Abstract: Hemoglobin conjugates useful as a hemoglobin-based oxygen carriers are prepared by reacting hemoglobin with oxidatively ring-opened polysaccharides such as hydroxyethyl starch or dextran, and storing the resultant conjugate under conditions which allow it to transform to a lower molecular weight product, after conjugation. The conjugate is then reductively stabilized to form secondary amino bonds between the hemoglobin and the polysaccharide, and formulated as an BOC.

Abstract: The invention is directed to a method of in vivo selection for genetically modified hematopoietic progenitor cells from nonmodified hematopoietic cells in a subject. Ibis goal is accomplished by introducing mutant dihydrofolate reductase genes into hematopoietic progenitor cells and then administering to a subject harboring the resultant transformed cells an antifolate and a nucleoside transport inhibitor. The invention is also directed to nucleic acids encoding mutant dihydrofolate reductase genes and to hematopoietic cells transformed with such mutant genes.

Abstract: A process is disclosed for the preparation of an essentially tetramer-free, substantially stroma-free, polymerized, pyridoxylated hemoglobin. Also disclosed is an essentially tetramer-free, substantially stroma-free, polymerized, pyridoxylated hemoglobin product capable of being infused into human patients in an amount of up to about 5 liters.

Abstract: Non-naturally occurring mutant hemoglobins rHb (&bgr;N108Q) and rHb (&bgr;L105W) are provided that have a lower oxygen affinity than that of native hemoglobin, but high cooperativity in oxygen binding. rHb (&bgr;N108Q) also exhibits enhanced stability against autoxidation. The mutant hemoglobins are preferably produced by recombinant DNA techniques. Such mutant hemoglobins may be used as a component of a blood substitute and hemoglobin therapeutics.

Abstract: A method for separating hemoglobin A2 from a hemoglobin mixture according to a cation exchange liquid chromatography characterized that at least two types of eluents are used including an eluent (hereinafter referred to as “eluent A”) for elution of faster hemoglobins than hemoglobin A0 and an eluent (hereinafter referred to as “eluent B”) for elution of hemoglobin A0 and slower hemoglobins than hemoglobin A0, and that the eluent A has a pH of 4.0-6.0 and the eluent B is maintained at a pH of at least 0.5 higher than that of the eluent A.

Abstract: Non-naturally occurring mutant hemoglobins rHb (&bgr;N108Q) and rHb (&bgr;L105W) are provided that have a lower oxygen affinity than that of native hemoglobin, but high cooperativity in oxygen binding. rHb (&bgr;N108Q) also exhibits enhanced stability against autoxidation. The mutant hemoglobins are preferably produced by recombinant DNA techniques. Such mutant hemoglobins may be used as a component of a blood substitute and hemoglobin therapeutics.

Abstract: Construct-complexes of a hemoglobin, a hepatocyte modifying substance bound to the hemoglobin, and a haptoglobin bound to the hemoglobin, are provided, for administration to mammalian patients. The construct-complex may be formed ex vivo, or a hemoglobin-hepatocyte modifying substance combination may be administered to the patient so that haptoglobin in the mammalian body bonds thereto to form the construct-complex in vivo. Disorders of the liver may be diagnosed and treated using construct-complexes described herein.

Abstract: Red blood cells are purified by defibrinating whole blood and then filtering the defibrinated whole blood, whereby at least a portion of a plasma component is separated from the red blood cells to form a suspension of red blood cells, thereby purifying the red blood cells. Whole blood is defibrinated by, for example, using a chemical coagulating agent or mechanical agitation. Separation of the plasma component from red blood cells can be completed by, for example, diafiltration. The suspension of red blood cells can then be employed to produce a hemoglobin-based oxygen carrier.

Abstract: Humoral and cellular immune responses against tumor cells and infectious agents are induced in a mammal using an antibody that binds with an epitope of an antigen that is associated with a tumor or an infectious agent, and that contains at least one &agr;-galactosyl epitope. Such an antibody is capable of forming a complex with cells that express the target epitope and with antibodies that bind &agr;-galactosyl epitopes. Suitable antibodies include molecules that contain at least one engineered glycosylation site in the constant region of the heavy chain.

Abstract: Non-naturally occurring mutant hemoglobins rHb (&bgr;N108Q) and rHb (&bgr;L105W) are provided that have a lower oxygen affinity than that of native hemoglobin, but high cooperativity in oxygen binding. rHb (&bgr;N108Q) also exhibits enhanced stability against autoxidation. The mutant hemoglobins are preferably produced by recombinant DNA techniques. Such mutant hemoglobins may be used as a component of a blood substitute and hemoglobin therapeutics.

Abstract: Non-naturally occurring mutant hemoglobins rHb (&bgr;N108Q) and rHb (&bgr;L105W) are provided that have a lower oxygen affinity than that of native hemoglobin, but high cooperativity in oxygen binding. rHb (&bgr;N108Q) also exhibits enhanced stability against autoxidation, The mutant hemoglobins are preferably produced by recombinant DNA techniques. Such mutant hemoglobins may be used as a component of a blood substitute and hemoglobin therapeutics.

Abstract: Administration of low doses of hemoglobin minimizes damage to the myocardium after blockage and significantly reduces reperfusion injury. Hemoglobin exerts a pharmacological effect by increasing perfusion and blocking the molecular events leading to permanent injury following an ischemic episode. Additional benefits include a reduction in the number of post-ischemic arrhythmias, reduction in the incidence of restenosis, and improved contractile function in the area of risk.

Abstract: Compositions and processes to alleviate free radical toxicity are disclosed based on the use of nitroxides in association with physiologically compatible macromolecules. In particular, hemoglobin-based red cell substitutes are described featuring stable nitroxide free radicals for use in cell-free hemoglobin solutions, encapsulated hemoglobin solutions, stabilized hemoglobin solutions, polymerized hemoglobin solutions, conjugated hemoglobin solutions, nitroxide-labelled albumin, and nitroxide-labelled immunoglobulin. Formulations are described herein that interact with free radicals, acting as antioxidant enzyme-mimics, which preserve nitroxides in their active form in vivo. Applications are described including blood substitutes, radioprotective agents, imaging agents, agents to protect against ischemia and reperfusion injury, particularly in cerebral ischemia in stroke, and in vivo enzyme mimics among others.

Abstract: The invention relates to novel recombinant hemoglobins having reduced nitric oxide scavenging and/or increased high soluble expression. The invention further relates to methods of increasing the soluble expression of recombinant hemoglobin by adding exogenous hemin in molar excess of the heme binding sites of recombinant hemoglobin.

Abstract: Disclosed and claimed are methods for the isolation and use of stem cell inhibiting factors for regulating the abnormal stem cell cycle and for accelerating the post-chemotherapy peripheral blood cell recovery. Also disclosed and claimed are the inhibitors of stem cell proliferation.

Abstract: Compositions, and methods of use thereof, for use as blood substitute products comprise aqueous mixtures of oxygen-carrying and non-oxygen carrying plasma expanders and methods for the use thereof. The oxygen-carrying component may consist of any hemoglobin-based oxygen carrier, while the non-oxygen carrying plasma expander my consist of any suitable diluent.

Abstract: A method for determining hemoglobins is provided which is suitable for determination of stable hemoglobin A1c. A method for determining hemoglobins by cation exchange liquid chromatography wherein an eluent is used containing a chaotropic ion and further an inorganic acid, an organic acid and/or any salt thereof having a buffer capacity in the 4.0-6.8 pH range.

Abstract: The present invention employs a filtration step during the hemoglobin purification process that substantially decreases viral contamination of a hemoglobin solution. The filtration means can be used to separate hemoglobin and several endogenous antioxidant enzymes from red blood cell stroma and potential adventitious agents. The purified hemoglobin/antioxidant composition is then subjected to a chemical modification process. The resulting modified hemoglobin/antioxidant composition is then fractionated to remove unmodified hemoglobin species and residual reactants, formulated in electrolytes and rendered sterile. The resulting modified hemoglobin product is substantially free of viral contamination and contains at least one endogenous antioxidant enzyme that retains antioxidant activity.

Abstract: The present invention is directed to a method of prognosticating the illness development of patients with carcinoma of the breast and/or for diagnosing carcinoma of the breast, the method comprising a qualitative determination of T1 protein and/or T1-mRNA in a sample material obtained from the patient. The invention further pertains to kits for performing the methods according to the invention.

Abstract: An a cellular red blood cell substitute which comprises an essentially tetramer-free, substantially stroma-free, cross-linked, polymerized, pyridoxylated hemoglobin and a nontoxic, pharmaceutically acceptable carrier, its use and a process for preparing said a cellular red blood cell substitute.

Abstract: Isolated cDNA molecules which encode the tumor rejection antigen precursor MAGE-10, the protein itself, antibodies to it, and uses of these are part of the invention.

Abstract: Nitrosylation of proteins and amino acid groups enables selective regulation of protein function, and also endows the proteins and amino acids with additional smooth muscle relaxant and platelet inhibitory capabilities. Thus, the invention relates to novel compounds achieved by nitrosylation of protein thiols. Such compounds include: S-nitroso-t-PA, S-nitroso-cathepsin; S-nitroso-lipoprotein; and S-nitroso-immunoglobulin. The invention also relates to therapeutic use of S-nitroso-protein compounds for regulating protein function, cellular metabolism and effecting vasodilation, platelet inhibition, relaxation of non-vascular smooth muscle, and increasing blood oxygen transport by hemoglobin and myoglobin. The compounds are also used to deliver nitric oxide in its most bioactive form in order to achieve the effects described above, or for in vitro nitrosylation of molecules present in the body.

Abstract: Dried hemoactive materials comprise both a cross-linked biologically compatible polymer and a non-cross-linked biologically compatible polymer. The cross-linked polymer is selected to form a hydrogel when exposed to blood. The non-cross-linked polymer is chosen to solubilize relatively rapidly when exposed to blood. The non-cross-linked polymer serves as a binder for holding the materials in desired geometries, such as sheets, pellets, plugs, or the like. Usually, the cross-linked polymer will be present in a particulate or fragmented form. The materials are particularly suitable for hemostasis and drug delivery.

Abstract: Hemoglobin conjugates useful as a hemoglobin-based oxygen carriers are prepared by reacting hemoglobin with oxidatively ring-opened polysaccharides such as hydroxyethyl starch or dextran, and storing the resultant conjugate under conditions which allow it to transform to a lower molecular weight product, after conjugation. The conjugate is then reductively stabilized to form secondary amino bonds between the hemoglobin and the polysaccharide, and formulated as an BOC.

Abstract: Isolated proteins comprising the T-cell surface antigen CD97 &agr; are provided. Compositions and methods for making and detecting CD97 &agr; are also provided. Further, the invention provides diagnostic and therapeutic methods and compositions for medical conditions involving CD97.

Abstract: S-nitrosohemoglobin (SNO-Hb) can be formed by reaction of Hb with S-nitrosothiol and by other methods described herein which do not result in oxidation of the heme Fe. Other methods can be used which are not specific only for thiol groups, but which nitrosate Hb more extensively, and may produce polynitrosated metHb as a product or intermediate product of the method. SNO-Hb in its various forms and combinations thereof (oxy, deoxy, met; specifically S-nitrosylated, or nitrosated or nitrated to various extents) can be administered to an animal or human where it is desired to oxygenate, to scavenge free radicals, or to release NO+ groups to tissues. Thiols and/or NO donating agents can also be administered to enhance the transfer of NO+ groups. Examples of conditions to be treated by SNO-Hbs or other nitrosated or nitrated forms of Hb include ischemic injury, hypertension, angina, reperfusion injury and inflammation, and disorders characterized by thrombosis.

Abstract: There is disclosed a stable, virus-safe, pharmaceutical preparation comprising thiol-group-containing proteins which are heat-treated and processed such that at least 40% of the thiol groups are capable of being nitrosated, a method of preparing such preparations as well as the use of these preparations.

Abstract: A process is disclosed for the preparation of an essentially tetramerfree, substantially stromafree, polymerized, pyridoxylated hemoglobin. Also disclosed is an essentially tetramerfree, substantially stromafree, polymerized, pyridoxylated hemoglobin product capable of being infused into human patients in an amount of up to about 5 liters.

Abstract: The invention is directed to a method for the prophylaxis or treatment of an animal for deleterious physiological effects such as systemic hypotension caused by nitric oxide production induced by a biological response modifier. Examples of such biological response modifiers include but are not limited to a cytokine and an endotoxin. The invention is also directed to a method for the treatment of septic shock.

Abstract: A set of contiguous and partially overlapping RNA sequences and polypeptides encoded thereby, designated as PS190 and transcribed from prostate tissue is described. These sequences are useful for the detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, or determining the predisposition of an individual to diseases and conditions of the prostate, such as prostate cancer. Also provided are antibodies which specifically bind to PS190-encoded polypeptide or protein, and agonists or inhibitors which prevent action of the tissue-specific PS190 polypeptide, which molecules are useful for the therapeutic treatment of prostate diseases, tumors or metastases.

Abstract: An exsanguinous metabolic support system for maintaining an organ or tissue at a near normal metabolic rate is disclosed. The system employs an organ chamber comprising a container and a support member adapted to inhibit movement of the organ within the container during perfusion and/or transport. The organ chamber additionally comprises a conduit for receiving venous outflow of perfusion solution and preventing its contact with the outer surfaces of the organ. A conduit for receiving organ product enables the collection of organ product from a functional organ during perfusion. Use of the organ chamber supports de novo or continued synthesis of constituents necessary for long-term maintenance of organs for transplantation, for resuscitation and active repair of organs that have sustained warm ischemic damage, and for transportation of isolated organs is also disclosed.

Abstract: A selective complex of a lipoprotein with hemoglobin or a hemoglobin analogue; a method of assaying hemoglobin or a hemoglobin analogue in a sample, comprising reacting the sample with a lipoprotein which is capable of selectively forming a complex with hemoglobin or a hemoglobin analogue; and a kit for assaying hemoglobin or a hemoglobin analogue, comprising a lipoprotein which is capable of selectively forming a complex with hemoglobin or a hemoglobin analogue.

Abstract: The present invention provides compositions useful as antimicrobial agents which include reptilian hemoglobin, the &agr; and &bgr; chains of hemoglobin free of heme, fragments of said proteins or polypeptide fragments thereof and combinations thereof. The compositions exert antimicrobial activity against both bacteria and fungi that is comparable to known antimicrobial peptides from human neutrophils, cathepsin G and azurocidin. Sensitive organisms include Gram-negative bacteria such as Escherichia coli and Pseudomonas aerioginosa, and the fungus Candida albicans. Methods for preparing the compositions also are provided.

Abstract: The present invention provides compositions useful as antimicrobial agents which include mammalian hemoglobin, the &agr; and &bgr; chains of hemoglobin free of heme, fragments of the &agr; and &bgr; chains that result from cyanogen bromide cleavage of the &agr; and &bgr; chains, and synthetic peptides derived therefrom. The compositions exert antimicrobial activity against both bacteria and fungi that is comparable to known antimicrobial peptides from human neutrophils, cathepsin G and azurocidin. Sensitive organisms include Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, Gram-positive bacteria such as Staphylococcus aureus and Streptococcus faecalis, and the fungus Candida albicans. Methods for preparing the compositions also are provided.

Abstract: An acellular red blood cell substitute which comprises an essentially tetramer-free, substantially stroma-free, cross-linked, polymerized, pyridoxylated hemoglobin and a nontoxic, pharmaceutically acceptable carrier, its use and a process for preparing said acellular red blood cell substitute.

Abstract: Compositions and processes to alleviate oxygen toxicity are disclosed based on the addition of nitroxides to physio-logically compatible macromolecules. In particular, hemoglobin-based red cell substitutes are described featuring stable nitroxide free radicals for use in cell-free hemoglobin solutions, encapsulated hemoglobin solutions, stabilized hemoglobin solutions, polymerized hemoglobin solutions, conjugated hemoglobin solutions, nitroxide-labelled albumin, and nitroxide-labelled immunoglobulin. The formulations described herein interact with free radicals, act as antioxidant enzyme-mimics, and alleviate oxidative stress and oxygen-related toxicity.

Abstract: This invention relates to monoclonal antibodies that recognize modified &bgr;-tubulin isotypes, methods of using such antibodies to detect modified &bgr;-tubulin isotypes, methods of using such antibodies to monitor &bgr;-tubulin modifying agents administered to a patient, methods of using such antibodies to isolate modified &bgr;-tubulin, and methods of detecting the anti-modified &bgr;-tubulin antibodies.

Abstract: The present invention reates to a method for detecting activated ras protein. The method includes immobilizing a protein on a solid support, incubating the immobilized protein with lysates from cultured cells, where the lysates include activated ras protein, and determining the amount of activated ras protein bound to the immobilized protein. The present invention also relates to a method of detecting ras oncogenic related malignancy in a human subject.

Abstract: Nitrosylation of proteins and amino acid groups enables selective regulation of protein function, and also endows the proteins and amino acids with additional smooth muscle relaxant and platelet inhibitory capabilities. Thus, the invention relates to novel compounds achieved by nitrosylation of protein thiols. Such compounds include: S-nitroso-t-PA, S-nitroso-cathepsin; S-nitroso-lipoprotein; and S-nitroso-immunoglobulin. The invention also relates to therapeutic use if S-nitroso-protein compounds for regulating protein function, cellular metabolism and effecting vasodilation, platelet inhibition, relaxation of non-vascular smooth muscle, and increasing blood oxygen transport by hemoglobin and myoglobin. The compounds are also used to deliver nitric oxide in its most bioactive form in order to achieve the effects described above, or for in vitro nitrosylation of molecules present in the body.

Abstract: The invention relates to a method for preserving the stability of a hemoglobin blood substitute comprising maintaining the hemoglobin blood substitute in an atmosphere substantially free of oxygen. The invention also involves a method for producing a stable polymerized hemoglobin blood-substitute from blood. The method of this invention includes mixing blood with an anticoagulent to form a blood solution, washing the red blood cells in the blood solution and then separating the washed red blood cells from the white blood cells. This method also includes disrupting the red blood cells to release hemoglobin and form a hemoglobin solution, which is then treated by high performance liquid chromatography to form a hemoglobin eluate. The hemoglobin eluate is then deoxygenated, contacted with a first sulfhydryl compound to form an oxidation-stabilized deoxygenated hemoglobin solution, and mixed with a cross-linking agent to form a polymerization reaction mixture, which is then polymerized.

Abstract: A functional linker for a polypeptide in which two alpha or beta globin-like domains are genetically fused is determined by screening a library of genetically fused polypeptides, in which the linker region is varied, for the ability to participate in the formation of hemoglobin-like protein, as measured by the protein's response to carbon monoxide. In a preferred embodiment, cells expressing the protein turn red as a result of carbon monoxide pressure.

Abstract: The invention relates to a method for preserving the stability of a hemoglobin blood substitute comprising maintaining the hemoglobin blood substitute in an atmosphere substantially free of oxygen. The invention also involves a method for producing a stable polymerized hemoglobin blood-substitute from blood. The method of this invention includes mixing blood with an anticoagulent to form a blood solution, washing the red blood cells in the blood solution and then separating the washed red blood cells from the white blood cells. This method also includes disrupting the red blood cells to release hemoglobin and form a hemoglobin solution, which is then treated by high performance liquid chromatography to form a hemoglobin eluate. The hemoglobin eluate is then deoxygenated, contacted with a first sulfhydryl compound to form an oxidation-stabilized deoxygenated hemoglobin solution, and mixed with with a cross-linking agent to form a polymerization reaction mixture, which is then polymerized.

Abstract: A method of treatment for human patients with an acquired hypercoagulable state or acquired protein C deficiency associated with sepsis, purpura fulminans, meningococcal sepsis, bone marrow and other transplantations, severe burns, pregnancy, major surgery, severe trauma, or ARDS, which comprises administering activated protein C providing a highly selective therapeutic agent with a low potential for causing bleeding complications.

Abstract: The present invention relates to methods of producing hemoproteins comprising (a) introducing into a filamentous fungal cell, which is capable of producing the hemoprotein, (i) one or more first control sequences capable of directing the expression of a heme biosynthetic enzyme encoded by a first nucleic acid sequence endogenous to the filamentous fungal cell, wherein the one or more of the first control sequences are operably linked to the first nucleic acid sequence; and/or (ii) one or more copies of one or more second nucleic acid sequences encoding a heme biosynthetic enzyme; (b) cultivating the filamentous fungal cell in a nutrient medium suitable for production of the hemoprotein and the heme biosynthetic enzymes; and (c) recovering the hemoprotein from the nutrient medium of the filamentous fungal cell.

Abstract: A method for preventing adverse effects associated with the use of a medical device in a patient by introducing into the patient a device of which at least a portion includes a prophylactic or therapeutic amount of a nitric oxide adduct. The nitric oxide adduct can be present in a matrix coating on a surface of the medical device; can be coated per se on a surface of the medical device; can be directly or indirectly bound to reactive sites on a surface of the medical device; or at least a portion of the medical device can be formed of a material, such as a polymer, which includes the nitric oxide adduct. Also disclosed is a method for preventing adverse effects associated with the use of a medical device in a patient by introducing the device during a medical procedure and before or during said procedure locally administering a nitric oxide adduct to the site of contact of said device with any internal tissue.

Abstract: Described are preferred oxygen-binding heme proteins which include at least one hemoglobin molecule incorporating at least one circularly permuted globin, especially an alpha globin. More preferred heme proteins of the invention include high molecular weight hemoglobin multimers. Also described are polynucleotides encoding proteins of the invention, and vectors and host cells including the same.

Abstract: Fatty acid derivatives of sulfhydryl-containing compounds (for example, sulfhydryl-containing peptides or proteins) comprising fatty acid-conjugated products with a disulfide linkage are employed for delivery of the compounds to mammalian cells. This modification markedly increases the absorption of the compounds by mammalian cells relative to the rate of absorption of the unconjugated compounds, as well as prolonging blood and tissue retention of the compounds. Moreover, the disulfide linkage in the conjugate is quite labile in the cells and thus facilitates intracellular release of the intact compounds from the fatty acid moieties.

Abstract: The present invention relates to globins containing non-naturally occurring binding domains, In particular, the present invention is directed toward a binding domain comprising the oligomerizing domain GCN4 and GCN4 derivatives. Thc present invention also relates to multimeric hemoglobins comprised of at least one globin containing at least one non-naturally occurring binding domain.

Abstract: The present invention provides an antibody which specifically reacts with an epitope of a polypeptide comprising the amino acid sequence X-Y-CYS GLN GLU GLU GLU CYS PRO ASP PRO TYR LEU CYS SER PRO VAL THR ASN ARG CYS GLU CYS THR PRO VAL LEU CYS ARG MET TYR CYS LYS PHE TRP ALA LYS ASP GLU LYS GLY CYS GLU ILE CYS LYS CYS GLU GLU LEU CYS GLN ASN GLN ASN CYS THR LYS ASP MET LEU CYS SER SER VAL THR ASN ARG CYS ASP CYS GLN ASP PHE LYS CYS PRO GLN SER TYR CYS-Z (SEQ. ID NO. 1) wherein X is MET or absent; Y is 0-28 amino acids of the sequence LYS MET CYS TRP ASN LYS GLY CYS PRO CYS GLY GLN ARG CYS ASN LEU HIS ARG ASN GLU CYS GLU VAL ILE ALA GLU ASN ILE GLU, (SEQ. ID NO. 2) with the proviso that if part of the sequence is present, it is a carboxy-terminal part of the sequence including the carboxy-terminal GLU and wherein Val may be preceded by Gly; and Z is absent or all or a part of the sequence Pro110-Lys156 shown in FIG. 7 (SEQ. ID NO.

Abstract: This invention relates to recombinant hemoglobins containing mutations in or near the heme pocket of the hemoglobin molecule. This invention particularly relates to recombinant hemoglobins that have altered geometry or polarity around the distal portion of the heme pocket.