synovial membrane and a cartilage, and matrix metalloproteases are excessively detected in the articular portions. Matrix metalloproteases degrade many types of extracellular matrix, which is one cause of articular destruction. They are produced not only from inflamed synovial membranes, macrophages and neutrophils but also from chondrocytes. This production is controlled by various cytokines produced or secreted in the same articular portions, superoxide anion, nitric oxide, prostaglandins, growth factors and the like. It has been reported that these induce the production of matrix metalloproteases from synovial cells and chondrocytes to promote degradation of extracellular matrix.
From these reports, it is considered that osteoarthritis and rheumatoid arthritis as well as arthritic diseases such as systemic lupus erythematosus which is an cryptogenic disease with an inflammatory tissue disorder caused by appearance of autoantibody and tissue deposition of an antigen-antibody complex and in which arthropathy occurs at a high rate, arthropathy, psoriatic arthritis leading to bone destruction with synovial membrane growth complicated in psoriatic patients, discopathy in which destruction of extracellular matrix of the intervertebral disc disease is observed and acute crystalline synovitis (gout, pseudogout) (Ryumachi Gaku, compiled by Hirohata Kazushi et al., Dobun Shoin, 1989) can be treated by inhibiting growth of the synovial membrane or destruction of cartilages.
In the pharmacotherapy of rheumatoid arthritis, various non-steroidal antiinflammatory agents, steroidal agents such as prednisolone and antirheumatic agents such as methotrexate have been so far used mainly to reduce pains and inflammation of joints (Chiryo, 78, 3553-3558, Nanzando, 1996). In osteoarthritis, various non-steroidal antiinflammatory agents, analgesic agents, hyaluronic acid pharmaceutical preparations as an intraarticular injection and the like have been administered to remove pains and inflammation. Hyaluronic acid which inhibits destruction of cartilages has been used as an agent for protecting cartilage (Creamer P., J. Rheum., 20, 1461-1464, 1993, Arthritis Rheum., 43, 1905-1915, 2000). Further, physical therapy and operative treatments such as osteotomy and artificial joint replacement have been carried out. Non-steroidal antiinflammatory agents and steroidal agents such as prednisolone are used in systemic lupus erythematosus, non-steroidal antiinflammatory agents and sulfasalazine as an antirheumatic drug in ankylotic arthropathy, non-steroidal antiinflammatory agents, antirheumatic drugs and steroidal intraarticular injections in psoriatic arthritis which involves synovial membrane growth complicated in psoriatic patients and leads to bone destruction, non-steroidal antiinflammatory agents and analgesic agents in intervertebral disc disease in which destruction of extracellular matrix of the intervertebral disk is observed, and non-steroidal antiinflammatory agents, colchicine and the like in acute crystalline synovitis respectively (Ryumachi Gaku, compiled by Hirohata Kazushi et al., Dobun Shoin, 1989). However, such a pharmacotherapy is a symptomatic therapy, and it has hardly inhibited the destruction of joints sufficiently.
In the therapy of rheumatoid arthritis, the selection of the positive therapy to prevent the destruction of joints as much as possible is currently being accepted. The point of this therapy is that a disease is diagnosed as rheumatoid arthritis at the earliest possible stage and antirheumatic drugs such as methotrexate are properly selected. However, sufficient diagnosis has not yet been provided.
A fibroblast growth factor (hereinafter abbreviated as FGF), one of various growth factors existing in vivo, has been known as a heparin-binding growth factor that affects vascular endothelial cells. Further, the FGF family involves 19 types or more, and FGF-2 (basic FGF), FGF-1 (acidic FGF) and the like have been long known. As an FGF receptor, seven types have been to date found, and encode a tyrosine kinase in the intracellular region.
FGF-8 is a factor isolated from a culture supernatant of mouse breast cancer cell line SC-3 (Nakamura N. et al., J. Steroid Biochem., 27, 459-464, 1987) showing sex hormone-dependent growth as an androgen-induced growth factor (AIGF). It is a growth factor which is inductively produced by androgen stimulation and enhances the growth of SC-3 cells in an autocrine manner (Tanaka A. et al., Proc. Natl. Acad. Sci. USA, 89, 8928-8932, 1992). It is reported that FGF-8 accelerates the growth of cells of prostate cancer or fibroblasts (Tanaka A. et al., FEBS Lett., 363, 226-230, 1995). It is reported that FGF-8 bound to three receptors, FGF receptor-2IIIc, FGF receptor-3IIIc and FGF receptor-4 (Ornitz D. M. et al., J. Biol. Chem., 271, 15292-15297, 1996). Moreover, binding to membrane type heparan sulfate proteoglycan such as syndecan is required for the function of FGF. Binding to heparan sulfate is necessary to the stable and local accumulation of FGF. In the situation of tissue remodelingion such as inflammation, it is considered that heparan sulfate is degraded to liberate FGF from extracellular matrix to exhibit its activity. A strong angiogenesis factor such as FGF-2 is comprised in cartilages (Satoh H. et al., J. Biol. Chem., 273, 12307-12315, 1998). In the arthritis, synovial cells, chondrocytes and inflammatory cells invaded synthesize FGF-1 or FGF-2 at an extremely high level (Sano H. et al., J. Cell Biol., 110, 1417-1426, 1990, Remmers E. F., Growth factors, 2, 179-188, 1990), and the FGF-2 concentration in a synovial fluid of rheumatic patients correlates with arthritis (Manabe N. et al., Rheumatology, 38, 714-720, 1999). FGF-2 is involved in osteophyte formation in osteoarthritis (Uchino M. et al., Clin. Orthop., 377, 119-125, 2000). These reports prove that FGF-1 or FGF-2 is involved in arthritis.
In the report using FGF-8 knockout mice, FGF-8 expressed at the stage of the development of joints (Haraguchi R. et al., Development, 127, 2471-2479, 2000; Lewandoski M. et al., Nat. Genet., 26, 460-463, 2000). Nevertheless, it is unknown that FGF-8 is involved in arthritis.


A method for a system and method for morphometric detection of malignancy associated change (MAC) is disclosed including the acts of obtaining a sample; imaging cells to produce 3D cell images for each cell; measuring a plurality of different structural biosignatures for each cell from its 3D cell image to produce feature data; analyzing the feature data by first using cancer case status as ground truth to supervise development of a classifier to test the degree to which the features discriminate between cells from normal or cancer patients; using the analyzed feature data to develop classifiers including, a first classifier to discriminate normal squamous cells from normal and cancer patients, a second classifier to discriminate normal macrophages from normal and cancer patients, and a third classifier to discriminate normal bronchial columnar cells from normal and cancer patients.

Lung cancer is the second most prevalent cancer in the United States and is the most lethal (4). Over 31 million patients in the United States (US) are at high risk for the development of lung cancer, primarily due to a smoking history, and approximately 160,000 US patients die of lung cancer each year (4). Many attempts have been made to develop screening procedures for lung cancer. Traditional cytologic methods on sputum or bronchial wash specimens have been shown to be relatively insensitive, with only about 40-60% of patients with cancer having positive results and the majority of those being from endobronchial squamous carcinomas (5, 6, 7). One bright spot related to the cytology examination is the relatively high positive predictive value of this procedure—generally above 98%—meaning that a positive test is a reliable indicator of disease (7).
Early detection remains by far the most reliable and potent strategy in curing cancer. Many approaches, however, are limited by poor sensitivity or specificity that increase health care costs and potentially risk patient health through unneeded, invasive procedures. The association between cell morphology and cancer has been established and well documented in the literature of cell cytology. On the other hand, the tumor field effect potentially introduces subtle changes into cells that are cytologically normal that reside in tumor microenvironment. This phenomenon has given rise to the malignancy associated change (MAC) hypothesis1.
In pursuit of such an early lung cancer detection system, advances in 3D imaging of biological cells using optical tomography have been developed by Nelson as disclosed, for example, in U.S. Pat. No. 6,522,775, issued Feb. 18, 2003, and entitled "Apparatus and method for imaging small objects in a flow stream using optical tomography," the full disclosure of which is incorporated by reference. Further major developments in the field are taught in Fauver et al., U.S. Pat. No. 7,738,945, issued Jun. 15, 2010, entitled "Method and apparatus for pseudo-projection formation for optical tomography," (Fauver '945) and Fauver et al., U.S. Pat. No. 7,907,765, issued Mar. 15, 2011, entitled "Focal plane tracking for optical microtomography," (Fauver '765) the full disclosures of Fauver '945 and Fauver '765 are also incorporated by reference. Building on the teachings therein an early lung cancer detection technology has been developed by VisionGate, Inc. of Phoenix, Ariz. to provide measurement advantages that have the potential to greatly improve the operating characteristics of conventional morphologic cytology analyses. New systems related to such an early lung cancer detection test are described herein.
Processing in an optical tomography system begins with specimen preparation. Typically, specimens taken from a patient are received from a hospital or clinic and processed to remove non-diagnostic elements, fixed and then stained. Stained specimens are then mixed with an optical gel, inserted into a microcapillary tube and images of objects, such as cells, in the specimen are produced using an optical tomography system. The resultant images comprise a set of extended depth of field images from differing perspectives called "pseudoprojection images." The set of pseudoprojection images can be reconstructed using backprojection and filtering techniques to yield a 3D reconstruction of a cell of interest. The ability to have isometric or roughly equal resolution in all three dimensions is an advantage in 3D tomographic cell imaging, especially for quantitative image analysis. The LuCED® test as implemented by Visiongate, Inc. of Phoenix Arizona is a promising new lung cancer detection method that produces high (92%) sensitivity and high (95%) specificity detection of lung cancer by imaging cells in true 3D3. In the LuCED® test, detection is managed through detection of abnormal cells in sputum. Imaging is provided by the Cell-CT™ platform, also manufactured by Visiongate, Inc., with sub-micron, isometric spatial resolution in 3D.
The 3D reconstructions produced remain available for analysis in order to enable the quantification and the determination of the location of structures, molecules or molecular probes of interest. An object such as a biological cell may be labeled with at least one stain or tagged molecular probe, and the measured amount and location of this biomarker may yield important information about the disease state of the cell, including, but not limited to, various cancers such as lung, breast, prostate, cervical, stomach and pancreatic cancers.
In this document a method to detect the presence of cancer by analyzing subtle changes in cellular morphology imparted to non-cancer cells through the cancer field effect is disclosed. The method can be implemented in the Cell-CT™ platform. A process is disclosed that circumvents the need for the presence of cancer cells in the patient specimen. This is important especially for early cancer detection, as cancer cells are typically rare and thus difficult to detect in patient samples such as sputum, blood or biopsy material. This invention not only simplifies detection but also allows for more expedient processing of patient samples, as it does not require processing of cancer cells.

Morphometric detection of malignancy associated change