WO2000013019A9 - Receptor for underivatized, aqueous soluble beta-(1,3)-glucan - Google Patents
Receptor for underivatized, aqueous soluble beta-(1,3)-glucanInfo
- Publication number
- WO2000013019A9 WO2000013019A9 PCT/US1999/018995 US9918995W WO0013019A9 WO 2000013019 A9 WO2000013019 A9 WO 2000013019A9 US 9918995 W US9918995 W US 9918995W WO 0013019 A9 WO0013019 A9 WO 0013019A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- glucan
- receptor
- underivatized
- aqueous soluble
- binding
- Prior art date
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
Definitions
- aqueous soluble ⁇ (l,3)-glucan also known as PGG glucan, triple helix glucan (TH-glucan) or Betafectin®
- PGG glucan triple helix glucan
- Betafectin® a novel and unique soluble ⁇ - glucan manufactured through a proprietary process. The biological activity of this molecule is clearly distinguishable from particulate or other soluble ⁇ -glucans.
- Numerous laboratories have reported direct induction of arachidonic acid metabolites (Czop et al, J. Immunol. 141(9):3 ⁇ 10-3 ⁇ 16 (1988)), cytokines (Abel and Czop, Intl. J. Immunopharmacol 14(8): ⁇ 363- ⁇ 313 (1992); Doita et al, J.
- ⁇ :A216 (1994) cytokine secretion
- cytokine secretion Putsiaka et al.Blood 82:3695-3700 (1993)
- proliferation Wakshull et al, J. Cell. Biochem. suppl 18A:22 (1994)
- aqueous soluble ⁇ (l,3)-glucan primes cells for activation by secondary stimuli (Mackin et al (1994); Brunke-Reese and Mackin, FASEB J. 5:A488 (1994); and Wakshull et al (1994)).
- the biological activity of ⁇ -glucans is mediated through specific receptors located on target cells.
- the leukocyte complement receptor 3 (CR3, also known as MAC 1 or CDl lb/CD 18) has been reported to bind both particulate and some soluble ⁇ -glucans, as well as other polysaccharides (Thornton et al, J. Immunol. 756:1235-1246 (1996)).
- a soluble aminated ⁇ -glucan preparation has been shown to bind to murine peritoneal macrophages (Konopski et al, Biochim. Biophys. Acta 7227:61-65 (1994)), and a phosphorylated ⁇ -glucan derivative has been reported to bind to monocyte cell lines (Muller et al, J. Immunol. 75(5:3418- 3425 (1996)).
- This invention pertains to the discovery that underivatized, aqueous soluble ⁇ (l,3)-glucan specifically activates a novel receptor located on rat NR8383 macrophages.
- a radiolabeled underivatized, aqueous soluble ⁇ (l,3)-glucan was used to measure the binding of this ⁇ -glucan to membrane receptors derived from rat NR8383 cells.
- the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan is a protein, and shows specific and saturable binding to membranes and is highly selective for a subclass of soluble ⁇ -glucans.
- the present invention relates to isolated preparations containing the receptor described herein.
- the present invention pertains to methods of isolating, concentrating or purifying ⁇ (l ,3)-glucans, as well as ⁇ (l ,3)-glucan-containing organisms, and also to assays to quantify the amount of ⁇ (l,3)-glucans, or ⁇ (l,3)- glucan-containing organisms in a test sample.
- the test sample can be a liquid or a solid, and can originate from various sources, including complex animal sources (e.g., a biological fluid, such as blood, serum, plasma, urine, feces, mucus, sputum, bile, ascites fluid, wound secretions, vaginal excretions, synovial fluid, cerebrospinal fluid, peritoneal lavage fluid, lung lavage fluid, ocular fluid, saliva or whole tissue extract; alternatively, the test sample can be solid specimen, such as skin or other tissue); plant sources (plant tissue, plant tissue extract, fruit or fruit extracts, seeds or seed extracts, sap, or homogenates); bacterial cells; viral cells; fungal cells; tissue cultures; environmental sources; food sources; or fermentation process sources.
- complex animal sources e.g., a biological fluid, such as blood, serum, plasma, urine, feces, mucus, sputum, bile, ascites fluid, wound secretions, vaginal excretions, synovial fluid, cerebro
- the receptor described herein is used to capture or purify ⁇ (l,3)-glucans and/or ⁇ (l,3)-glucan-containing organisms from a test sample.
- the ⁇ (l,3)-glucan receptor, or the test sample can be coupled or attached to a solid phase or a fluid phase.
- the test sample is contacted with the receptor under conditions that are suitable for binding of any ⁇ (l ,3)-glucan that may be present in the test sample to the receptor; resultant complexes of ⁇ (l,3)-glucan and receptor ("primary complexes") can then be isolated, in order to isolate ⁇ (l,3)- glucan.
- the primary complexes can be detected, in order to detect ⁇ (l,3)-glucan or ⁇ (l,3)-glucan-containing organisms in the test sample: the presence of primary complexes is indicative of the presence of ⁇ (l,3)-glucan or ⁇ (l,3)-glucan-containing organisms.
- the presence of ⁇ (l,3)-glucan can be detected through a competition assay, in which labeled underivatized, aqueous soluble ⁇ (l,3)-glucan and receptors are incubated with the test sample; the amount of ⁇ (l,3)-glucan in the test sample is inversely proportional to the amount of complexes of labeled underivatized, aqueous soluble ⁇ (l,3)-glucan and receptors.
- the presence of ⁇ (l,3)-glucan (or of ⁇ (l,3)-glucan-containing organisms) in the test sample is indicative of a fungal infection or fungal contamination.
- Antibodies that can be used in these methods and assays, as well as kits that can be used in these methods and assays, are also within the scope of the invention.
- This invention also pertains to a method of altering (e.g., activating or deactivating) signal transduction pathways, for example through modulation of one or more transcriptional regulatory factors in receptor-positive cells, i.e., cells which contain the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan.
- the signal transduction pathway is modulated or regulated by one or more transcriptional regulatory factors from the NF- ⁇ B and/or NF-IL6 and/or AP-1 families of transcriptional regulatory factors.
- signal transduction pathways which can be altered by the methods of the present invention include the ras/raf-1/MAP kinase (ERK) pathway, the G- protein/phospholipase C/protein kinase C pathway, the non-G-protein/phospholipase C/protein kinase C pathway, the JAK/STAT pathway, the phospholipase A pathway, G-protein/phospholipase D/phosphatidic acid pathway, the c-AMP-dependent pathway, the c-Jun N-terminal kinase (INK, also known as stress-activated protein kinase (SAPK)) pathway, the I ⁇ B kinase ⁇ (LKK ⁇ ) pathway, the protein tyrosine kinase pathway, and the oxygen radical pathway.
- ERK ras/raf-1/MAP kinase
- G- protein/phospholipase C/protein kinase C pathway the non-G-protein/phospho
- the activity of the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan is activated through binding of an underivatized, aqueous soluble ⁇ (l,3)-glucan, or an agent that mimics the activity of underivatized, aqueous soluble ⁇ (l,3)-glucan, whereby a signal transduction process is activated such that one or more transcriptional regulatory factors (e.g., from the NF- ⁇ B, NF-IL6 and/or AP-1 families) are activated.
- transcriptional regulatory factors e.g., from the NF- ⁇ B, NF-IL6 and/or AP-1 families
- Activation of these transcriptional regulatory factors or of the enzymes in the pathway of transcription factor activation can be used to measure the activation of the associated signal transduction pathway.
- Activation of the receptor can comprise, among others, an alteration in the receptor conformation, formation of a ligand-receptor complex, or alteration of the ligand-receptor complex.
- the activity of the receptor can be initiated by an agent which mimics the binding and activation ability of an underivatized, aqueous soluble ⁇ (l,3)-glucan, such as another conformer of the glucan.
- the transcriptional regulatory factor is activated as a result of ligand binding.
- the activity of the transcriptional regulatory factor is decreased, either partially or totally, by the binding of an agent to the receptor (and thus excludes the underivatized, aqueous soluble ⁇ (l,3)-glucan), but lacks the ability to activate the receptor.
- the invention also pertains to an assay for identifying agents which alter (e.g., increase or decrease) the binding of underivatized, aqueous soluble ⁇ (l,3)- glucan to its receptor.
- the assay comprises combining radiolabeled underivatized, aqueous soluble ⁇ (l,3)-glucan, the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan, and an agent to be tested, under conditions suitable for binding of underivatized, aqueous soluble ⁇ (l,3)-glucan to its receptor.
- the extent of binding of underivatized, aqueous soluble ⁇ (l,3)-glucan to its receptor in the presence of the agent to be tested is determined and compared with the extent of binding in the absence of the agent to be tested; a difference in the extent of binding indicates that the agent alters the binding of underivatized, aqueous soluble ⁇ (l,3)-glucan to its receptor.
- An increase in the extent of binding in the presence of the agent indicates that the agent enhances, i.e., prolongs or increases, binding or is an agonist of the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan.
- a decrease in the extent of binding in the presence of the agent indicates that the agent diminishes, i.e., shortens or decreases, binding or is an antagonist of the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan.
- the invention also relates to agents identified by assays described herein, and accordingly, relates to agonists and antagonists of underivatized, aqueous soluble ⁇ (l,3)-glucan activity.
- the present invention also pertains to a novel assay for identifying agents which alter (e.g., increase or decrease) the effect of underivatized, aqueous soluble ⁇ (l,3)-glucan on cellular signal transduction pathways, such as activation of transcriptional regulatory factors.
- This assay comprises combining underivatized, aqueous soluble ⁇ (l,3)-glucan, the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan and an agent to be tested under conditions in which binding of underivatized, aqueous soluble ⁇ (l,3)-glucan to its receptor occurs (i.e., conditions suitable for binding of underivatized, aqueous soluble ⁇ (l,3)-glucan to the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan).
- Binding of underivatized, aqueous soluble ⁇ (l,3)-glucan to its receptor activates the receptor, which in turn activates a signal transduction as exemplified or measured by a modulation of one or more transcriptional regulatory factors such as those from the NF- ⁇ B, NF-IL6 and/or AP-1 families or the enzymes in the respective signal transduction pathways of the transcriptional regulatory pathways.
- a transcriptional regulatory factors such as those from the NF- ⁇ B, NF-IL6 and/or AP-1 families or the enzymes in the respective signal transduction pathways of the transcriptional regulatory pathways.
- the extent of activation of the selected transcriptional regulatory factor, or pathway enzyme, in the presence of an agent to be tested is determined and compared with the extent of activation of the selected transcriptional regulatory factor, or pathway enzyme, in the absence of the agent to be tested; a difference in the extent of activation indicates that the agent alters the effect of underivatized, aqueous soluble ⁇ (l,3)-glucan on activation of the transcriptional regulatory factor or pathway enzyme.
- An increase in the activation of the transcriptional regulatory factor or pathway enzyme in the presence of the agent indicates that the agent enhances, i.e., prolongs or increases, the activation.
- a decrease in the activation of the transcriptional regulatory factor or pathway enzyme in the presence of the agent indicates that the agent diminishes, i.e., shortens or decreases, the activation.
- the assays and methods of the present invention can be used to identify agents and drugs for use in treatment of infectious disease, inflammation, autoimmune diseases, ischemia reperfusion injury, cancer, asthma and hypersensitivity disorders.
- the assays and methods described herein can also be used to identify drugs which prolong or mimic the underivatized, aqueous soluble ⁇ (l,3)-glucan effect, and therefore can be used in any therapeutic or prophylactic application in which underivatized, aqueous soluble ⁇ (l,3)-glucan can be used, such as for immunomodulation, hematopoiesis, prevention and treatment of infectious disease, platelet production, peripheral blood precursor cell mobilization and wound healing.
- agents or drugs act to enhance the effects of underivatized, aqueous soluble ⁇ (l,3)-glucan by, for example, prolonging the binding of the glucan to its receptor or the effects thereof.
- the present invention also relates to agents or drugs, such as, but not limited to, peptides or small organic molecules designed with reference to the binding site for underivatized, aqueous soluble ⁇ (l,3)-glucan on the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan.
- agents or drugs can be designed to mimic the activity of the receptor binding site in that they bind underivatized, aqueous soluble ⁇ (l,3)-glucan, thus decreasing the amount of the ⁇ (l,3)-glucan which is available for binding to the receptor and decreasing the activation of downstream events such as signal transduction.
- the present invention also pertains to an agonist or mimic of underivatized, aqueous soluble ⁇ (l,3)-glucan activity with respect to its binding and activation of the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan, such as another conformation of glucan.
- the drug or agent can be designed to bind the receptor binding site, rendering it unavailable for binding by underivatized, aqueous soluble ⁇ (l,3)- glucan; the present invention also relates to antagonists of underivatized, aqueous soluble ⁇ (l ,3)-glucan binding activity.
- the work described herein has application to many areas. For example, it can be used in the monitoring of the underivatized, aqueous soluble ⁇ (l,3)-glucan manufacturing process and product characterization for commercial release, to measure ⁇ -glucans in fluids, to assess and determine structure-activity relationships of agents that interact with the receptor for the underivatized aqueous soluble ⁇ (l,3)- glucan. Additionally, this work has application to the targeted delivery of various agents, including drugs and small molecules, to receptor-positive cells such as peripheral polymorphonuclear leukocytes, monocytes, macrophages and epithelial cells.
- results described herein can also be used in purification schemes to enrich for both receptor-positive cells and receptor-negative cells, as well as in the generation of anti-receptor antibodies for diagnostic purposes. Furthermore, the methods and assays of the invention allow for specific, highly sensitive detection of ⁇ (l,3)-glucans in a test sample, facilitating diagnosis of fungemias and detection of fungal contamination, without requiring expensive equipment or trained personnel.
- Fig. 1 is a graphic representation of dose-dependent binding of 3 H-TH-glucan to NR8383 cells. Solid circles, total binding; open circles, specific binding; solid squares, non-specific binding.
- Fig. 2A is a graphic representation of ⁇ -glucan selectivity of 3 H-TH-glucan binding to NR8383 cells, determined by competition binding assays using various concentrations of unlabeled competitor low molecular weight ⁇ -glucan. Solid circles, TH-glucan; open squares, low molecular weight (LMW)-glucan.
- LMW low molecular weight
- Fig. 2B is a graphic representation of ⁇ -glucan selectivity of 3 H-TH-glucan binding to NR8383 cells, determined by competition binding assays using various concentrations of unlabeled competitor very high molecular weight ⁇ -glucan. Solid circles, TH-glucan; open squares, very high molecular weight (VHMW)-glucan.
- Fig. 2C is a graphic representation of ⁇ -glucan selectivity of 3 H-TH-glucan binding to NR8383 cells, determined by competition binding assays using various concentrations of unlabeled competitor ⁇ -glucan, scleroglucan. Solid circles, TH- glucan; open squares, scleroglucan.
- Fig. 3 A is a graphic representation of ⁇ -glucan selectivity of 3 H-TH-glucan binding to lactosylceramide, determined by competition binding assays using various concentrations of unlabeled competitor ⁇ -glucan, LMW-glucan. Closed circles, TH-glucan; open squares, LMW-glucan.
- Fig. 3B is a graphic representation of ⁇ -glucan selectivity of 3 H-TH-glucan binding to lactosylceramide, determined by competition binding assays using various concentrations of unlabeled competitor ⁇ -glucan, VHMW-glucan. Closed circles, TH-glucan; open squares, VHMW-glucan.
- Fig. 3C is a graphic representation of ⁇ -glucan selectivity of 3 H-TH-glucan binding to lactosylceramide, determined by competition binding assays using various concentrations of unlabeled competitor ⁇ -glucan, scleroglucan. Closed circles, TH-glucan; open squares, scleroglucan.
- This invention pertains to the discovery that underivatized, aqueous soluble ⁇ (l,3)-glucan (see U.S. Serial No. 07/934,015, filed August 21, 1992, the teachings of which are incorporated herein by reference) specifically binds to a novel receptor located on rat NR8383 macrophages. Underivatized, aqueous soluble ⁇ (l,3)-glucans are also described in U.S. Serial Nos. 08/400,488, 08/432,303, 07/934,015, and 08/469,233 and U.S. Patent Nos. 5,322,841, 5,488,040, 5,532,223 and 5,783,569 (the teachings of all of which are incorporated herein by reference).
- results of work described herein characterize this receptor for underivatized, aqueous soluble ⁇ (l,3)- glucan (also known as TH-glucan) and clearly differentiate it from previously described ⁇ -glucan receptors, while revealing important information about the mechanism of underivatized, aqueous soluble ⁇ (l,3)-glucan biological activity.
- the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan is located primarily in rat NR8383 macrophages, particularly alveolar macrophages.
- receptor is intended to encompass a traditional receptor molecule as well as a binding site; such a binding site can have an effect of its own or may induce or activate a second molecule or binding site to produce an effect (also known as "unmasking" of a second site; Sandberg et al, Infect. Immun. 63(l):2625-263 ⁇ (1995)).
- receptor is also intended to include compounds which have an affinity for underivatized, aqueous soluble ⁇ (l,3)-glucan; these compounds can be receptor mimics or receptor analogues, and can be isolated from a naturally- occurring source or can be chemically or synthetically produced.
- NR8383 receptor refers to a receptor as defined above, which has certain characteristics of the receptor identified on NR8383 cells as described below, such as a proteinaceous composition, an association with a membrane, a certain binding affinity, temperature sensitivity, ⁇ -glucan selectivity, trypsin sensitivity, and/or activation of transcription factors.
- the receptor also referred to herein as a “protein receptor” or “proteinaceous receptor” can be present on NR8383 cells, as well as on other types of cells, and can also be isolated (i.e., separated from cells).
- NR8383 cells see, for example, Helmke, R.J.
- Binding of labeled, underivatized, aqueous soluble ⁇ (l,3)-glucan in the triple helix conformation to the receptor in NR8383 cells was specific, saturable, and fit by a two-site model, with affinities of 45 pM and 170 pM.
- binding of underivatized, aqueous soluble ⁇ (l,3)-glucan to the receptor on NR8383 cells occurred at either 4°C or 37°C.
- TH-glucan aqueous soluble ⁇ (l,3)- glucan
- LMW low molecular weight
- VHMW very high molecular weight
- NR8383 receptor Binding of underivatized, aqueous soluble ⁇ (l,3)-glucan to the receptor on NR8383 cells was destroyed by pre- treatment of cells with trypsin, and recovery of binding activity was dependent upon protein synthesis, indicating that the NR8383 receptor is a protein.
- LMW-glucan and VHMW-glucan (but not scleroglucan) induced activation of an NF- ⁇ B-like factor; underivatized, aqueous soluble ⁇ (l,3)-glucan stimulated protein kinase activity of the MAP kinase JNK and the signal some kinase LKK ⁇ , but not of the MAP kinases ERK1/2 or p38.
- the receptor in NR8383 cells differ in binding affinity, temperature sensitivity, ⁇ -glucan selectivity, and trypsin sensitivity, from other previously identified underivatized, aqueous soluble ⁇ (l,3)-glucan receptors, as described below in the Examples.
- the receptor on NR8383 cells represents a previously unidentified receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan.
- the invention therefore pertains to isolated preparations containing the proteinaceous receptor described herein, that binds (e.g., specifically and selectively), to a ligand that is an underivatized, aqueous soluble ⁇ (l,3)-glucan, such as underivatized, aqueous soluble ⁇ (l,3)-glucan in a triple helix conformation.
- Methods are now available to capture, concentrate or purify ⁇ (l,3)-glucans, including yeast or fungal soluble ⁇ (l,3)-glucans, yeast or fungal insoluble ⁇ (l,3)- glucans, as well as to capture, concentrate or purify ⁇ (l,3)-glucan-containing organisms, using the receptor from NR8383 cells.
- assays to quantify the amount of ⁇ (l,3)-glucan in a test sample are described herein; these assays can be used for diagnosis of fungal infections. Kits useful in the assays and methods are also described.
- the methods, assays and kits take advantage of the affinity of the ⁇ (l,3)-glucan receptor, as described above, for soluble ⁇ (l,3)-glucans, such as ⁇ (l,3)-glucan and other conformations of glucan (e.g., LMW-glucan, HMW-glucan and VHMW-glucan).
- soluble ⁇ (l,3)-glucans such as ⁇ (l,3)-glucan and other conformations of glucan (e.g., LMW-glucan, HMW-glucan and VHMW-glucan).
- a sample known to contain, or thought to contain, fungus or ⁇ (l,3)-glucan-containing organisms is obtained.
- the test sample can be a liquid or a solid, and can originate from various sources, including complex animal sources (e.g., mammalian cells or tissues), plant cells or tissues, bacterial cells, viral cells, fungal cells, tissue cultures, environmental sources, food sources, or fermentation process sources.
- complex animal sources e.g., mammalian cells or tissues
- plant cells or tissues e.g., bacterial cells, viral cells, fungal cells, tissue cultures, environmental sources, food sources, or fermentation process sources.
- the test sample can be a biological fluid, such as blood (whole blood, whole blood serum or whole blood plasma), urine, feces, mucus, sputum, bile, ascites fluid, wound secretions, vaginal excretions, synovial fluid, cerebrospinal fluid, peritoneal lavage fluid, lung lavage fluid, ocular fluid, saliva or whole tissue extract; alternatively, the test sample can be solid specimen, such as skin or other tissue. If the individual suspected of having a fungal infection is immunocompromized, the preferred test sample is a urine sample.
- blood whole blood serum or whole blood plasma
- urine feces, mucus, sputum, bile, ascites fluid, wound secretions, vaginal excretions, synovial fluid, cerebrospinal fluid, peritoneal lavage fluid, lung lavage fluid, ocular fluid, saliva or whole tissue extract
- the test sample can be solid specimen, such as skin or other tissue. If the individual suspected of having a fungal infection is
- the sample is from a plant source, it can be a fluid or a solid specimen, such as plant tissue, plant tissue extract, fruit or fruit extracts, seeds or seed extracts, sap, or plant homogenates.
- the test sample can also be an environmental source such as water, soil, or soil extracts, or a food source such as prepared foodstuffs, that may be fungally contaminated.
- the test sample can be a sample from a fermentation process, such as fermentation broth.
- a de-pro teinating step such as standard trichloroacetic acid or perchloric acid precipitation methods, or precipitation using a standard 70% ethanol precipitation method can be performed prior to performing the assay of the invention (Methods in Enzymology Vol. 182: Guide to Protein Purification (Deutscher, M.P., Ed., Academic Press, Inc, San Diego CA, 1990), Chapter 22, pp. 285-306).
- a step is not necessary to perform the methods of the invention.
- the proteinaceous receptor as described herein is used to capture or purify ⁇ (l,3)-glucans and or ⁇ (l,3)-glucan-containing organisms from a test sample.
- the receptor can be coupled to a solid phase (e.g., filter, membrane such as cellulose or nitrocellulose, plastic (e.g., microtiter plate, dipstick), glass (e.g., slide), bead (e.g., latex beads), particle, organic resin, or other organic or non-organic solid phase) or a fluid (e.g.,TRIS buffer or phosphate buffer) phase.
- a solid phase e.g., filter, membrane such as cellulose or nitrocellulose, plastic (e.g., microtiter plate, dipstick), glass (e.g., slide), bead (e.g., latex beads), particle, organic resin, or other organic or non-organic solid phase) or a fluid (e.g.,TRIS buffer or phosphate buffer) phase.
- Coupling the receptor to the solid or fluid phase can be accomplished by standard methods, including, for example, air drying, heat drying, or chemical reaction.
- the test sample is contacted with a receptor coupled to a solid or fluid phase, under conditions that are suitable for binding of any ⁇ (l,3)-glucan (or ⁇ (l,3)-glucan- containing organisms) that may be present in the test sample to the receptor.
- ⁇ (l ,3)- glucan or a ⁇ (l,3)-glucan-containing organism that is bound to a receptor is referred to herein as a "primary complex".
- the formation of primary complexes indicates that ⁇ (l,3)-glucan, or a ⁇ (l,3)-glucan-containing organism, has been "captured" from the test sample.
- test sample can be attached (e.g., adsorbed, coated, coupled, covalently attached, attached by affinity binding) to a solid support, such as by dipping the solid support into the test sample, by dropwise application of a fluid test sample with the solid support, or by smearing a solid test sample onto the solid support.
- test sample can be mixed with a liquid, such as saline or any other appropriate biological buffer, prior to attachment to the solid support, such as, for example, in the case of solid test samples.
- a liquid such as saline or any other appropriate biological buffer
- a receptor is contacted with the attached test sample, under conditions that are suitable for binding of any ⁇ (l,3)-glucan that may be present in the attached test sample to the receptor.
- Receptor that is bound to any ⁇ (l,3)-glucan or ⁇ (l,3)-glucan-containing organism in the attached test sample is also referred to herein as a "primary complex".
- any primary complexes that have formed can be isolated, in order to obtain, to isolate, to concentrate or to purify the ⁇ (l,3)-glucan (or ⁇ (l,3)-glucan containing organisms) from the test sample.
- the primary complexes can be obtained or isolated using standard methods, such as by immunoprecipitation or other means, resulting in concentrated or purified primary complexes, from which concentrated or purified ⁇ (l,3)-glucan (or ⁇ (l,3)-glucan- containing organisms) can be separated using standard methods (see Current Protocols in Molecular Biology, Vol. II, Ch. 10 (Ausubel, F. et al, eds., John Wiley & Sons, 1997).
- the primary complexes can be detected, in an assay to determine whether ⁇ (l,3)-glucan (or ⁇ (l,3)-glucan- containing organisms) is present in the test sample.
- the amount of primary complexes can be determined: the concentration of the primary complexes correlates positively with the quantity of ⁇ (l,3)-glucan (or ⁇ (l,3)-containing organisms) present in the test sample.
- a variety of methods can be used to detect, and/or to determine the quantity of, the ⁇ (l,3)-glucan, including detection of the ⁇ (l,3)-glucan using an agent that selectively binds the primary complex, such as anti- ⁇ -glucan antibodies (monoclonal or polyclonal) or antibody fragments; detection of enzymatic reaction precipitated by ⁇ (l,3)-glucan, such as by Limulus amebocyte lysate or Limulus lysate Factor G assay (Mori, T. et al, Eur. J. Clin. Chem. Biochem 35(7): 553-560 (1997); Hossai, M.A. et al, J. Clin. Lab.
- the primary complexes can be detected through the use of a detectable label on the receptor, such as a radionucleotide (e.g., radioummunoassay), dye, fluorescent compound, biotin or streptavidin.
- a detectable label on the receptor such as a radionucleotide (e.g., radioummunoassay), dye, fluorescent compound, biotin or streptavidin.
- the quantity of primary complexes can be compared to a standard curve generated with a known ⁇ (l,3)-glucan standard.
- a competition assay can be used to determine the quantity of ⁇ (l,3)-glucan (or ⁇ (l,3)-glucan containing organisms) that is present in the test sample.
- aqueous soluble ⁇ (l,3)- glucan for example, labeled (for example, with radionucleotides, dyes, or fluorescent labels) underivatized, aqueous soluble ⁇ (l,3)- glucan, or another soluble ⁇ (l,3)-glucan, such as low molecular weight (LMW)- glucan (approximately 8,000-120,000 daltons, as determined by MALLS), high molecular weight (HMW)-glucan (approximately >210,000 - ⁇ 1,000,000, as determined by MALLS) or very high molecular weight (VHMW)-glucan
- LMW low molecular weight
- HMW high molecular weight
- VHMW very high molecular weight
- This amount is compared with the amount of primary complexes generated by incubation of labelled underivatized, aqueous soluble ⁇ (l,3)-glucan with the receptor in a control sample (i.e., a sample having a known amount of unlabeled ⁇ (l,3)-glucan) or a series of control samples (e.g., a standard curve).
- a control sample i.e., a sample having a known amount of unlabeled ⁇ (l,3)-glucan
- a series of control samples e.g., a standard curve
- Detection and quantitation of the presence of ⁇ (l,3)-glucan, or of ⁇ (l,3)- glucan containing organisms is useful in the diagnosis of fungemias or fungal contamination.
- a sample such as a biological fluid sample, is obtained from the individual.
- the sample is assayed for the presence of ⁇ (l,3)-glucan, as described in detail above.
- the presence of ⁇ (l,3)-glucan in the test sample is indicative of the presence of a component of fungal cell walls, and is therefore indicative of fungal presence, contamination, or infections.
- the presence of ⁇ (l,3)-glucan in a sample from an environmental source, or a food source is indicative of fungal presence or contamination.
- quantitation of the amount of ⁇ (l,3)-glucan, or of ⁇ (l,3)-glucan containing organisms can provide information regarding the severity of the infection or contamination.
- the methods and assays of the invention provide accurate and specific identification of the presence of ⁇ (l,3)-glucans in a test sample, thereby facilitating diagnosis and treatment of fungal infection and contamination.
- test samples can be included in a standard competition receptor binding assay to allow characterization of the molecule in terms of relative affinity for the receptor. This characterization will yield information with respect to some or all of the following characteristics: batch to batch quality, identification of product as a ⁇ (l,3)-glucan of a particular conformation and purity of the product.
- Test samples can also be included in standard capture or competition assays and compared with a standard sample to elucidate structure-activity relationships. Alternatively, the test sample binding can be tested directly after radiolabeling. Samples can also be tested in an underivatized, aqueous soluble ⁇ (l,3)-glucan receptor-mediated assay to test for inhibition or stimulation of these functions. For example, such assays can be used in the development of polymer or small molecule receptor agonists or to develop a receptor antagonist to inhibit an inappropriate immunoenhancement, such as to prevent a transplant rejection which might occur as a result of an opportunistic fungal infection during immunosuppressive therapy.
- various agents can be conjugated (e.g., chemically conjugated, cross-linked or covalently bonded) to underivatized, aqueous soluble ⁇ (l,3)-glucan to produce a conjugate molecule which can be targeted to receptor-positive cells such as macrophages.
- Such a targeted delivery system can be used to enhance the delivery of agents such as antimicrobials for resistant intracellular pathogens (e.g., mycobacterium, leishmania, malaria), cytotoxins for receptor-positive leukemias, genes for gene therapy (e.g., for enhanced cytokine production or replacement for dysfunctional enzymes), or antigens for enhanced presentation and production of specific antibodies or T-cell activation.
- agents such as antimicrobials for resistant intracellular pathogens (e.g., mycobacterium, leishmania, malaria), cytotoxins for receptor-positive leukemias, genes for gene therapy (e.g., for enhanced cytokine production or replacement for dysfunctional enzymes), or antigens for enhanced presentation and production of specific antibodies or T-cell activation.
- the binding specificity of the underivatized, aqueous soluble ⁇ (l,3)-glucan to its receptor provides a method for purification of both receptor-positive and receptor-negative cells (i.e., cells which do not contain the receptor); for example, underivatized, aqueous soluble ⁇ (l,3)-glucan can be affixed to a solid matrix and used as an affinity matrix to positively select receptor-positive cells or to negatively select receptor-negative cells.
- anti-receptor antibodies can be used in place of the immobilized underivatized, aqueous soluble ⁇ (l,3)-glucan. Cells which are purified by this method can subsequently be expanded for use in cell therapy.
- the present invention makes possible the generation of anti- receptor antibodies for diagnostic purposes.
- Monoclonal or polyclonal antibodies can be produced using enriched or purified receptor preparations by standard techniques.
- the present invention also relates to antibodies which bind the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan.
- polyclonal and monoclonal antibodies which bind to the described receptor are within the scope of the invention.
- a mammal such as a mouse, hamster, goat or rabbit, can be immunized with an immunogenic form of the receptor (i.e., an antigenic portion of the receptor which is capable of eliciting an antibody response).
- Techniques for conferring immunogenicity include, for example, conjugation to carriers or other techniques well known in the art.
- the antigen can be administered in the presence of an adjuvant, and the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
- Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibody.
- Antibodies to the proteinaceous receptor described herein can be produced by standard techniques such as, for example, those described in Current Protocols in Immunology (John Wiley & Sons, 1997). Such antibodies can be used to identify alterations in receptor-positive or receptor-negative cell populations which reflect disease pathology (e.g., response to cryptic fungal infection or leukemia).
- This invention also pertains to a method of altering (e.g., activating or deactivating) signal transduction pathways, for example through modulation of transcriptional regulatory factors or signal transduction enzymes (pathway enzymes) in receptor-positive cells, i.e., cells which contain the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan described herein.
- the transcriptional regulatory factor is from the NF- ⁇ B and/or NF-IL6 and/or AP-1 families of transcriptional regulatory factors.
- the transcription factor can be NF- ⁇ B, NF-IL6 or AP-1.
- the activity of the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan is activated through binding of an underivatized, aqueous soluble ⁇ (l,3)-glucan, whereby a signal transduction pathway (e.g., consisting of a cascade of enzymes) is activated and thereby regulated by a transcriptional regulatory factor (e.g., from the NF- ⁇ B, NF-IL6 and/or AP-1 families) is activated.
- Activation of the receptor can comprise, among others, an alteration in the receptor conformation, formation of a ligand-receptor complex, or alteration of the ligand-receptor complex.
- the activity of the receptor can be activated by an agent which mimics the binding and activation ability of an underivatized, aqueous soluble ⁇ (l,3)-glucan (e.g., another conformation of glucan).
- the transcriptional regulatory factor or pathway enzyme is activated as a result of ligand binding.
- the activity of the transcriptional regulatory factor or pathway enzyme is decreased, either partially or totally, by the binding of an agent which binds the receptor (and thus excludes the underivatized, aqueous soluble ⁇ (l,3)-glucan), but lacks the ability to activate the receptor.
- signal transduction pathways which can be altered by the methods of the present invention include the ras/raf-1/MAP kinase (ERK) pathway, the G- protein/phospholipase C/protein kinase C pathway, the non-G-protein/phospholipase C/protein kinase C pathway, the JAK/STAT pathway, the phospholipase A pathway, G-protein/phospholipase D/phosphatidic acid pathway, the c-AMP-dependent pathway, the c-Jun N-terminal kinase (JNK, also known as stress-activated protein kinase (SAPK)) pathway, the I ⁇ B kinase ⁇ (LKK ⁇ ) pathway, the protein tyrosine kinase pathway, and the oxygen radical pathway.
- ERK ras/raf-1/MAP kinase
- G- protein/phospholipase C/protein kinase C pathway the non-G-protein/phospho
- the present invention also pertains to a novel assay for identifying agents which alter the effect of underivatized, aqueous soluble ⁇ (l,3)-glucan on signal transduction pathways, such as activation of transcriptional regulatory factors or pathway enzymes.
- This assay comprises combining underivatized, aqueous soluble ⁇ (l,3)-glucan, the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan and an agent to be tested under conditions in which binding of underivatized, aqueous soluble ⁇ (l,3)-glucan to its receptor occurs (i.e., conditions suitable for binding of underivatized, aqueous soluble ⁇ (l,3)-glucan to the receptor for underivatized, aqueous soluble ⁇ (l,3)-glucan).
- Binding of underivatized, aqueous soluble ⁇ (l,3)- glucan to its receptor activates the receptor, which in turn activates a signal transduction pathway as shown by transcriptional regulatory factors such as those of the NF- ⁇ B and/or NF-IL6 and/or AP-1 families.
- the extent of activation of the selected transcriptional regulatory factor or pathway enzyme in the presence of an agent to be tested is determined (e.g., using radiolabeled DNA oligonucleotides specific for the transcriptional regulatory factor, as in the Examples) and compared with the extent of activation of the selected transcriptional regulatory factor or pathway enzyme in the absence of the agent to be tested; a difference in the extent of activation indicates that the agent alters the effect of underivatized, aqueous soluble ⁇ (l,3)-glucan on activation of the transcriptional regulatory factor or pathway enzyme.
- An increase in the activation of the transcriptional regulatory factor or pathway enzyme in the presence of the agent indicates that the agent enhances, i.e., prolongs or increases, the activation.
- Kits useful in the methods and assays described above are also contemplated in the invention.
- Such a kit comprises primarily the components and reagents described for the methods and assays, such as a solid phase, detectable labels, ⁇ (l,3)-glucan binding agent, and/or labeled, underivatized, aqueous soluble, ⁇ (l,3)- glucan.
- the kit comprises the receptor described herein as a ⁇ (l,3)-glucan binding agent.
- the ⁇ (l,3)-glucan binding agent is attached to a solid support, such as a filter, a membrane such as cellulose or nitrocellulose, a plastic support (e.g., microtiter plate, dipstick), a glass support (e.g., slide), beads (e.g., latex beads), particles, an organic resin, or other organic or non- organic solid phase.
- a solid support such as a filter, a membrane such as cellulose or nitrocellulose, a plastic support (e.g., microtiter plate, dipstick), a glass support (e.g., slide), beads (e.g., latex beads), particles, an organic resin, or other organic or non- organic solid phase.
- TH-glucan low molecular weight- glucan (LMW-glucan, approximately 8,000-120,000 daltons), high molecular weight-glucan (HMW-glucan, approximately >210,000 - ⁇ 1, 000,000 daltons), very high molecular weight-glucan (VHMW-glucan, approximately >1, 000,000 daltons) were prepared at Alpha-Beta Technology, Inc. according to methods described in U.S. Patent Nos.
- Scleroglucan (ACTIGUMTM) was obtained from Sanofi Bio-Industries (Paris, France). Scleroglucan used for assays in this report was modified by mild formic acid hydrolysis, neutralization and size fraction on gel permeation chromotography to achieve the equivalent hydrodynamic volume as TH-glucan. All ⁇ -glucan samples tested negative for endotoxin contamination by Limulus amoebocyte lysate assay. All other reagent grade materials were purchased from either Sigma or Boehringer Mannheim (Indianapolis, IN).
- GST fusion proteins ATF-2, ELK-1, c-jun were obtained from New England Biolabs (Beverly, MA); anti-ERKl/2, -p38, -JNK, and -IKK antibodies, as well as GST fusion protein I ⁇ B ⁇ , were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
- NR8383 cells see, for example, Helmke, R.J. et al, In Vitro Cell Develop. Biol. 23:567-574 (1987); Helmke, R.J. et al, In Vitro Cell Develop. Biol. 25:44-48 (1989). Molecular weights were determined by MALLS.
- TH-glucan (Alpha-Beta Technology, Worcester, MA) was incubated with NaIO 4 (10 mg/ml; Sigma) in sterile pyro gen- free (SPF) water for 72 hours at room temperature. The periodate was quenched by the addition of 0.5 ml ethylene glycol. The oxidized TH-glucan was dialyzed against SPF water, and then reductively labeled with 100 mCi of NaB 3 H 4 (New England Nuclear, Boston, MA). ⁇ -TH- glucan was separated from tritiated low molecular weight degradation products by dialysis and ultrafiltration. Purity of the labeled product was assessed by gel permeation chromatography.
- NR8383 cells were grown in Ham's F-12 medium containing 15% fetal bovine serum (F-12/fcs) using standard tissue culture techniques. Cells were harvested at a cell density of approximately 3x10 5 cells/ml by scraping and centrifugation (400 x g, 5 minutes, room temperature). Cells were resuspended in PBS and combined with 3 H-TH-glucan (1 ⁇ g/ml final) and either saline or competitor as indicated in the figures. Cell concentration was 0.25-1.0 xlO 6 cells/well/200 ⁇ l. The binding reaction was allowed to proceed for 60 minutes at 37 °C.
- Cells were harvested as described above and resuspended in sterile PBS containing 5 mM EDTA. Cells were aliquoted into separate tubes, re-centrifuged, and then resuspended into 1 ml PBS/EDTA with or without 0.25% trypsin (w/v). Cells were incubated at 35 °C for 45 minutes. At the end of the incubation period, 0.5 ml soybean trypsin inhibitor (0.25%, w/v) was added, followed by 10 ml F- 12/fcs.
- Cells were centrifuged as above and resuspended in either 0.7 ml PBS for immediate assay of binding activity, or in 10 ml F-12/fcs with or without cycloheximide (1 ⁇ g/ml) and cultured for an additional 24 hours. For assay of binding activity, cells were counted in a hemocytometer and cell numbers equalized. Cells were then assayed as described above except that the binding reaction was done at 4°C in the presence or absence of 100 ⁇ g/ml unlabeled TH-glucan. Cells were processed and radioactivity quantitated as described.
- lactosylceramide (Sigma Chemical Co.) was dissolved in ethanol at 1 mg/ml. Aliquots (20 ⁇ l) were applied in triplicate to the wells of a 96-well polystyrene plate (Costar) and air dried. Plates were blocked by incubation with 300 ⁇ l of 1% gelatin (w/v) in PBS at 37 °C for 1-2 hours. Plates were then rinsed with PBS (2 x 200 ⁇ l).
- NR8383 cells were incubated in F-12 + 0.1% fcs overnight at 5xl0 6 cells/ml x2 ml in 6-well plates. Cells were then incubated with either LPS (1 ⁇ g/ml) or TH- glucan (3 ⁇ g/ml) for the indicated times at 37°C. Cells were placed on ice, scraped from the wells and added to 10 ml ice cold PBS. Cells were centifuged at 400 x g for 5 minutes at 4°C, then resuspended in 1 ml PBS at 4°C.
- lysis buffer (20 mM Tris pH 7.4, 137 mM NaCl, 2 mM EDTA, 25 mM b-glycerophosphate, 2 mM Na pyrophosphate, 10% glycerol, 1% Triton X-100) containing 1 ⁇ g/ml leupeptin, aprotinin, pepstatin, 1 mM NaVO 4 , 1 mM PMSF, incubated for 10 minutes at 4°C, then centrifuged at 15,000 x g for 20 minutes. Protein content was determined by the Bradford assay (Pierce).
- Equal protein loads (100-500 ⁇ g) were added to protein A sepharose beads (Pierce) pre- bound with kinase-specific antibody (see figures) as described below.
- the immunoprecipitation reaction mixture was incubated 2-4 hours with rotation at 4°C. Beads were then washed 3 x 1 ml lysis buffer and 1 x 1 ml with kinase reaction buffer (25 mM HEPES pH 7.4, 25 mM b-glycerophosphate, 25 mM MgCl 2 , 2 mM NaVO 4 ) and finally resuspended in 50 ⁇ l kinase reaction buffer containing 2 mM DTT.
- kinase reaction buffer 25 mM HEPES pH 7.4, 25 mM b-glycerophosphate, 25 mM MgCl 2 , 2 mM NaVO 4
- the kinase reaction was then initiated by combining 20 ⁇ l beads, 22 ⁇ l reaction mix (20 ⁇ l kinase reaction buffer, 1 ⁇ l 1 mM ATP, 1 ⁇ l/10 ⁇ Ci g- 32 P-ATP), 0.5 ⁇ l of 2 ⁇ g/ml kinase substrate. Reaction proceeded for 15-20 minutes at room temperature and the reaction stopped by addition of 10 ⁇ l 4x Laemmli buffer and boiling for 5 minutes. Samples were centrifuged and an aliquot of the reaction mixture run on a 10% SDS-PAGE followed by autoradiography.
- kinase-specific antibodies were pre-bound to protein A sepharose beads as follows: beads were washed twice in 1 ml lysis buffer, centrifuged (15,000 x g for 30 seconds), and resuspended in 0.5 volumes lysis buffer. Twenty microliters of bead suspention were aliquoted into microfuge tubes, and 2 ⁇ l antibody (approximately 0.4 ⁇ g) were added. The bead/antibody mixture was allowed to incubate for 20-30 minutes on ice before addition of cell lysates.
- ESA Electrophoretic mobility shift assays
- EMS A were performed essentially as described in Adams, et al, J. Leuk. Biol. 62 (6): 865-873 (1997).
- Cell Stimulations LPS (1 ⁇ g/ml), TH-glucan (3 ⁇ g/ml), low molecular weight-glucan (LMW- glucan; 3 or 30 ⁇ g/ml), high molecular weight-glucan (VHMW-glucan; 3 ⁇ g/ml), or scleroglucan (3 ⁇ g/ml) were added to the medium and 37 °C incubations continued for 0.5-1.0 hr. After stimulation cells were scraped into ice cold PBS, rinsed once with PBS, then used for nuclear extractions.
- PMN were prepared from donor blood anti-coagulated with acid citrate dextrose. Following sedimentation in 1.5% dextran (w/v), cells were centrifuged (400 x g, 7 minutes) and resuspended in autologous plasma. Cells were then layered over a Ficoll gradient (Lymphocyte Separation Medium; Organon Teknika, Durham, NC) and centrifuged (400 x g, 30 minutes). Cells recovered from the pellet were hypotonically lysed to remove residual RBC. The remaining cells were >95% neutrophils as judged by morphological criteria, and were then stimulated as described above prior to preparation of nuclear extracts.
- Ficoll gradient Lymphocyte Separation Medium
- Nuclear extracts for EMSA were prepared as described previously (Adams, et al, 1997)) using 1.0-2.0 x 10 7 cells per sample. All buffers were freshly supplemented with DTT (0.5 mM), protease inhibitor cocktail, and phosphatase inhibitor cocktail. Protein concentrations were determined by Bradford assay (Pierce) against a BSA standard.
- Electrophoretic Mobility Shift Assays Transcription factor activations were assayed using an EMSA as described previously (Adams, et al, (1997)). NF- ⁇ B consensus synthetic duplex probe was as described (Adams, et al, (1997); see also Lenardo, M.J. and Baltimore, D., Cell 58:221-229 (1989)). 32 P-labeled duplex probes were prepared with polynucleotide kinase.
- Labeled probe (0.5 pmol) was mixed with 3 ⁇ g of nuclear extract protein in a solution containing 10 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 5% glycerol, 0.02% ⁇ -mercaptoethanol, 0.1-1.0 ⁇ g of poly(dI/dC) (Pharmacia). Reactions were incubated at 25 °C for 20 min then electrophoresed under non-denaturing conditions through 4% polyacrylamide gels in 0.5X TBE buffer. Bands were visualized by autoradiography.
- NR8383 cells were incubated with 3 H-TH-glucan at increasing concentrations in the presence (non-specific) or absence (total) of 1000-fold excess unlabeled TH- glucan for 60 minutes at 4°C. At the end of the incubation, unbound radioactivity was removed by washing, and the amount of cell-associated radioactivity determined as described above. Specific binding was determined by subtracting non-specific from total radioactivity. The results are shown in Fig. 1 ; the specific binding curve was fit by a nonlinear regression analysis of a two-site model as described above. The data in Fig. 1 demonstrate that 3 H-TH-glucan binding to the NR8383 cells was dose-dependent, specific, and saturable.
- NR8383 cells bound increasing amounts of 3 H-TH-glucan as the concentration of ligand was increased (total). Addition of unlabeled TH-glucan to the incubation mixture inhibited the amount of 3 H-TH-glucan associated with the cells (non-specific). After subtraction of the non-specific bound from the total bound, the specific binding was found to be saturable at about 30 fm/10 6 cells.
- Non-linear regression analysis of the specific binding curve fit a two-site binding model, with apparent dissociation constants Kdl and Kd2 of 45 pM and 170 pM, respectively. The number of binding sites was 3,200/cell and 18,000/cell for the high and low affinity sites, respectively.
- the binding activity observed at 4°C for NR8383 cells contrasts with that observed for lactosylceramide, which shows no binding activity at 4°C (data not shown).
- NR8383 cells were co-incubated with 3 H -TH-glucan and various concentrations of unlabeled competitor ⁇ -glucan (LMW-glucan, VHMW-glucan; or scleroglucan) for 60 minutes at 37 °C. Unbound radioactivity was removed and cell-associated radioactivity determined as described above. The results of this type of experiment are shown in Fig. 2A-C (Fig. 2A: LMW-glucan; Fig. 2B: VHMW-glucan; Fig. 2C: scleroglucan).
- LMW-glucan unlabeled competitor ⁇ -glucan
- VHMW-glucan VHMW-glucan
- Fig. 2C scleroglucan
- TH-glucan was included as a positive control in each experiment. Unlabeled TH-glucan, LMW-glucan, and VHMW-glucan were able to compete for 3 H-TH-glucan binding, whereas scleroglucan was not an effective competitor in the concentration range tested.
- Lactosylceramide has been identified as the TH-glucan receptor on human neutrophils (U.S. Patent application number 08/990,125, attorney docket no. ABY97-04, filed on December 12, 1997; the entire teachings of which are incorporated herein by reference). The ability of these same ⁇ -glucans to compete for binding of 3 H-TH-glucan to lactosylceramide was therefore investigated.
- Lactosylceramide was coated onto 96-well plates as described above, and incubated with 3 H-TH-glucan (1 ⁇ g/ml) in the presence of various concentrations of unlabeled ⁇ -glucans (LMW-glucan; VHMW-glucan; scleroglucan) for 60-90 minutes at 37 °C. Wells were rinsed and the remaining radioactivity was measured as described above. The results are shown in Fig. 3A-C (Fig. 3 A: LMW-glucan; Fig. 3B: VHMW- glucan; Fig. 3C: scleroglucan).
- Activation of an NF-kB-like transcription factor TH-glucan-induced signal transduction through lactosylceramide on human PMN and murine BMC2.3 macrophages has been shown to activate an NF- ⁇ B-like nuclear transcription factor (U.S. Patent application number 08/990,125, attorney docket no. ABY97-04, filed on December 12, 1997; the entire teachings of which are incorporated herein by reference).
- the ability of TH-glucan, LMW- glucan,VHMW-glucan, and scleroglucan to activate NF- ⁇ B-like DNA binding activity in NR8383 cells and human PMN was compared using Electrophoretic Mobility Shift Assays (EMSA).
- ESA Electrophoretic Mobility Shift Assays
- ⁇ -glucans control; LPS, 1 ⁇ g/ml; TH-glucan, 3 ⁇ g/ml; scleroglucan, 3 ⁇ g/ml; LMW-glucan, 3 ⁇ g/ml or 30 ⁇ g/ml; VHMW-glucan, 3 ⁇ g/ml).
- Nuclear extracts were prepared for determination of NF- ⁇ B activation by EMSA as described above.
- TH-glucan, VHMW-glucan, and LMW-glucan induce NF- ⁇ B-like DNA binding activity, but scleroglucan does not (data not shown).
- TH-glucan and scleroglucan induce the NF-kB-like DNA binding activity, while LMW-glucan does not (data not shown: VHMW-glucan was not tested in these cells).
- signal transduction initiated by these various ⁇ -glucans in rat NR8383 cells and human PMN correlates with their ability to compete for 3 H-TH-glucan binding.
- NR8383 cells were incubated with LPS (1 ⁇ g/ml, positive control), TH-glucan (3 ⁇ g/ml), or no addition for 30 minutes at 37°C. Cells were lysed, immunoprecipitated with kinase-specific antibody, and a kinase assay performed as described above. Equal amounts of protein were used for immunoprecipitations within each experiment. TH-glucan does not activate the
- MAP kinases extracellular mitogen-regulated kinase kinase substrate was ATF-2-GST; or p38 (kinase substrate with ELK- 1 -GST) (data not shown).
- TH-glucan rapidly and transiently activates the MAP kinase c-Jun N- terminal kinase (JNK, also known as stress-activated protein kinase or S APK; kinase substrate was c-jun-GST) (data not shown).
- TH-glucan also rapidly and transiently activates the I ⁇ B kinase alpha (LKKa; kinase substrate was I ⁇ B ⁇ -GST), the kinase that phosphorylates I ⁇ Ba, leading to activation of NF- ⁇ B-like transcription factors (data not shown).
- LKKa kinase substrate was I ⁇ B ⁇ -GST
- NF- ⁇ B-like transcription factors data not shown.
- TH-glucan and Conformers TH-glucan (Alpha-Beta Technology) and certain conformers were used in studies of their effect on an infection model.
- Table 2 illustrates the molecular weights of TH-glucan and the conformers used in this study. Because the molecular weight of conformers can change with the temperature in the GPC column, conformers separated at room temperature (25 °C) and at a higher temperature (37°C) were tested.
- the average molecular weights were measured by a gel permeation chromatography/multi-angle laser light scattering (GPC/MALLS). HTH and LTH were prepared by fractionating samples of TH-glucan.
- Virus-antibody free Wistar rats were purchased from Charles River Breeding Laboratories (Wilmington, MA). Animals were housed according to the National Institutes of Health guidelines and were provided food and water ad libitum. Rats were quarantined for 7 days prior to being entered into experiments. Body weights of rats ranged from 160 g to 210 g at the time of experimentation. Drug Administration
- TH-glucan and conformers, as well as whole glucan particles were dosed intramuscularly (IM) using a 25-gauge needle at 1 mg/kg at 48 h, 24 h and 4 h before bacterial challenge and at 4 h after bacterial challenge.
- WGP was also given intraperitoneally (IP) at a dose of 5 mg/kg at 24 h and 4 h before bacterial challenge.
- IP intraperitoneally
- Rats were anesthetized by im injection of a mixed anesthetic cocktail consisting of Ketamine (Fort Dodge Laboratores Inc., Fort Dodge, IA), PromAce (Ayerst Laboratores Inc., Rouses Point, MY), Xylazine (Phoenix Scientific Inc., St. Joseph, MO) and saline (750 mg, 10 mg, 100 mg and saline to 20 ml) using a 25 gauge needle. The anesthesia was adjusted for each rat based on body weight by administering 0.0019 ml/g body weight.
- Results are expressed at the mean ⁇ standard error of the mean (SEM) of data obtained from replicate experiments (2 -6 experiments, containing 20-80 animals). Unpaired t-tests (treatment group vs. saline) were performed using EXCEL 5.0 software (Microsoft Corporation, Redmond, WA) and differences were considered significant at p ⁇ 0.05.
- the S. aureus dose used in these studies was a 50% lethal infective dose. Although all animals were euthanized on day 2 for CFU and blood cell evaluations, a significantly lower mortality was observed in the rats treated with WGP (IP) (p ⁇ 0.01) and LMW (p ⁇ 0.05) compared to saline-treated rats. Slightly lower mortality was also seen in the rats treated with WGP (IM) (31% vs saline 48%) and LTH 25 °C (31% vs saline 48%). A slightly higher mortality (63% vs saline 48%) was observed in the LTH 37 0 C-treatment group. However, these differences did not reach statistical significance.
- TH-glucan as well as conformers (agents which mimic the activity of TH-glucan), can be used to augment host defenses, and thus can be used in the treatment of diseases. It was interesting to note that although the conformer molecular weights changed up to approximately 100 fold (e.g., VHMW vs LMW), the anti-infective activity remained constant.
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EP99943790A EP1105733A1 (en) | 1998-08-26 | 1999-08-19 | Receptor for underivatized, aqueous soluble beta-(1,3)-glucan |
AU56819/99A AU749716B2 (en) | 1998-08-26 | 1999-08-19 | Receptor for underivatized, aqueous soluble beta-(1,3)-glucan |
MXPA01002022A MXPA01002022A (en) | 1998-08-26 | 1999-08-19 | Receptor for underivatized, aqueous soluble beta-(1,3)-glucan. |
JP2000567952A JP2002523522A (en) | 1998-08-26 | 1999-08-19 | Non-derivatized water-soluble beta- (1,3) -glucan receptor |
CA002341668A CA2341668A1 (en) | 1998-08-26 | 1999-08-19 | Receptor for underivatized, aqueous soluble beta-(1,3)-glucan |
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1999
- 1999-08-19 MX MXPA01002022A patent/MXPA01002022A/en active IP Right Grant
- 1999-08-19 EP EP99943790A patent/EP1105733A1/en not_active Ceased
- 1999-08-19 CA CA002341668A patent/CA2341668A1/en not_active Abandoned
- 1999-08-19 AU AU56819/99A patent/AU749716B2/en not_active Ceased
- 1999-08-19 WO PCT/US1999/018995 patent/WO2000013019A1/en not_active Application Discontinuation
- 1999-08-19 JP JP2000567952A patent/JP2002523522A/en not_active Withdrawn
-
2011
- 2011-01-21 JP JP2011011128A patent/JP2011102323A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
AU749716B2 (en) | 2002-07-04 |
MXPA01002022A (en) | 2002-04-24 |
CA2341668A1 (en) | 2000-03-09 |
EP1105733A1 (en) | 2001-06-13 |
WO2000013019A1 (en) | 2000-03-09 |
AU5681999A (en) | 2000-03-21 |
JP2011102323A (en) | 2011-05-26 |
JP2002523522A (en) | 2002-07-30 |
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