Patent Application
20120195875
The present invention relates to assessing immune modulation and susceptibility to infection in individuals, including those with sepsis and chronic infections. The present invention further relates to the identification of individuals who are at risk for cancer.
All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
The detection of pathogens by innate immune cells triggers a robust and essential inflammatory reaction. However, uncontrolled inflammation leads to excessive tissue destruction and manifestation of pathological states like sepsis, autoimmune diseases, and cancer, making a tight control of the inflammatory reaction necessary. One important mechanism to prevent over-exuberant inflammation is the induction of endotoxin tolerance. On the other hand prolonged and severe immune tolerance can increase the risk for secondary bacterial infections. Despite numerous studies the molecular mechanisms underlying LPS tolerance in vitro and in vivo remain largely elusive (12). In particular, the effect of LPS-induced tolerance on protein phosphatase activity remains largely unknown.
Currently there is no diagnostic test available to assess the level of immune suppression in septic patients, and there is no treatment available to reverse the immune modulation. Further, although infection has been shown to be associated with cancer development, there are no effective prevention strategies. Therefore, there is a need in the art to identify a diagnostic marker to assess the level of immune modulation in septic patients, and in patients with chronic infections, to provide a method of prevention and treatment of secondary infections, as well as prevention and treatment of cancerous conditions.
In some embodiments, the invention teaches a method of determining an individual's susceptibility to an infection, including: obtaining a biological sample from the individual; measuring a level of protein phosphatase 2A (PP2A) activity in the biological sample; and determining the individual's susceptibility to the infection based upon the level of PP2A activity, wherein a higher than average risk of developing the infection is determined if abnormally low PP2A activity is measured, and an average risk of developing the infection is determined if normal PP2A activity is measured. In some embodiments, the level of PP2A activity is measured indirectly by measuring the level of phosphorylation of its upstream and/or downstream interacting/target molecules in the biological sample. In some embodiments, the individual is immune suppressed. In some embodiments, the individual has sepsis. In some embodiments, the infection is a superinfection. In some embodiments, the infection is selected from the group consisting of: bacterial, fungal, viral, and combinations thereof. In some embodiments, the individual has a history of infection. In some embodiments, the infection is a gram-negative and/or gram-positive bacterial infection.
In some embodiments, the invention teaches a method of reducing a likelihood of an individual developing an infection, including: providing a prophylactic dose of protein phosphatase 2A (PP2A), and administering the dose to the individual. In some embodiments, the individual is immune suppressed. In some embodiments, the infection is a superinfection. In some embodiments, the infection is selected from the group consisting of: bacterial, fungal, viral, and combinations thereof. In some embodiments, the infection is a gram-negative and/or gram-positive bacterial infection. In some embodiments, the individual has sepsis and the infection is a secondary infection.
In some embodiments, the invention teaches a method of treating an infection in an individual, including: providing a therapeutic dose of protein phosphatase 2A (PP2A), and administering the dose to the individual. In some embodiments, the individual is immune suppressed. In some embodiments, the infection is a superinfection. In some embodiments, the infection is selected from the group consisting of: bacterial, fungal, viral, and combinations thereof. In some embodiments, the infection is a gram-negative and/or gram-positive infection.
In certain embodiments, the invention teaches a kit for determining an individual's susceptibility to infection, including: a means for measuring a level of protein phosphatase 2A (PP2A) activity in a biological sample of the individual, and instructions for use thereof to determine an individual's susceptibility to infection. In some embodiments, the level of PP2A activity is measured indirectly by measuring the level of phosphorylation of its upstream and/or downstream interacting/target molecules in the biological sample. In some embodiments, the infection is selected from the group consisting of: viral, bacterial, fungal, and combinations thereof.
In certain embodiments, the invention teaches a kit for treating an infection in an individual, including: a quantity of protein phosphatase 2A (PP2A), and instructions for use thereof to treat an infection in an individual. In some embodiments, the infection is selected from the group consisting of: viral, bacterial, fungal, and combinations thereof.
In certain embodiments, the invention teaches a kit for reducing the likelihood of an individual contracting an infection, including: a quantity of protein phosphatase 2A (PP2A), and instructions for use thereof for reducing the likelihood of an individual contracting an infection. In some embodiments, the infection is selected from the group consisting of: viral, bacterial, fungal, and combinations thereof.
In certain embodiments, the invention teaches a method of determining an individual's susceptibility to cancer, including: obtaining a biological sample from the individual; measuring a level of protein phosphatase 2A (PP2A) activity in the biological sample; and determining the individual's susceptibility to cancer based upon the level of PP2A activity, wherein a higher than average risk of developing cancer is determined if abnormally low PP2A activity is measured, and an average risk of developing cancer is determined if normal PP2A activity is measured. In some embodiments, the level of PP2A activity is measured indirectly by measuring the level of phosphorylation of its upstream and/or downstream interacting/target molecules in the biological sample. In some embodiments, the cancer is the result of repeated infections and/or reduced PP2A activity.
In some embodiments, the invention teaches a kit for determining an individual's susceptibility to cancer, including: a means for measuring a level of protein phosphatase 2A (PP2A) activity in a biological sample of the individual, and instructions for use thereof to determine an individual's susceptibility to cancer. In some embodiments, the level of PP2A activity is measured indirectly by measuring the level of phosphorylation of its upstream and/or downstream interacting/target molecules in the biological sample. In some embodiments, the cancer is the result of repeated infections and/or reduced PP2A activity.
In some embodiments, the invention teaches a method of treating an individual after determining the individual has a higher than average risk of developing an infection resulting from abnormally low PP2A activity, including: obtaining a biological sample from the individual; measuring a level of protein phosphatase 2A (PP2A) activity in the biological sample; determining the individual's susceptibility to the infection based upon the level of PP2A activity, wherein a higher than average risk of developing the infection is determined if abnormally low PP2A activity is measured, and an average risk of developing the infection is determined if normal PP2A activity is measured; and treating the individual with a prophylactic dose of a type of medication selected from the group consisting of: antiviral, antifungal, antibacterial, and combinations thereof if it is determined the individual has a higher than average risk of developing the infection resulting from abnormally low PP2A activity. In some embodiments, the level of PP2A activity is measured indirectly by measuring the level of phosphorylation of its upstream and/or downstream interacting/target molecules in the biological sample.
In certain embodiments, the invention teaches a method of monitoring an individual after determining the individual has a higher than average risk of developing cancer resulting from abnormally low PP2A activity, including: obtaining a biological sample from the individual; measuring a level of protein phosphatase 2A (PP2A) activity in the biological sample; determining the individual's susceptibility to cancer based upon the level of PP2A activity measured, wherein a higher than average risk of developing cancer is determined if abnormally low PP2A activity is measured, and an average risk of developing cancer is determined if normal PP2A activity is measured; and testing the individual for one or more forms of cancer more frequently than an individual having an average risk of developing cancer if it is determined the individual has a higher than average risk of developing cancer resulting from abnormally low PP2A activity. In some embodiments, the level of PP2A activity is measured indirectly by measuring the level of phosphorylation of its upstream and/or downstream interacting/target molecules in the biological sample.
In certain embodiments, the invention teaches a method of treating an individual after determining the individual has a higher than average risk of developing cancer of the digestive tract resulting from abnormally low PP2A activity, including: obtaining a biological sample from the individual; measuring a level of protein phosphatase 2A (PP2A) activity in the biological sample; determining the individual's susceptibility to cancer of the digestive tract based upon the level of PP2A activity, wherein a higher than average risk of developing cancer of the digestive tract is determined if abnormally low PP2A activity is measured, and an average risk of developing cancer of the digestive tract is determined if normal PP2A activity is measured; and treating the individual with antibiotics if it is determined the individual has a higher than average risk of developing cancer of the digestive tract resulting from abnormally low PP2A activity. In some embodiments, the individual has been diagnosed with inflammatory bowel disease. In some embodiments, the level of PP2A activity is measured indirectly by measuring the level of phosphorylation of its upstream and/or downstream interacting/target molecules in the biological sample.
Exemplary embodiments are illustrated in the referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.
All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, N.Y. 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled in the art with a general guide to many of the terms used in the present application.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.
As used herein:
The acronym “LPS” means lipopolysaccharide.
The acronym “PBMC” means peripheral blood mononuclear cell.
The acronym “MDM” means monocyte-derived macrophages.
The acronym “SOCS” means suppressor of cytokine signaling.
The acronym “PP2A” means protein phosphatase 2A.
As used herein, “beneficial results” may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a subject developing the disease condition and prolonging a subject's life or life expectancy.
“Conditions” and “disease conditions,” as used herein may include, but are in no way limited to any form of acute infection, chronic infection, sepsis, super-infection, immune suppression, cancerous conditions, or combinations thereof.
“Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.
“Treatment” and “treating,” as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to slow down (lessen) the targeted pathologic condition, prevent the pathologic condition, pursue or obtain beneficial results, or lower the chances of the individual developing the condition even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented.
In some embodiments, the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the application are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
By way of background, macrophages exposed to enteric gram-negative lipopolysaccharide (LPS) become refractory to subsequent challenge with LPS. This refractory phenotype, referred to as LPS-tolerance, is characterized by a decline in the production of inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12 (1-3). LPS-tolerance was first demonstrated in animal models of endotoxemia (4), and this phenotype can also be induced in primary monocytes, monocyte-derived macrophages (MDM), and mature macrophages as well as in myeloid cell lines (5-8). In humans, this condition is observed in peripheral blood mononuclear cells (PBMC) isolated from subjects given a single intravenous dose of Escherichia coli LPS (9) and in monocytes isolated from patients with sepsis (10,11) where it contributes to “immunosuppression”, secondary infections, and mortality.
Although the molecular mechanisms underlying tolerance to microbial products have been studied extensively they are still largely unknown (12-14). Interaction of LPS with its cellular receptor TLR4 triggers a multitude of signaling events including the phosphorylation of MAPKs and IκB leading to the activation of NF-κB responsive proinflammatory cytokines such as TNF-α. (15,16). The activation of MAPKs and NF-κB are tightly regulated and dysregulation of these signaling pathways have been implemented in LPS tolerance. Decreased expression of the LPS receptor complex TLR4/MD2 (17), suppressed IRAK-1-MyD88 interactions and IRAK-1 activation (14, 18-22), as well as decreased TLR4 tyrosine phosphorylation and consequently poor MyD88 recruitment to TLR4 (23) were suggested to mediate LPS tolerance. Others have shown an increase in negative regulators of TLR4 signaling, such as Toll-interacting protein (Tollip), suppressor of cytokine signaling (SOCS)-1, IL-1R-associated kinase-M (IRAK-M), ST2, an alternatively spliced short version of MyD88 (MyD88s), and SH2-containing inositol phosphatase (SHIP-1) expression in tolerized cells (24-28).
Following initiation of the above mentioned signaling pathways de-phosphorylation plays a central role in regulating and limiting intracellular signaling. Since phosphorylation of several signaling molecules, such as IRAK-1, MAPKs, PKR, and IKB is impaired in LPS tolerant cells (23,29) the inventors explored the possible role of phosphatases in LPS-induced tolerance in macrophages. Furthermore, SH2-containing inositol phosphatase (SHIP-1) has been implicated in endotoxin tolerance (28) but the involvement of other phosphatases has not been studied.
The serine-threonine protein phosphatase PP2A is abundantly expressed in cells and has been implicated in the regulation of many signaling pathways including those implicated in endotoxin tolerance such as JNK, and MAPK pathways (30,31). Inhibition of PP1 and PP2A by calyculin A has been shown to inhibit LPS-induced TNF-α, IL-1β, IFN-β, IP-10, IRF-1, and TNFR-2 release in macrophages (32). In addition to tyrosine kinases, serine-threonine kinases have been proposed to regulate LPS-induced signaling in macrophages (27). Briefly, PP2A associates with IKK and leads to the modulation of NF-KB transcriptional activity (33,34) by directly interacting with IKKβ (34).
Toll-like receptors are sensors for microbial components leading to the secretion of pro-inflammatory mediators by monocytes/macrophages. This process is tightly regulated by various mechanisms. Repetitive exposure of macrophages to bacterial lipopolysaccharide (LPS) is known to tolerize them to further stimulation and inhibit pro-inflammatory cytokine release.
The inventors demonstrate herein that PP2A activity is suppressed in vitro in lipopolysaccharide (LPS)-tolerized human THP-1 macrophages, and in vivo in peritoneal macrophages obtained from mice repeatedly exposed to LPS. These observations have important clinical implications in understanding the mechanisms associated with tolerization and consequences of repeated LPS exposure.
In certain embodiments, the present invention teaches a method of determining an individual's susceptibility to infection by obtaining a biological sample from the individual and determining a level of PP2A activity in the biological sample. In certain embodiments, a higher than average risk of developing an infection is determined if abnormally low PP2A activity is measured, and an average risk of developing an infection is determined if normal PP2A activity is measured. In some embodiments, PP2A activity is determined indirectly by measuring the level of phosphorylation of its upstream and/or downstream interacting/target molecules in the biological sample. In certain embodiments, the susceptibility determined is susceptibility to a bacterial, fungal or viral infection, or combinations thereof. In some embodiments, the susceptibility determined is susceptibility to a superinfection. In certain embodiments, the susceptibility determined is susceptibility to a gram-negative and/or gram-positive bacterial infection. In various embodiments, the individual tested for susceptibility has a history of a gram-negative and/or gram-positive bacterial infection, fungal infection, or viral infection. In certain embodiments, the individual is a mammal. In certain embodiments, the individual is a human.
One of skill in the art would readily appreciate there are many ways to measure PP2A activity. In certain embodiments, PP2A activity is measured according to the protocols set forth in Examples 6 and 10, disclosed herein. In certain embodiments, PP2A activity is measured utilizing an immunologically-based assay to analyze the biological sample. In certain embodiments, PP2A activity is measured using a spectroscopy-based assay to analyze the biological sample. In certain embodiments PP2A activity may be measured alone. In other embodiments, PP2A activity may be measured in combination with other phosphatases. In certain embodiments, PP2A activity may be measured indirectly by measuring the level of phosphorylation of its upstream and/or downstream interacting/target molecules.
In certain embodiments, the present invention teaches a method of reducing an individual's likelihood of developing an infection by administering to the individual a prophylactic dose of PP2A. In some embodiments, the individual is immune suppressed. In some embodiments, the infection is a superinfection. In some embodiments, the PP2A is administered in combination with prophylactic doses of other phosphatases and/or other therapeutic agents. In certain embodiments, the PP2A administered is recombinant PP2A. In yet other embodiments, a substitute agent for PP2A with substantially similar physical properties and/or physiological effect with respect to the ability of PP2A to reduce an individual's likelihood of developing an infection is administered to the individual. In certain embodiments, the source of the immune suppression in the individual is prior exposure to LPS from a gram-negative bacterial infection or antigens from gram-positive bacterial infections, fungal, or viral infections. In certain embodiments, the individual is a mammal. In certain embodiments, the individual is a human.
In certain embodiments, the present invention teaches a method of treating an infection in an individual by administering to the individual a therapeutic dose of PP2A. In certain embodiments, the individual is immune suppressed. In some embodiments, improved immune function results from treatment. In certain embodiments, the PP2A administered is recombinant PP2A. In yet other embodiments, a substitute agent for PP2A with substantially similar physical properties and/or physiological effect with respect to the ability of PP2A to treat an infection in an individual is administered to the individual. In some embodiments, endogenous expression of PP2A is upregulated in order to treat, prevent or reduce the likelihood of an infection. Upregulation of PP2A can be achieved by any number of methods, as will be readily appreciated by one of skill in the art. For example, a vector may be used to introduce additional copies of PP2A, in order to increase expression thereof. In some embodiments, the vector is an adenovirus. One of skill in the art would readily appreciate that any number of other viruses with substantially similar capacities for expression could be used. Alternatively, endogenous expression of PP2A may be upregulated by treating the individual with one or more substances that naturally induce greater expression of PP2A, either directly, or indirectly. In certain embodiments, the infection is a superinfection. In some embodiments, the infection is caused by gram-negative bacteria, gram-positive bacteria, fungi, virus, or combinations thereof. In some embodiments, the administration of PP2A is part of a treatment protocol that includes one or more additional therapeutic agents.
In certain embodiments, the present invention teaches a method of determining an individual's susceptibility to cancer secondary to chronic infection by obtaining a biological sample from the individual, measuring a level of PP2A activity in the sample, and determining the individual's risk of developing cancer, based upon the level of PP2A activity, alone or in combination with other phosphatases, with or without the presence of other risk factors and with or without other laboratory test results. In certain embodiments, a higher than average risk of developing cancer is determined if abnormally low PP2A activity is measured, alone or in combination with other phosphatases, and an average risk of developing cancer is determined if normal PP2A activity is measured, alone or in combination with other phosphatases. In some embodiments, PP2A activity is determined indirectly by measuring the level of phosphorylation of its upstream and/or downstream interacting/target molecules in the biological sample.
In certain embodiments, the invention discloses a method of reducing an individual's likelihood of developing cancer by administering PP2A to the individual, alone or in combination with other phosphatases. In some embodiments, the individual has been determined to have abnormally low PP2A activity, alone or in combination with other phosphatases. In certain embodiments, the PP2A administered is recombinant PP2A. In certain embodiments PP2A is administered via viral vectors. In some embodiments, the viral vector is an adenovirus. One of skill in the art would readily appreciate that any number of other viruses with substantially similar capacities for expression could be used. In yet other embodiments, a substitute agent for PP2A with substantially similar physical properties and/or physiological effect with respect to the ability of PP2A to reduce an individual's likelihood of developing cancer is administered to the individual by one or more methods described herein. In certain embodiments, the individual is a mammal. In certain embodiments, the individual is a human.
In some embodiments, the invention teaches a method of treating an individual after determining the individual has a higher than average risk of developing an infection resulting from abnormally low PP2A activity, including: obtaining a biological sample from the individual; measuring a level of protein phosphatase 2A (PP2A) activity in the biological sample; determining the individual's susceptibility to the infection based upon the level of PP2A activity, wherein a higher than average risk of developing the infection is determined if abnormally low PP2A activity is measured, and an average risk of developing the infection is determined if normal PP2A activity is measured; and treating the individual with a prophylactic dose of a type of medication selected from the group consisting of: antiviral, antifungal, antibacterial, and combinations thereof if it is determined the individual has a higher than average risk of developing the infection resulting from abnormally low PP2A activity.
In certain embodiments, the invention teaches a method of monitoring an individual after determining the individual has a higher than average risk of developing cancer resulting from abnormally low PP2A activity, including: obtaining a biological sample from the individual; measuring a level of protein phosphatase 2A (PP2A) activity in the biological sample; determining the individual's susceptibility to cancer based upon the level of PP2A activity measured, wherein a higher than average risk of developing cancer is determined if abnormally low PP2A activity is measured, and an average risk of developing cancer is determined if normal PP2A activity is measured; and testing the individual for one or more forms of cancer more frequently than an individual having an average risk of developing cancer if it is determined the individual has a higher than average risk of developing cancer resulting from abnormally low PP2A activity.
In certain embodiments, the invention teaches a method of treating an individual after determining the individual has a higher than average risk of developing cancer of the digestive tract resulting from abnormally low PP2A activity, including: obtaining a biological sample from the individual; measuring a level of protein phosphatase 2A (PP2A) activity in the biological sample; determining the individual's susceptibility to cancer of the digestive tract based upon the level of PP2A activity, wherein a higher than average risk of developing cancer of the digestive tract is determined if abnormally low PP2A activity is measured, and an average risk of developing cancer of the digestive tract is determined if normal PP2A activity is measured; and treating the individual with antibiotics if it is determined the individual has a higher than average risk of developing cancer of the digestive tract resulting from abnormally low PP2A activity. In some embodiments, the individual has been diagnosed with inflammatory bowel disease.
In certain embodiments, the gram-negative and gram-positive bacteria associated with one or more types of infection disclosed herein, include: Enterobacter, Staphylococcal, and Streptococcal species. In certain embodiments, specific organisms include but are not limited to Escherichia coli, Salmonella, Shigella, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, Legionella, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Hemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, Acinetobacter species, Klebsiella species, Burkholderia, Staphylococcus aureus (including Methicillin resistant staphylococcus aureus), Streptococcus pneumonia, Group A streptococcus, Group B streptococcus, Coagulase negative staphylococcus, Listeria monocytogeneses, and combinations thereof. In certain embodiments, the virus or viruses associated with viral infections include but are not limited to Influenza, parainfluenza, respiratory syncytial virus, adenovirus, rhinovirus, measles, metapneumovirus, SARS and combinations thereof. In certain embodiments the yeast associated with infections may include but are not limited to species of Candida. In certain embodiments, the mold associated with infection may include but are not limited to species of Aspergillus and mucormycosis.
In various embodiments, the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a prophylactically or therapeutically effective amount of PP2A, recombinant PP2A, or a substitute agent with substantially similar physical properties and/or physiological effect as those attributed to PP2A as described herein, alone or in combination with a prophylactically or therapeutically effective amount of other phosphatases. “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. “Route of administration” may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral. “Transdermal” administration may be accomplished using a topical cream or ointment or by means of a transdermal patch. “Parenteral” refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. Via the enteral route, the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Via the topical route, the pharmaceutical compositions based on compounds according to the invention may be formulated for treating the skin and mucous membranes and are in the form of ointments, creams, milks, salves, powders, impregnated pads, solutions, gels, sprays, lotions or suspensions. They can also be in the form of microspheres or nanospheres or lipid vesicles or polymer vesicles or polymer patches and hydrogels allowing controlled release. These topical-route compositions can be either in anhydrous form or in aqueous form depending on the clinical indication. Via the ocular route, they may be in the form of eye drops.
The pharmaceutical compositions according to the invention can also contain any pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, viral vector or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its prophylactic or therapeutic benefits.
The pharmaceutical compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, an elixir, an emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
The pharmaceutical compositions according to the invention may be delivered in a prophylactically or therapeutically effective amount. The precise prophylactically or therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the compound and/or viral vector (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a prophylactically or therapeutically effective amount through routine experimentation, for instance, by monitoring a subject's response to administration of a compound/vector and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).
Typical dosages can be in the ranges recommended by the manufacturer where known prophylactic or therapeutic compounds are used, and also as indicated to the skilled artisan by the in vitro responses or responses in animal models. Such dosages typically can be reduced by up to about one order of magnitude in concentration or amount without losing the relevant biological activity. Thus, the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the prophylactic or therapeutic method based, for example, on in vitro responsiveness or the responses observed in the appropriate animal models.
The present invention is also directed to a kit with an intended function selected from the group consisting of: preventing, lessening, diagnosing, prognosing, and treating immune suppression, infection, sepsis and the associated risk of cancer development. The kit can be configured in numerous ways to be useful for practicing any of the inventive methods disclosed herein, including: preventing, lessening, diagnosing, prognosing, and treating infection, sepsis, immune suppression, and the associated risk of cancer development secondary to infection. The kit is an assemblage of materials or components, including at least one of the inventive compositions. Thus, in some embodiments the kit contains a composition including PP2A, with or without other phosphatases, as described above. In other embodiments, the kit contains a composition and/or components useful for measuring the level of PP2A activity directly and/or indirectly, and/or other phosphatases in a subject, as described herein.
The exact nature of the components configured in the inventive kit depends upon its intended purpose. For example, some embodiments are configured for the purpose of diagnosing or treating infection or determining the risk thereof. Other embodiments are configured for the purpose of diagnosing or treating immune suppression or determining the risk thereof. Other embodiments are configured for the purpose of treating cancerous conditions or determining the risk thereof. Some embodiments are configured for determining the aforementioned risks by proving a means for measuring PP2A activity directly or indirectly, alone or together with the activity of other phosphatases. In the event the kit is configured for treating one or more of the conditions described above, it includes a quantity of PP2A, or a functionally equivalent substitute with the same physiological effect. In one embodiment, the kit is configured particularly for use with mammalian subjects. In another embodiment, the kit is configured particularly for use with human subjects. In further embodiments, the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals.
Instructions for use may be included in the kit. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit to affect a desired outcome, such as to treat an infection, prevent an infection, or to diagnose susceptibility to an infection, sepsis, or a cancerous condition secondary to infection. Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia as will be readily recognized by those of skill in the art.
The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. The packaging materials employed in the kit are those customarily utilized in treating, detecting or preventing infection, or cancer secondary thereto. As used herein, the term “package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain suitable quantities of an inventive composition containing PP2A and/or other phosphatases and/or functional equivalents thereof, or a substance useful in detecting the activity of PP2A directly and/or indirectly in a subject. The package may also contain materials to measure the level of other phosphatases. The packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.
The following examples are for illustrative purposes only and are not intended to limit the scope of the disclosure or its various embodiments in any way.
Ultrapure LPS from E. coli 0111:B4 was purchased from InvivoGen (San Diego, Calif.). PP2A Immunoprecipitation Phosphatase Assay Kit was purchased from Upstate (Lake Placid, N.Y.).
Six-week-old C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, Me.). Mice were maintained under specific pathogen-free conditions in the Animal Care Facility at Cedars-Sinai Medical Center. The mice used in all experiments were handled according to the guidelines and approved protocols of the Cedars-Sinai Medical Center Animal Care and Use Committees (IACUC 2700).
The human acute monocytic leukemia cell line, THP-1, was obtained from the American Type Culture Collection and maintained in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10 U/ml penicillin G, 10 μg/ml streptomycin, 2 mM L-glutamine, and 10% FBS at 37° C. and 5% CO2 in a humidified incubator. LPS-tolerant THP-1 cells were made and are characterized in detail as described previously (35). Cells were rendered LPS tolerant by preincubation over 16 h with LPS (10 ng/ml LPS from Escherichia coli 0111:B4). Low passage number and log-phase cells were used for all experiments.
Resident peritoneal macrophages were obtained from C57BL/6 mice by peritoneal lavage. Cells were washed with PBS and seeded in 96-well plates. After 2 h cells were washed again and adherent cells were used for experiments. Cells were cultured in RPMI supplemented with 10% FBS and preincubated with LPS (10 ng/ml) overnight. On the next day, cells were washed with PBS and stimulated with 100 ng/ml LPS or medium for 5 h. Supernatants were harvested and stored at −80° C.
Human TNF-α, mouse TNF-α, and mouse IL-6 were quantified by ELISA (eBioscience) in supernatants or serum samples.
The activity of PP2A in cell lysates was determined as recommended by the manufacturer (Upstate). In brief, cells were incubated for the indicated time-points, placed on ice and washed with ice-cold PBS/orthovanadate (1 mM). Cells were lysed in phosphate extraction buffer (20 mM imidazole, 2 mM EDTA, 2 mM EGTA, pH 7.0 containing protease inhibitor cocktail) for 20 min. on ice. Cellular lysates were cleared by centrifugation (20 min, 4° C., 2000×g). Protein concentrations were determined by Bradford assay and equal amounts of protein were incubated with anti-PP2A and Protein A agarose beads for 2 h at 4° C. with constant rocking Phosphopeptide substrate was added (final concentration 750 μM) and incubated for 10 min. at 30° C. in a shaking incubator. Samples were centrifuged and added to a microtiter plate. 10 to 15 min. after the addition of Malachite Green Phosphate Detection Solution the plate was read at 650 nm in a microplate reader. The PP2A activity was calculated using a phosphate standard curve.
LPS tolerance was induced in mice as described previously (36). In brief, mice were i.p. injected with either 25 μg LPS or PBS. 20 h after the first injection, mice were i.p. injected with either 25 μg LPS or PBS. Serum was collected from mice 1.5, 6, and 24 h after the 2nd LPS injection. Peritoneal macrophages were collected 24 h after the 2nd LPS injection and assessed for PP2A activity.
Data shown are the mean±SD of three or more independent experiments. Student's 2-tailed t-test was used to compare groups. Unless otherwise noted, p<0.05 was considered significant.
Serine-threonine phosphatases play an important role in LPS-induced signal transduction. Treatment of macrophages with the PP1/PP2A inhibitor calyculin A inhibits LPS-induced macrophage responses (32).
The inventors hypothesized that serine-threonine phosphatases may have a role in LPS-induced macrophage tolerance. In order to test this hypothesis, the inventors used the THP-1 human macrophage cell line and treated with either 10 ng/ml LPS or media for various durations followed by a second stimulation with 100 ng/ml LPS for 5 h. PP2A activity was determined 5 h after 2nd LPS treatment. The inventors observed that repeated LPS stimulation of THP-1 cells led to significantly reduced PP2A activity compared to THP-1 cells that only received the 2nd LPS treatment (
In order to confirm the in vitro findings in an in vivo animal model the inventors first determined whether murine peritoneal macrophages could be tolerized under the same conditions. Peritoneal macrophages from C57BL/6 mice were isolated by peritoneal lavage and stimulated with 10 ng/ml LPS overnight. After the second LPS stimulation (100 ng/ml for 5 h) IL-6 and TNF-α release into the supernatants were determined by ELISA (
Next, the inventors tolerized C57BL/6 mice in vivo by repeated injections of LPS. LPS (25 μg/mouse) was injected i.p. followed by a 2nd injection of LPS (25 μg/mouse) 20 h later. Serum was collected at 1.5, 6, and 24 h later, and TNF-α and IL-6 levels were measured to confirm induction of tolerance (
Next, inventors determined the effect of in vivo LPS tolerization of C57BL/6 mouse on PP2A activity. Peritoneal macrophages of tolerized and non-tolerized mice were harvested 24 h after the 2nd injection of LPS and PP2A activity was determined. As shown in
The inventors also determined that exogenous recombinant PP2A is able to restore LPS responsiveness in THP-1 cells. THP-1 cells were tolerized to LPS by overnight stimulation with low-dose LPS (10 ng/ml). The next day, cells were stimulated with 100 ng/ml LPS for 5 h. TNF-α release as measured by ELISA confirmed that THP-1 cells lost LPS responsiveness after overnight exposure to LPS (
As disclosed herein, the inventors show that the serine/threonine protein phosphatase 2 A (PP2A) activity is suppressed in in vitro tolerized human macrophage cell line as well as in peritoneal macrophages isolated from in vivo LPS-tolerized mice.
In tolerized THP-1 cells, suppression of PP2A activity was time-dependent. The inventors observed that 24 and 48 h of pretreatment with LPS led to the greatest reduction in PP2A activity. Pretreatment with LPS for longer than 48 h (i.e. 72 h) resulted in only slightly reduced PP2A activity after the 2nd LPS stimulation. This transient down-regulation of PP2A activity correlates with previous observations on the duration of immune suppression in LPS tolerized macrophages. Zuckerman et al. have shown that LPS responsiveness was restored in macrophages after 48 h of LPS stimulation (8).
Phosphatases play a crucial role in the tight regulation of LPS-induced signaling events. The transient phosphorylation of signaling molecules that leads to the activation of transcription factors such as NF-κB guarantees protection against over-stimulation of the signaling pathways that induce inflammation. Phosphatases have been implemented to play an important role in LPS tolerance (37). Expression of the SH2-containing inositol phosphatase 1 (SHIP-1) for instance is required for the development of an LPS-refractory state in macrophages and mice (38) as macrophages from SHIP-deficient mice do not develop endotoxin tolerance. Serine/threonine phosphatases play an important role in LPS-induced signal transduction. Treatment of macrophages with the PP1/PP2A inhibitor calyculin A inhibits LPS-induced macrophage responses (32).
The serine/threonine phosphatase PP2A has been shown to inactivate PKC by dephosphorylation (39,40) and regulate the MAPK cascade (41). Both pathways have been implicated in endotoxin tolerance (12,42). Ropert et al. have shown that treatment with okadaic acid (OA), an inhibitor of serine/threonine phosphatases PP1 and PP2A 1 h before the first LPS stimulation or 16 h before the second LPS stimulation reversed LPS tolerance in murine peritoneal macrophages (43). OA treatment restored and prolonged the phosphorylation of the ERK-1/ERK-2, JNK/SAPK and p38/SAPK-2 in response to second LPS stimulation in macrophages (43). Interestingly OA treatment led to inhibition of p38 phosphorylation in response to primary LPS stimulation of macrophages (43).
Protein kinase R(PKR) is a serine-threonine kinase that is activated upon LPS stimulation of TLR4 (44). PKR phosphorylates the B56a regulatory subunit of PP2A to increase the activity of trimeric PP2A holoenzyme (45). Recently Vogel et al. have shown that PKR activity is inhibited in LPS-tolerant macrophages (29). These data support the inventors' observations that PP2A activity is suppressed in LPS-tolerized macrophages.
In conclusion the inventors' study demonstrates that in vivo, peritoneal macrophages isolated from tolerized mice had reduced PP2A activity supporting the inventors' in vitro findings of reduced PP2A activity in THP-1 cells.
Finally, repeated infections have been thought to be associated with development of cancers (46), while inhibition of PP2A activity has been suggested to play a role in cancer development (47).
The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features.
Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.
Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.
In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.
Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.
All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail.
In closing, it is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.
This application claims priority from U.S. Provisional Patent Application No. 61/436,859 filed on Jan. 27, 2011, which is incorporated herein by reference in its entirety.
The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant No. AI51216 awarded by the National Institutes of Health.
| Number | Date | Country | |
|---|---|---|---|
| 61436859 | Jan 2011 | US |
