RU2587853C2 - Modified immunomodulating particles - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions concerns methods of reducing duration or severity of the inflammatory immune response in an individual, as well as a pharmaceutical composition containing carboxylated particles. Whereupon attached biologically active substances or attached antigenic molecules in the above particles are absent. Particles are particles of polylactic-co-glycolic acid (PLGA), polystyrene particles or diamond particles. Diameter of said particles is between approximately 0.1 mcm to approximately 10 mcm. Individual can have an autoimmune disorder, such as multiple sclerosis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, systemic lupus erythematosus, Reynaud syndrome, Sjogren's syndrome, autoimmune uveitis, autoimmune myocarditis or Crohn's disease, has ischemic reperfusion injury, atherosclerosis, suffered from myocardial infarction, is recipient of transplant, has psoriasis or dermatitis, or suffers from allergic disorders, such as eczema, asthma, allergic rhinitis or skin hypersensitivity. Individual can have a viral or bacterial infection, which can induce development of encephalitis or meningitis.

EFFECT: group of inventions provides reduction or elimination of inflammatory, immune response in an individual without the need to attach any biologically active substances, antigens to particles.

12 cl, 13 dwg, 7 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of provisional applications for US patent No. 61/413016 and 61/413018, filed November 12, 2011 and which are incorporated in their entirety by reference.

BACKGROUND

[0002] Inflammatory diseases and disorders are conditions in which an abnormal or otherwise unregulated inflammatory response contributes to the etiology or severity of the disease. Examples include autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and diabetes, infectious diseases such as tuberculosis and various forms of meningitis and encephalitis, including West Nile viral encephalitis and other disorders, including atherosclerosis and ischemic reperfusion.

[0003] Many of these diseases are characterized by infiltration of mononuclear cells at the site of tissue damage or other injury. Examples of mononuclear cells that have been detected in these infiltrates include lymphocytes, especially T lymphocytes, and mononuclear phagocyte system cells (SMF cells), such as monocytes, macrophages, dendritic cells, microglial cells and others.

[0004] It is believed that many of the cells found in infiltrates of mononuclear cells play a role in these abnormal inflammatory responses. For example, in diseases such as multiple sclerosis, CD4 ⁺ T cells are known to play a central role in the pathological autoimmune response. Dendritic cells and other SMF cells may be responsible for the activation of CD4 ⁺ T cells at an early moment of T cell activation. SMF cells can also contribute to inflammation by phagocytosis, although at least in some inflammatory diseases it is unclear whether such cells are capable of this in the absence of CD4 ⁺ T cells.

[0005] Peripheral blood monocytes can be classified into two groups according to the expression or absence of certain cell surface molecules. In particular, human "resident monocytes" or "mature monocytes" are considered to have the CD14 ^lo CD16 ⁺ phenotype (the mouse analogue is CX ₃ CR1 ^hi CCR2 ^- Gr1 ^- ). Another group of cells - "inflammatory monocytes" or "immature monocytes" - are considered as having the phenotype CD14 ⁺ CD16 ^- (the mouse analogue is CX ₃ CR1 ^lo CCR2 ⁺ Gr1 ⁺ ). (Geissmann F. et al. 2003 Immunity 19: 71-82)

[0006] It is important to note that while the latter are understood to be “inflammatory” in the sense that they migrate into the inflamed tissue from peripheral blood cells formed from bone marrow, it has not been shown that these cells cause inflammation either directly or through the action of other cells. In addition, it was not shown that various SMF cells, which can be formed by differentiation of these cells, cause inflammation.

[0007] Traditional clinical strategies for nonspecific long-term immunosuppression in disorders associated with an undesirable immune response are based on the long-term use of broad-acting immunosuppressants, for example, signal blocking agents 1, such as cyclosporin A (CsA), FK506 (tacrolimus) and corticosteroids. Long-term use of high doses of these drugs can have toxic side effects. Moreover, even in those patients who are able to tolerate these drugs, the need for lifelong immunosuppressive drug therapy carries a significant risk of serious side effects, including tumors, severe infections, nephrotoxicity and metabolic disorders.

[0008] Methods have been developed to induce antigen-specific tolerance, including binding of an antigen or peptide to a cell. For example, in one of the methods, the peptide induced binding mediated cell tolerance, including collecting, separating and treating peripheral blood cells with disease specific antigens and the binding reagent Ethylene Carbodiimide (ECDI) under sterile conditions, followed by repeated administration to the donor / patient. This process is expensive and must be carried out under strict control of conditions by qualified specialists, and is limited by the number of centers that can carry out the procedure. The use of red blood cells as a type of donor cell expands the potential source, considering allogeneic donors, thereby significantly increasing the supply of source cells and potentially expanding the provision of this therapy with any equipment that is certified for blood transfusion. These methods have significant limitations associated with the supply of source cells and the need for matching tissue types to minimize the immune response to donor cells. In addition, local processing of EDCI cells to bind autoantigens is a significant challenge for quality control. In addition, these approaches also require at least some knowledge of the pathological antigen that requires immune tolerance.

[0009] Recently, peptide-bound particles have been described, which eliminates the need for a supply of source cells and circumvents the requirement for tissue typing of preliminary methods, see WO 2010/085509, incorporated herein by reference in its entirety. However, these approaches are still based on antigen-specific immune tolerance.

[0010] Generally, antigen-specific tolerance is not ideal, since, as a rule, specific antigens / epitopes of human diseases are not known. In addition, antigens can vary from subject to subject, so in order for an antigen-specific approach to be effective, it is necessary to determine which antigens each patient will recognize, or it will be necessary to link a library of possible peptides to particles before administration. The synthesis and individual binding of these peptides is both time consuming and expensive. Thus, there is a need for a treatment method that solves both of these problems, thereby eliminating the need for a suitable tissue source of cells and at the same time eliminating the need for synthesis and binding of large panels of peptides.

SUMMARY OF THE INVENTION

[0011] The present invention describes the surprising discovery of the fact that modified particles by themselves, that is, without the peptide associated with them, are effective in eliminating the inflammatory immune response in patients in need thereof. Surprisingly, all that is needed to attenuate the inflammatory immune response and also to treat an inflammatory disease is the administration of carboxylated particles without the need for peptide (s) to be attached to them.

[0012] In one embodiment, the present invention provides a pharmaceutical composition comprising carboxylated particles. In a further embodiment of the present invention, the carboxylated particles do not contain an attached peptide or antigenic moieties. In some embodiments of the present invention, the carboxylated particles are polystyrene particles. In other embodiments of the present invention, the carboxylated particles are diamond particles. In other embodiments, the carboxylated particles are poly (lactic-co-glycolic acid) (PLGA) particles.

[0013] In one embodiment of the present invention, a pharmaceutical composition comprising carboxylated particles induces immune tolerance when administered to a subject in need thereof. In a further embodiment of the present invention, a pharmaceutical composition comprising carboxylated particles eliminates the inflammatory immune response when administered to a subject in need thereof.

[0014] In one embodiment of the present invention, the carboxylated particles containing the pharmaceutical composition of the present invention have a diameter of from about 0.1 μm to about 10 μm. In a further embodiment of the present invention, the carboxylated particles have a diameter of from about 0.3 microns to about 5 microns. In yet a further embodiment of the present invention, the carboxylated particles have a diameter of from about 0.5 microns to about 3 microns. In yet a further embodiment of the present invention, the carboxylated particles have a diameter of about 0.5 microns.

[0015] In one embodiment, the present invention provides a method for reducing the duration or severity of an inflammatory immune response in a subject, comprising administering to the subject a pharmaceutical composition comprising carboxylated particles. In a further embodiment of the present invention, the carboxylated particles are free of bound peptides or antigenic fragments. In some embodiments of the present invention, the carboxylated particles are polystyrene particles. In other embodiments of the present invention, the carboxylated particles are diamond particles. In other embodiments, the carboxylated particles are poly (lactic-co-glycolic acid) (PLGA) particles.

[0016] In one embodiment of the present invention, the method of the present invention induces immune tolerance when administered to a subject in need thereof. In a further embodiment of the present invention, the method eliminates the inflammatory immune response when administered to a subject in need thereof.

[0017] In one embodiment of the present invention, the method according to the present invention uses carboxylated particles having a diameter of from about 0.1 μm to about 10 μm. In a further embodiment of the present invention, the carboxylated particles have a diameter of from about 0.3 microns to about 5 microns. In yet a further embodiment of the present invention, the carboxylated particles have a diameter of from about 0.5 microns to about 3 microns. In yet a further embodiment of the present invention, the carboxylated particles have a diameter of about 0.5 microns.

[0018] In one embodiment of the present invention, the subject has an autoimmune disorder. In a further embodiment of the present invention, the autoimmune disorder is multiple sclerosis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, systemic lupus erythematosus, Raynaud's syndrome, Sjogren's syndrome, autoimmune uveitis, autoimmune myocarditis or Crohn's disease. In a specific embodiment, an autoimmune disorder is multiple sclerosis.

[0019] In another embodiment of the present invention, the subject has an allergic disorder. In a further embodiment of the present invention, the allergic disorder is eczema, asthma, allergic rhinitis or skin hypersensitivity.

[0020] In another embodiment of the present invention, the subject is a transplant recipient. In yet another embodiment of the present invention, the subject suffered a myocardial infarction. In yet another embodiment of the present invention, the patient has ischemic reperfusion. In yet another embodiment of the present invention, the patient has atherosclerosis.

[0021] In one embodiment of the present invention, the method comprises administering carboxylated particles by any suitable means. In one embodiment of the present invention, the composition is administered orally, nasally, intravenously, intramuscularly, into the eyes, transdermally or subcutaneously. In a particular embodiment of the present invention, carboxylated particles are administered nasally. In yet another embodiment of the present invention, the particles are administered intravenously.

[0022] In one embodiment, the present invention provides a method for treating a bacterial or viral infection in a subject, comprising administering to the subject a pharmaceutical composition comprising carboxylated particles. In a further embodiment of the present invention, the carboxylated particles are free of bound peptide or antigenic fragments. In some embodiments of the present invention, the carboxylated particles are polystyrene particles. In other embodiments of the present invention, the carboxylated particles are diamond particles. In other embodiments, the carboxylated particles are poly (lactic-co-glycolic acid) (PLGA) particles.

[0023] In one embodiment of the present invention, the method of the present invention induces immune tolerance when administered to a subject with a bacterial or viral infection. In a further embodiment of the present invention, the method eliminates or attenuates the inflammatory immune response when administered to a patient with a bacterial or viral infection.

[0024] In one embodiment of the present invention, methods for treating a bacterial or viral infection according to the present invention use carboxylated particles having a diameter of from about 0.1 μm to about 10 μm. In a further embodiment of the present invention, the carboxylated particles have a diameter of from about 0.3 microns to about 5 microns. In yet a further embodiment of the present invention, the carboxylated particles have a diameter of from about 0.5 microns to about 3 microns. In yet a further embodiment of the present invention, the carboxylated particles have a diameter of about 0.5 microns.

[0025] In one embodiment of the present invention, the subject has a viral infection. In a further embodiment of the present invention, the viral infection is a herpes virus infection, hepatitis B infection, West Nile virus infection, flavivirus infection, influenza infection, rhinovirus infection, papillomavirus infection or parainfluenza virus infection. In a further embodiment of the present invention, a viral infection infects the central nervous system of said subject. In yet a further embodiment of the present invention, a viral infection causes viral encephalitis or viral meningitis.

[0026] In one embodiment of the present invention, the subject has a bacterial infection. In a further embodiment of the present invention, a bacterial infection affects the central nervous system of said subject. In yet a further embodiment of the present invention, a bacterial infection causes septic bacterial encephalitis or bacterial meningitis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 describes (A) the percentage of survival of mice after infection with a high dose or a low dose of WNV; (B) weight loss associated with infection of mice with a high dose of WNV; (C) virus titers in the brains of mice killed by infection; (D) weight loss in mice infected with a high and low dose of WNV 0-7 days after infection; (E) virus titers in the brain of mice infected with a high and low dose of WNV on the 7th day after infection and the correlation between the percentage of weight loss on day 7 and the titer of the virus, (F) the correlation between the percentage of weight loss and the presence of CD45 ⁺ leukocytes in the brain mice 7 days after infection with a high and low dose of WNV; (G) the correlation between the titer of the virus in the brain and the presence of CD45 ⁺ leukocytes in the brain of mice 7 days after infection with a high and low dose of WNV; (H) correlation between the percentage of weight loss and the presence of CD45 ^hi macrophages in the brain of mice 7 days after infection with a high and low dose of WNV.

[0028] Figure 2 describes the correlation between the percentage of mass loss and the presence of (A) CD45 ^int CD11b ⁺ immigrated microglia; (C) CD3 ⁺ T cells, (D) CD11b ^hi Ly6G ⁺ neutrophils and (E) NK1.1 ⁺ CD11b ^{lo / -} natural killer cells, while the number of CD45 ^lo resident microglia (B) remained unchanged after 7 days after infection with a high or low dose of WNV; (F) describes the correlation between weight loss and virus titer in the brain at the time of killing of mice infected with a low dose of WNV; (G) describes the correlation of leukocyte infiltration and percent weight loss in mice infected with a low dose of WNV and (H) shows the absence of a correlation between the titer of the virus and leukocyte infiltration after infection with a low dose of WNV.

[0029] Figure 3 (A) describes the long-term survival of mice treated with carboxylated polystyrene granules in PBS on day 6 after infection with a high dose of WNV; (B) demonstrates that treating mice infected with low doses of WNV carboxylated with polystyrene granules starting 6 days after infection is ineffective in prolonging the survival of mice; (C) demonstrates that treatment of mice infected with low doses of WNV carboxylated with polystyrene granules starting from day 6 after infection is ineffective in preventing weight loss in mice compared to mice of the control group, (D) demonstrates that treatment of mice infected low WNV, is effective in prolonging the survival of mice when granules are introduced during weight loss in mice. (E-G) describes the weight loss recorded for these mice up to 20 days after infection.

[0030] Figure 4 (AD) is an example of the treatment of mice infected with low doses of WNV, carboxylated polystyrene granules with mouse weight loss, the mice in (AB) require granule treatment only for 5 days, and the mass remains stable and they survive without further granule treatment, while mice in (CD) begin to lose weight again after discontinuation of the granule treatment after 5 days, so treatment continues until the mass stabilizes, (E) describes the infiltration of CD45 ⁺ CD11b ⁺ macrophages into the brain of mice infected with neither vigorous doses of WNV, 9 days after infection, which either lost weight or did not lose weight and received either PBS or carboxylated granules on day 8 after infection; (F) is a graphical demonstration of the types of cells that infiltrate the brain of mice infected with WNV, 9 days after infection, which either lost weight or did not lose weight and received either PBS or carboxylated granules on day 8 after infection.

[0031] Figure 5 describes (A) the difference in survival between mice treated with carboxylated polystyrene granules, bare polystyrene granules or PBS after infection with a low dose of WNV; (B) describes the difference in percent weight loss in mice treated with carboxylated polystyrene granules, bare polystyrene granules or PBS after infection with a low dose of WNV; (C, D) describes the difference in the percentage of weight loss between the treatment of mice with carboxylated granules and bare granules after infection with a low dose of WNV; (E, O) describes the localization of FITC-conjugated carboxylated granules or naked granules on day 7 in mice infected with a high dose of WNV on day 0 and FITC-carboxylated granules, FITC-conjugated naked granules or PBS on day 6. (EG) represent the blood of 3 separate mice treated with PBS, (HJ) represent the blood of 3 separate mice treated with polystyrene beads, and (LN) represent the blood of 3 separate mice treated with carboxylated polystyrene beads, demonstrating that remains in the blood more simple granules than carboxylated granules.

[0032] Figure 6 shows (A-C) the absence of infiltration of FITC-conjugated polystyrene granules in the brain of infected and treated mice, as shown in Figure E-O; (D-E) describes a reduction in the infiltration of various leukocytes, macrophages and microglia in the brain of WNV-infected mice treated with carboxylated polystyrene granules or bare polystyrene granules, as shown in Figure 5 (E-O).

[0033] Figure 7 describes (A) the association of FITC-conjugated polystyrene carboxylated granules and FITC-conjugated naked polystyrene granules in the spleen with CD45 ⁺ leukocytes (A, B, F) in CD11 ^{b +} (C, G), CD11c ⁺ (D, H) Ly6 c ⁺ (E, I) cells; (JR) describes cell types that capture FITC-conjugated carboxylated granules and FITC-conjugated naked granules.

[0034] Figure 8 describes the ability to capture FITC-conjugated polystyrene carboxylated granules or FITC-conjugated bare polystyrene granules and an increase in the number of CD11b ⁺ CD11c ^- monocytes (A) and CD11b ⁺ CD11c ⁺ (B) or CD11b ^- CD11c ⁺ (C) dendritic cells in the spleen after infection with a high dose of WNV.

[0035] Figure 9 describes (AD) the ability to capture FITC-conjugated polystyrene carboxylated granules or FITC-conjugated naked polystyrene granules and the increase in the number of subpopulations of CD19 ⁺ B cells and CD3 ⁺ T cells in the spleen after infection with a high dose of WNV.

[0036] Figure 10 describes (AL) the ability to capture FITC-conjugated polystyrene carboxylated granules or FITC-conjugated bare polystyrene granules CD11b ⁺ (C, G), CD11c ⁺ (D, H), and Ly6 c ⁺ (E, I) cells, in particular, CD11b ⁺ CD11c ^- monocytes (J) and CD11b ⁺ CD11c ⁺ (K) or CD11b ^- CD11c ⁺ (L) dendritic cells in the liver after infection with a high dose of WNV.

[0037] Figure 11 describes (AG) the ability to capture FITC-conjugated polystyrene carboxylated granules or FITC-conjugated naked polystyrene granules CD11b ⁺ , (C, F), CD11c ⁺ and Ly6C ⁺ (D, G) cells in the bone marrow after infection high dose of WNV.

[0038] Figure 12 describes (A) the survival rate of mice infected with a low dose of WNV receiving a high or low dose of carboxylated polystyrene granules of various sizes; (B) describes the survival rate of mice infected with a low dose of WNV receiving FITC-conjugated carboxylated granules, naked FITC-conjugated granules, carboxylated PLGA spheres or naked PLGA spheres; (C) describes the infiltration / activation of various populations of monocytes in the brain of mice infected with a low dose of WNV and receiving carboxylated FITC granules, carboxylated FITC PLGA spheres or carboxylated nanodiamonds.

[0039] Figure 13 describes (A) percent survival and (B) weight loss in wild-type mice and T-cell deficient mice infected with a high or low dose of WNV; (C) the correlation between weight loss and virus titers in the brain of wild-type mice and T-cell deficient mice infected with a high or low dose of WNV; (D) weight loss (E) and immune cell infiltration of the brain of wild-type mice and mice with T-cell deficiency infected with a high dose of WNV on day 8 after infection; (F) percent survival (G) and weight loss of wild-type and T-cell deficient mice infected with high or low dose WNV and treated with carboxylated granules or PBS with significant weight loss.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present inventors have unexpectedly found that when carboxylated particles, such as carboxylated polystyrene, PLGA or diamond particles of a certain size are administered to subjects, inflammatory immune responses are eliminated. Additionally, the authors of the present invention also unexpectedly found that the same carboxylated particles when administered to patients with active viral or bacterial infections, especially infections that permanently affect the central nervous system, lead to a significant reduction in the symptoms of these infections and increased survival. Therefore, these particles can be useful in the treatment of any disease or condition characterized by an excessive inflammatory immune response, for example, autoimmune diseases, as well as in the treatment of bacterial and viral infections.

[0041] "Particle" in this context refers to any non-tissue minute composition of a substance, it can be a sphere, sphere-like or granule. The term “particle” and the term “granule” can be used interchangeably. Additionally, the term “particle” can be used to encompass granules and spheres.

[0042] "Carboxylated particles" or "carboxylated granules" or "carboxylated spheres" includes any particle that has been modified and contains a carboxyl group on its surface. In some embodiments, the addition of a carboxyl group enhances phagocytic / monocytic uptake of particles from the bloodstream, for example, by interaction with phagocytic receptors, for example, MARCO.

[0043] "Antigenic fragment" as used herein refers to any fragment, such as a peptide, that is recognized by the host immune system. Examples of antigenic fragments include, but are not limited to, autoantigens and / or bacterial or viral proteins, peptides or components. Without being bound by any theory, while carboxylated granules themselves can be recognized by the immune system, carboxylated granules without any attached particle are not considered an “antigenic fragment” for the purposes of the present invention.

[0044] “Naked granules”, or “naked particles” or “naked spheres” as used herein, refer to granules, particles or spheres that have not been carboxylated.

[0045] The particle may have any particle shape or conformation. However, in some embodiments of the present invention, it is preferable to use particles that are less likely to agglutinate in vivo. Examples of particles in these embodiments of the present invention are those that are spherical in shape.

[0046] It is not necessary that each particle is uniform in size, although, in general, the particles must be of sufficient size to trigger phagocytosis by an antigen-presenting cell or other SMF cell. Preferably, the particles are microscopic or nanoscopic in order to increase solubility, in order to avoid possible complications caused by in vivo aggregation and to alleviate pinocytosis. Particle size may be a capture factor from the interstitial space in the area of maturation of lymphocytes. A particle having a diameter of from about 0.1 μm to about 10 μm is capable of triggering phagocytosis. Thus, in one embodiment of the present invention, the particle has a diameter within these limits. In another embodiment of the present invention, the particle has a diameter of from about 0.3 microns to about 5 microns. In yet another embodiment of the present invention, the particle has a diameter of from about 0.5 microns to about 3 microns. In a preferred embodiment of the present invention, the particle has a size of about 0.5 microns. Particles in the composition should not be uniform in diameter. For example, a pharmaceutical composition may contain many particles, some of which have a diameter of about 0.5 microns, while others have a diameter of about 1.0 microns. Any mixture of particle sizes in these given ranges will be helpful.

[0047] In some embodiments of the present invention, the particle is non-metallic. In these embodiments of the present invention, the particle may be made of a polymer. In a preferred embodiment of the present invention, the particle is biodegradable in an individual. In this embodiment of the present invention, the particles can be administered to the individual in multiple doses without the accumulation of particles in the individual. Examples of suitable particles include polystyrene particles, PLGA particles and diamond particles.

[0048] Preferably, the surface of the particle consists of a material that minimizes non-specific or undesirable biological interactions. Interactions between the particle surface and interstitium can be a factor that plays an important role in lymphatic uptake. The surface of the particle may be coated with material to prevent or reduce non-specific interactions. Steric stabilization by coating the particles with hydrophilic layers such as polyethylene glycol (PEG) and its copolymers such as PLURONICS (including poly (ethylene glycol) -bl-poly (propylene glycol) -bl-poly (ethylene glycol) copolymers) can reduce non-specific interactions with proteins interstitium, as demonstrated by improved lymphatic uptake after subcutaneous administration. All these facts indicate the importance of the physical properties of particles for lymphatic uptake. Biodegradable polymers can be used to make all or some of the polymers and / or particles and / or layers. Biodegradable polymers can undergo degradation, for example, as a result of the reaction of functional groups with water in solution. The term "degradation" in this context refers to the acquisition of solubility properties, either by reducing the molecular weight or by converting hydrophobic groups to hydrophilic groups. Polymers with ester groups are generally susceptible to spontaneous hydrolysis, such as polylactides and polyglycolides.

[0049] Particles according to the present invention may also contain additional components. For example, carriers may have imaging agents incorporated or conjugated to the carrier. An example of a carrier nanosphere that has a visualizing agent and which is currently commercially available are Kodak X-sight nanospheres. It was found that inorganic quantum-sized luminescent nanocrystals, known as quantum dots (QDs), are ideal donors in FRET methods: their high quantum yield and modifiable size-dependent Stokes shifts allow different sizes to emit radiation from blue to infrared when excited when single wavelength of the ultraviolet spectrum (Bruchez, et al., Science, 1998, 281, 2013; Niemeyer, C. M Angew. Chem. Int. Ed. 2003, 42, 5796; Waggoner, A. Methods Enzymol. 1995, 246, 362; Brus, LEJ Chem. Phys. 1993, 79, 5566). Quantum dots, for example hybrid organic / inorganic quantum dots based on a class of polymers known as dendrimers, can be used in biological labeling, image processing, and optical biosensor systems (Lemon, et al., J. Am. Chem. Soc. 2000, 122, 12886). Unlike traditional synthesis of inorganic quantum dots, the synthesis of these hybrid quantum dot nanoparticles does not require high temperatures or high toxicity, unstable reagents (Etienne, et al., Appl. Phys. Lett. 87, 181913, 2005).

[0050] Particles can be made from a wide variety of materials. Preferably, the particle consists of a material suitable for biological use. For example, the particles may be composed of glass, silica, polyesters of hydroxycarboxylic acids, polyanhydrides of dicarboxylic acids or copolymers of hydroxycarboxylic acids and dicarboxylic acids. In general, the carrier particles may consist of straight or branched chain polyesters, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl or alkoxyhydroxy straight or branched chain polyanhydrides, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl or alkoxycarboxylic lot. Additionally, the carrier particles can be quantum dots or consist of quantum dots, such as polystyrene particles of quantum dots (Joumaa et al. (2006) Langmuir 22: 1810-6). Carrier particles, including mixtures of ester and anhydride bonds (e.g., glycolic and sebacic acid copolymers) can also be used. For example, carrier particles may contain materials including polyglycolic acid (PGA) polymers, polylactic acid (PLA) polymers, polysebacic acid polymers (PSA), polylactic-co-glycolic acid copolymers (PLGA), lactic-co-sebacic acid copolymers ( PLSA), polyglycolic-co-sebacic acid copolymers (PGSA), etc. Other biocompatible, biodegradable polymers used in the present invention include polymers or copolymers of caprolactones, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates and decomposable urethanes, as well as their straight or branched chain copolymers, substituted or unsubstituted, alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl or aromatic hydroxy or dicarboxylic acids. Additionally, biologically important amino acids with reactive side chain groups such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine and cysteine or their enantiomers can be included in copolymers with any of the above materials to provide reaction groups for conjugation with antigenic peptides and proteins or conjugating fragments. Biodegradable materials suitable for the present invention include diamond, PLA, PGA and PLGA polymers. Biocompatible, but not biodegradable, materials can also be used in carrier particles in accordance with the present invention. For example, there may be used non-biodegradable polymers of acrylates, ethylene vinyl acetate, acyl substituted cellulose acetates, non-degradable urethanes, styrenes, vinyl chloride, vinyl fluoride, vinylimidazoles, chlorosulfonated olefin, ethylene oxide, vinyl alcohol, TEFLON ® (DuPont, Wilmington, DE), and nylon.

[0051] Suitable granules that are currently commercially available include polystyrene granules such as FluoSpheres (Molecular Probes, Eugene, Oregon).

[0052] Physical properties are also related to the usefulness of nanoparticles after being captured and held in locations with immature lymphocytes. These include mechanical properties such as stiffness or elasticity. Some embodiments of the present invention are based on an elastic core, for example a polypropylene sulfide (PPS) core with a top layer, for example a hydrophilic top layer, such as in a PEG, such as in a PPS-PEG system, recently developed and described for a systemic (but not directed or immune) delivery. An elastic core is opposed to a substantially rigid core as in a system of polystyrene or metal nanoparticles. The term "elastic" refers to some elastic materials, in addition to natural or synthetic rubbers, "elastic" is a term known in the field of polymers. For example, crosslinked PPS can be used to make a hydrophobic elastic core. PPS is a polymer that decomposes under oxidizing conditions to polysulfoxide and finally polysulfone, passing from hydrophobic rubber to a hydrophilic water-soluble polymer. Other sulfide polymers can be adapted for use with the term “sulfide polymer” referring to a polymer with sulfur in the base of the monomer unit. Other elastic polymers that can be used are polyesters with a glass transition temperature under hydrated conditions that is less than about 37 ° C. The hydrophobic core can preferably be used with a hydrophilic top layer, since the core and top layer will not mix, as a result of which the top layer will tend to sterically expand from the core. The core refers to a particle having a top layer. A layer refers to a material covering at least a portion of a core. The layer may be adsorbed or covalently linked. The particle or core may be solid or hollow. Elastic hydrophobic nuclei have advantages over hard hydrophobic nuclei, such as crystalline or glassy (as in the case of polystyrene) nuclei, in which large doses of hydrophobic drugs can be transferred by particles with elastic hydrophobic nuclei.

[0053] Another physical property is surface hydrophilicity. A hydrophilic material may have a solubility in water of at least 1 gram per liter when it is not cross-linked. Steric stabilization of particles by hydrophilic polymers can improve capture from interstitium by reducing non-specific interactions; however, the increased nature of the stealth of particles can also reduce the internalization of phagocytic cells in areas with immature lymphocytes. Balancing these competing characteristics has proven problematic, however, as documented in this application, nanoparticles have been created for efficient lymphatic delivery to DCs and other AICs in the lymph nodes. Some embodiments of the present invention include a hydrophilic component, for example a layer of hydrophilic material. Examples of suitable hydrophilic materials are one or more of polyalkylene oxides, polyethylene oxides, polysaccharides, polyacrylic acids and polyethers. The molecular weight of the polymers in the layer can be adjusted to provide the desired degree of in vivo steric mismatch, for example, from about 1000 to about 100000 or even more; it will immediately be apparent to those skilled in the art that all ranges and values are considered within the ranges indicated in a clear manner, for example, between 10,000 and 50,000.

[0054] Nanoparticles may include functional groups for further reaction. Functional groups for further reaction include electrophiles or nucleophiles; they are suitable for interaction with other molecules. Examples of nucleophiles are primary amines, thiols and hydroxyls. Examples of electrophiles are succinimidyl esters, aldehydes, isocyanates and maleimides.

[0055] The particles of the present invention can be administered at any dose effective to reduce the inflammatory immune response in a subject in need thereof, or to treat a bacterial or viral infection in a subject in need thereof. In some embodiments of the present invention, from about 10 ² to about 10 ²⁰ particles are administered to the individual. In yet another embodiment of the present invention, between about 10 ³ to about 10 ¹⁵ particles are added. In yet another embodiment of the present invention, about 10 ⁶ to about 10 ¹² particles are administered. In yet another embodiment, between about 10 ⁸ to about 10 ¹⁰ particles are added. In one embodiment of the present invention, the preferred dose is 0.1% solids / ml. Thus, for granules of 0.5 μm, the preferred dose is about 4x10 ⁹ granules, for granules of 0.05 μm, the preferred dose is about 4x10 ¹² granules, for granules of 3 μm, the preferred dose is 2x10 ⁷ granules. However, any effective dose in the treatment of a particular condition requiring treatment is encompassed by the present invention.

[0056] The present invention is useful for the treatment of immune disorders, such as autoimmune disease, transplant rejection and allergic reactions. Replacing a synthetic biocompatible particle system to induce immune tolerance can lead to simplified production, widespread availability of therapeutic agents, increased homogeneity between samples, an increase in the number of potential treatment sites, and a significant reduction in the possibility of allergic responses to the host cell.

[0057] As used herein, the term "immune response" includes T cell and / or B cell mediated immune responses. Examples of immune responses include T-cell responses, such as cytokine production and cellular cytotoxicity. In addition, the term immune response includes immune responses that are indirectly mediated by T-cell activation, for example, antibody production (humoral response) and activation of cytokine-sensitive cells, for example, macrophages. Immune cells involved in the immune response include lymphocytes such as B cells and T cells (CD4 ⁺ , CD8 ⁺ , Th1 and Th2 cells); antigen-presenting cells (for example, professional antigen-presenting cells, such as dendritic cells, macrophages, B-lymphocytes, Langerhans cells and non-professional antigen-presenting cells, such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells, myeloid cells such as macrophages, eosinophils, mast cells, basophils and granulocytes. In some embodiments of the present invention, the modified particles of the present invention are effective in reducing the migration of inflammatory cells to the site of inflammation.

[0058] In this context, the terms "anergy", "tolerance" or "antigen-specific tolerance" refer to the insensitivity of T cells to stimulation through a T cell receptor. Such insensitivity, as a rule, is antigen-specific and persists after termination of exposure to the antigenic peptide. For example, T cell anergy is characterized by a lack of cytokine production, such as IL-2. T cell energy occurs when antigens are exposed to T cells and they receive the first signal (T cell receptor or CD-3 mediated signal) in the absence of a second signal (co-stimulating signal). Under these conditions, repeated exposure to cells of the same antigen (even if repeated exposure occurs in the presence of a co-stimulating molecule) leads to an inability to produce cytokines and subsequently inability to proliferate. Thus, the inability to produce cytokines inhibits proliferation. Vigorous T cells, however, proliferate upon culturing with cytokines (e.g., IL-2). For example, T-cell anergy can also be observed in cases of insufficient production of IL-2 by T-lymphocytes, determined by ELISA or proliferation analysis, using an indicator cell line. Alternatively, a reporter gene construct may be used. For example, anergic T cells cannot initiate transcription of a DL-2 gene induced by a heterologous promoter under the control of an enhancer of the 5'-IL-2 gene or a multimer API sequence that can be detected in an enhancer (Kang et al. 1992 Science. 257: 1134) .

[0059] In this context, the term "immunological tolerance" refers to methods implemented on the part of subjects who received treatment, compared with subjects who did not receive treatment, where: a) the level of specific immunological response is reduced (considered to be mediated, at least partially antigen-specific effector T-lymphocytes, B-lymphocytes, antibody or their equivalents), b) delay the onset or progression of a specific immune response, or c) the risk of a onset or development of a specific immune response is reduced. “Specific” immunological tolerance occurs when immunological tolerance is predominantly directed against certain antigens in comparison with others. “Nonspecific” immunological tolerance occurs when immunological tolerance is indiscriminately directed against antigens leading to an inflammatory immune response. A “quasi-specific” immunological tolerance occurs when immunological tolerance is semi-selectively directed against antigens leading to a pathogenic immune response, but not against others leading to a protective immune response.

[0060] The transfer of tolerogenic activity is the ability of a particle to stimulate the production of the corresponding cytokine in the desired location. The immunoregulatory cytokine produced by T-suppressor cells at the target site is believed to be TGF-β (Miller et al., Proc. Natl. Acad. Sci. USA 89: 421, 1992). Other factors that may be produced in tolerance are the cytokines IL-4 and IL-10 and the mediator PGE. Conversely, lymphocytes in tissues undergoing active immune destruction secrete cytokines such as IL-1, IL-2, IL-6 and IFNγ. Therefore, the effectiveness of a modified particle can be assessed by measuring its ability to stimulate an appropriate type of cytokine.

[0061] With this in mind, a quick screening test of modified particles, effective mucosal binding components, effective combinations, or effective mucosal administration regimens and schedules using animal model systems can be performed. The animals are tested on the surface of the mucous membrane with a composition of particles, and after a while they are injected with a pathogenic antigen or an infectious agent. Spleen cells are isolated and cultured in vitro in the presence of a pathogenic antigen or antigen obtained from an infectious agent at a concentration of about 50 μg / ml. The secretion of cytokines into the medium can be quantified by standard immunoassay.

[0062] The ability of particles to suppress cell activity can be determined using cells isolated from animals immunized with modified particles, or by creating a cell line that responds to a pathogenic antigen or viral antigen to a target (Ben-Nun et al., Eur. J. Immunol. 11: 195, 1981). In one variant of this experiment, the suppressor cell population is weakly irradiated (from about 1000 to 1250 rad) to prevent proliferation, the suppressors are cultured together with immunoreactive cells, and then tritium-labeled thymidine (or MTT) is used to quantify the proliferative activity of immunoreactive cells. In another embodiment, the suppressor cell population and the immunoreactive cell population are cultured at the upper and lower levels of the Transwell bicameral culture system (Costar, Cambridge Mass.), Which allows for joint incubation of populations within 1 mm of each other, separated by a polycarbonate membrane (WO 93/16724) . In this method, irradiation of a population of suppressor cells is not necessary, since the proliferative activity of immunoreactive cells can be measured separately.

[0063] The effectiveness of the compositions and methods of administration for the treatment of a particular disease can also be achieved on the corresponding animal model of the disease. The ability of treatment to weaken or delay the symptoms of the disease is controlled at the level of circulating biochemical and immunological signs of the disease, immunohistological examination of the affected tissue and total clinical signs depending on the model used. Non-limiting examples of animal models that may be used for testing are included in the following section.

[0064] The invention contemplates modeling tolerance by modeling a TH1 response, TH2 response, TH17 response, or a combination of these responses. Modeling the TH1 response encompasses a change in expression of, for example, interferon gamma. The TH2 response modeling encompasses a change in expression, for example, of any combination of IL-4, IL-5, IL-10, and IL-13. Typically, an increase (decrease) in the TH2 response will include an increase (decrease) in the expression of at least one of IL-4, IL-5, IL-10, or IL-13; more typically, an increase (decrease) in the TH2 response will include an increase in the expression of at least two of IL-4, IL-5, IL-10, or IL-13; most typically, an increase (decrease) in the TH2 response will include an increase in at least three IL-4, IL-5, IL-10 or IL-13, and ideally an increase (decrease) in the TH2 response will include an increase ( decrease) in the expression of all IL-4, IL-5, IL-10 and IL-13. TH17 modeling involves altering the expression of, for example, TGF-beta, IL-6, IL-21, and IL-23 and affecting the levels of IL-17, IL-21, and IL-22.

[0065] Tolerance to autoantigens and autoimmune disease is achieved through a variety of mechanisms, including negative selection of self-reactive T cells in the thymus and peripheral tolerance mechanisms for those auto-reactive T cells that avoid thymic deletion and can be detected at the periphery. Examples of mechanisms that provide peripheral T-cell tolerance include “ignoring” their own antigens, anergy or lack of response to autoantigen, cytokine immune deviation, and activation-induced death of self-reacting T cells. In addition, it has been shown that regulatory T cells are involved in ensuring peripheral tolerance. See, for example, Walker et al. (2002) Nat. Rev. Immunol. 2: 11-19; Shevach et al. (2001) Immunol. Rev. 182: 58-67. In some situations, peripheral autoantigen tolerance is lost (or impaired) and an autoimmune response follows. For example, in an animal model of EAE, it has been shown that activation of antigen-presenting cells (ARCs) through TLRs by innate immune receptors impairs auto-tolerance and leads to EAE induction (Waldner et al. (2004) J. Clin. Invest. 113: 990-997).

[0066] In this regard, in some embodiments, the present invention describes methods for enhancing antigen presentation while suppressing or decreasing TLR7 / 8, TLR9 and / or TLR 7/8/9 dependent cell stimulation. As described herein, the administration of specific modified particles results in the presentation of a DC or APC antigen while simultaneously suppressing TLR 7/8, TLR9 and / or \ TLR7 / 8/9-dependent cellular responses associated with immunostimulatory polynucleotides. Such suppression may include lowering the levels of one or more TLR-associated cytokines.

[0067] As described above, the present invention provides new compounds that have biological properties useful in the treatment of disorders mediated by Mac-1 and LFA-1.

[0068] In this regard, in another aspect of the present invention, pharmaceutical compositions are provided comprising carboxylated particles and optionally containing a pharmaceutically acceptable carrier. In some embodiments, embodiments of the present invention, these compositions optionally contain one or more additional therapeutic agents. Alternatively, the modified particles of the present invention may be administered to a patient in need thereof in combination with the administration of one or more other therapeutic agents. For example, additional therapeutic agents for co-administration or incorporation into a pharmaceutical composition with a compound of the present invention may be an approved anti-inflammatory agent or may be any of the agents approved by the Food and Drug Administration that will ultimately be approved for the treatment of any disorder characterized by an uncontrolled inflammatory immune response or a bacterial or viral infection. You should also take into account that some of the modified particles according to the present invention may exist in free form for treatment or, if necessary, as their pharmaceutically acceptable derivative.

[0069] The pharmaceutical compositions of the present invention further comprise a pharmaceutically acceptable carrier, which, in this context, includes any and all solvents, diluents or other liquid carrier, dispersing or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives , solid binders, lubricants, and the like, suitable for the particular desired dosage form. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) describes various carriers used in the development of pharmaceutical compositions and known methods for their preparation. Unless the standard carrier medium is incompatible with the compounds of the present invention, for example, creating any undesirable biological effect or interacting in any other detrimental way with any other component (s) of the pharmaceutical composition, its use is contemplated within the scope of the present invention. Some examples of substances that can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut butter, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethyl alcohol and phosphate buffered saline, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavoring agents, flavoring agents, preservatives and antioxidants that may also be present in the composition in accordance with the decision of the recipe developer.

[0070] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, liquid dosage forms may contain inert diluents customary in the art, such as, for example, water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular cottonseed, peanut, corn, germ, olive, castor and sesame oils), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and flavoring agents.

[0071] Injectable forms, for example sterile injectable aqueous or oily suspensions, may be formulated according to the prior art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable carriers and solvents that can be used are water, Ringer's solution (according to the US Pharmacopoeia) and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose any bland fixed oil may be used, including synthetic mono- or diglycerides. In addition to this, fatty acids such as oleic acid are used to produce injectable forms.

[0072] Injectable dosage forms can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents into the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0073] To prolong the effect of the drug, it will often be desirable to slow the absorption of the drug by subcutaneous or intramuscular administration. This can be achieved by using a liquid suspension or crystalline or amorphous material with poor solubility in water. Then the absorption rate of the drug depends on its dissolution rate, which, in turn, may depend on the size of the crystals and crystalline form. Alternatively, delayed absorption of a parenterally administered dosage form is achieved by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are obtained by forming microcapsule drug matrices in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, it is possible to control the rate of release of the drug. Examples of other biodegradable polymers include polyorthoesters and polyanhydrides. Injectable drug depot forms are also prepared by incorporating the drug into liposomes or microemulsions that are compatible with body tissues.

[0074] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles are mixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and / or a) fillers or diluents, such as starches, lactose, sucrose, glucose, mannitol and silicic acid, b) binders, such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic, c) humectants, such as glycerin, d) disintegrating in substances such as agar-agar, calcium carbonate, potato starch or cassava starch, alginic acid, certain silicates and sodium carbonate, e) dissolution retarding agents, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite clay, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, lauryl sodium sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents.

[0075] Solid dosage forms of a similar type can also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and coatings, such as enteric coatings and other coatings well known in the pharmaceutical art. They may optionally contain opacifiers and may also be a composition releasing the active ingredient (s) only or preferably in a certain part of the intestinal tract, optionally in a delayed manner. Examples of immersion compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

[0076] The modified particles can also be in microencapsulated form with one or more excipients, as already noted. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and coatings, such as enteric coatings, release control coatings, and other coatings well known in the pharmaceutical art. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose and starch. Such dosage forms may also contain, as in normal practice, additional substances other than inert diluents, for example, tabletted lubricants and other tabletting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents. They may optionally contain radiopaque substances and may also be a composition releasing the modified particles only or preferably in a certain part of the intestinal tract, optionally in a delayed manner. Examples of immersion compositions that can be used include polymeric substances and waxes.

[0077] The present invention encompasses pharmaceutically acceptable dosage forms for external use of the modified particles of the present invention. The term "pharmaceutically acceptable dosage form for external use" in this context means any dosage form that is pharmaceutically acceptable for intradermal administration of the modified microparticles of the present invention by applying the dosage form to the epidermis. In some embodiments of the present invention, a pharmaceutically acceptable dosage form for external use comprises a carrier system. Pharmaceutically effective carriers include, but are not limited to, solvents (e.g., alcohols, polyalcohols, water), creams, lotions, ointments, oils, plasters, liposomes, powders, emulsions, microemulsions and buffered solutions (e.g., hypotonic or buffered saline) or any another carrier known in the field of topical medications. A more complete list of carriers known in the art is provided in reference texts that are standard in the art, for example, Remington's Pharmaceutical Sciences, 16th Edition, 1980 and 17th Edition, 1985, both published by Mack Publishing Company, Easton, Pa., The contents of which incorporated herein by reference in its entirety. In some other embodiments, the topical dosage forms of the present invention may contain excipients. Any pharmaceutically acceptable excipient known in the art can be used to prepare pharmaceutically acceptable external dosage forms according to the present invention. Examples of excipients that may be included in the external dosage forms of the present invention include, but are not limited to, preservatives, antioxidants, humectants, emollients, buffers, solubilizers, other penetrating agents, skin protecting agents, surfactants and propellants and / or additional therapeutic agents used in combination with modified particles. Suitable preservatives include, but are not limited to, alcohols, quaternary amines, organic acids, parabens, and phenols. Suitable antioxidants include, but are not limited to, ascorbic acid and its esters, sodium bisulfite, butylated hydroxytoluene, butylated hydroxyanisole, tocopherols and chelating agents such as EDTA and citric acid. Suitable humectants include, but are not limited to, glycerin, sorbitol, polyethylene glycols, urea, and propylene glycol. Suitable buffering agents for use in the present invention include, but are not limited to, citric acid, hydrochloric acid, and lactic acid buffers. Suitable solubilizers include, but are not limited to, quaternary ammonium chlorides, cyclodextrins, benzyl benzoate, lecithin, polysorbates. Suitable skin protecting agents that can be used in the external dosage forms of the present invention include, but are not limited to, an oil solution of vitamin E, allatoin, dimethicone, glycerin, petrolatum, and zinc oxide.

[0078] In some embodiments, the pharmaceutically acceptable topical dosage forms of the present invention at least comprise modified particles of the present invention and a penetration enhancing agent. The choice of a dosage form for external use will depend on several factors, including the condition to be treated, the physicochemical characteristics of the compound of the present invention and other excipients present, their stability in the dosage form, available manufacturing equipment, and cost constraints. In this context, the term "penetration enhancing agent" means an agent capable of transporting a pharmacologically active compound through the stratum corneum and into the epidermis and dermis, preferably with little or no systemic absorption. A wide range of compounds has been evaluated for their effectiveness in increasing the rate of penetration of drugs through the skin. See, for example, Percutaneous Penetration Enhancers, Maibach H. I. and Smith H. E. (eds.), CRC Press, Inc., Boca Raton, Fla. (1995), which reviews the use and testing of various skin penetration enhancers, and Buyuktimkin et al., Chemical Means of Transdermal Drug Permeation Enhancement in Transdermal and Topical Drug Delivery Systems, Gosh TK, Pfister WR, Yum SI (Eds.) , Interpharm Press Inc., Buffalo Grove, Ill. (1997). In certain exemplary embodiments of the present invention, penetration enhancing agents for use in the present invention include, but are not limited to, triglycerides (e.g., soybean oil), aloe compositions (e.g., aloe vera gel), ethyl alcohol, isopropyl alcohol, octoliphenylpolyethylene glycol, oleic acid, polyethylene glycol 400, propylene glycol, N-decylmethyl sulfoxide, fatty acid esters (e.g. isopropyl myristate, methyl laurate, glycerol monooleate and propylene glycol monole r) and N-methylpyrrolidone.

[0079] In some embodiments, the compositions may be in the form of ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, or plasters. In some exemplary embodiments of the present invention, the dosage forms of the compositions of the present invention are creams, which may further comprise saturated or unsaturated fatty acids, such as stearic acid, palmitic acid, oleic acid, palmitooleic acid, cetyl or oleyl alcohols, wherein stearic acid is particularly preferred. The creams of the present invention may also contain a nonionic surfactant, for example, polyoxy-40-stearate. In some embodiments, the active ingredient is optionally mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers. An ophthalmic dosage form, ear drops and eye drops are also contemplated as being within the scope of the present invention. In addition, the present invention provides the use of transdermal patches, which have the added advantage of providing controlled delivery of the compound to the body. Such dosage forms are prepared by dissolving or dispersing the compound in an appropriate medium. As noted above, penetration enhancers can also be used to increase the flow of the compound through the skin. The speed can be controlled either by using a membrane that controls the speed, or by dispersing the compound in a polymer matrix or gel.

[0080] Modified particles may be administered as an aerosol. This is achieved by preparing an aqueous aerosol, liposome drug, or solid particles containing modified particles. A non-aqueous suspension (e.g., a fluorocarbon propellant) may be used.

[0081] Typically, an aqueous aerosol is prepared by combining an aqueous solution or suspension of an agent with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary depending on the requirements of the particular compound, but usually include nonionic surfactants (Tweens, Pluronics or polyethylene glycol), non-toxic proteins such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols are usually made from isotonic solutions.

[0082] It should be appreciated that the modified particles and pharmaceutical compositions of the present invention can be developed and used in combination therapies, that is, the compounds and pharmaceutical compositions can be developed with or simultaneously administered with, before or after, one or more desired therapeutic agents or medical procedures. A particular combination of treatment methods (therapeutic agents or procedures) for use in a combination regimen will take into account the compatibility of the desired therapeutic agents and / or procedures and the desired therapeutic effect to be achieved. It should be appreciated that the treatment methods used can achieve the desired effect for the same disorder (for example, the compound of the present invention can be administered simultaneously with another anti-inflammatory agent), or they can achieve various effects (for example, control of any side effects).

[0083] In some embodiments of the present invention, pharmaceutical compositions comprising modified particles of the present invention further comprise one or more additional therapeutically active ingredients (eg, anti-inflammatory and / or palliative). For the purposes of the present invention, the term “palliative” refers to a treatment that is focused on alleviating the symptoms of the disease and / or side effects of the therapeutic regimen, but is not therapeutic. For example, palliative care includes pain medications, antiemetics, and motion sickness medications.

[0084] The invention provides methods for regulating an immune response in an individual, preferably a mammal, more preferably a human, comprising administering to said individual the modified particles described herein. The immunoregulation methods of the present invention include methods that suppress and / or inhibit the innate immune response or adaptive immune response, including, but not limited to, an immune response activated by immunostimulatory polypeptides or viral or bacterial components.

[0085] The modified particles are administered in an amount sufficient to regulate the immune response. As described herein, the regulation of the immune response can be humoral and / or cellular and is measured using standard methods in this field and as described herein.

[0086] In some embodiments, an individual suffers from a disorder associated with unwanted immune activation, such as an allergic disease or condition, allergy, and asthma. An individual having an allergic disease or asthma is an individual with a recognizable symptom of an existing allergic disease or asthma.

[0087] In some embodiments of the present invention, the individual suffers from a disorder associated with unwanted immune activation, such as atherosclerosis, ischemic reperfusion injury, and myocardial infarction.

[0088] In some embodiments of the present invention, the individual suffers from a disorder associated with unwanted immune activation, such as an autoimmune disease and inflammatory disease. An individual having an autoimmune disease or inflammatory disease is an individual with a recognizable symptom of an existing allergic disease or inflammatory disease.

[0089] Autoimmune diseases can be divided into two broad categories: organ-specific and systemic. Autoimmune diseases include, but are not limited to, rheumatoid arthritis (PA), systemic lupus erythematosus (SLE), type I diabetes mellitus, type II diabetes mellitus, multiple sclerosis (MS), immune infertility such as premature extinction of ovarian function, scleroderma, Sjogren's disease , vitiligo, alopecia (baldness), polyglandular insufficiency, Graves disease, hypothyroidism, polymyositis, pemphigus vulgaris, exfoliative pemphigus, inflammatory bowel disease, including Crohn's disease and ulcerative colitis, autoimmune hepatitis it, including those associated with hepatitis B virus (HBV) and hepatitis C virus (HCV), hypopituitarism, graft-versus-host disease (GVHD), myocarditis, Addison’s disease, autoimmune skin diseases, uveitis, pernicious anemia and hypoparathyroidism.

[0090] Autoimmune diseases may include, but are not limited to, Hashimoto's thyroiditis, autoimmune polyglandular syndromes of type I and type II, paraneoplastic pemphigus, bullous pemphigoid, herpetiform dermatitis, IgA dependent linear dermatosis, acquired pulmonary tuberculosis, pulmonary tuberculosis, cicatricial pemphigoid, essential mixed cryoglobulinemia, chronic childhood bullous disease, hemolytic anemia, thrombocytopenic purpura, Goodpasture syndrome, autoimmune neutrop Eugenia, myasthenia gravis, Eaton-Lambert myasthenic syndrome, stiff person syndrome, acute disseminated encephalomyelitis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, multifocal motor neuropathy with conduction block, chronic neuropathy with monoclonal neuropathy, monoclonal neuropathy, encephalomyelitis, retinopathy, primary biliary sclerosis, sclerosing cholangitis, gluten-sensitive enteropathy, ankylosing spondylitis, react Willow arthritis, polymyositis / dermatomyositis, mixed connective tissue disease, Behcet’s syndrome, psoriasis, polyarteritis nodosa, allergic vasculitis and granulomatosis (Cherga-Strauss disease), multivasculitis with overlap syndrome, allergic vasculitis, Wegener's granulomatosis, temporal arteritis, Takasu arteritis, arteritis , isolated vasculitis of the central nervous system, obliterating thrombovasculitis, sarcoidosis, glomerulonephritis and cryopathy. These conditions are well known in the medical field and are described, for example, in Harrison's Principles of Internal Medicine, 14th ed., Fauci A S et al., Eds., New York: McGraw-Hill, 1998.

[0091] Animal models for research on autoimmune diseases are known in the art. For example, animal models that are most similar to human autoimmune disease include animal strains that spontaneously develop a single disease with a high incidence. Examples of such models as non-limiting examples are non-obese diabetic (NOD) mice that develop a disease similar to type I diabetes and animals prone to lupus disease such as New Zealand Hybrid, MRL-Fas ^lpr and BXSB mice. Animal models in which an autoimmune disease was induced include, but are not limited to, experimental autoimmune encephalomyelitis (EAE), which is a model of multiple sclerosis, collagen-induced arthritis (CIA), which is a model of rheumatoid arthritis, and experimental autoimmune uveitis (EAU) which is a model for uveitis. Animal models for autoimmune diseases have also been created by genetic manipulation and include, for example, IL-2 / IL-10 knockout mice for inflammatory bowel disease, knockout mice deficient in Fas or Fas ligand for SLE, and knockout mice deficient antagonist of the IL-1 receptor for rheumatoid arthritis.

[0092] In some embodiments of the present invention, the individual suffers from a bacterial or viral infection. An individual having a bacterial or viral infection is an individual with a recognizable symptom of an existing bacterial or viral infection.

[0093] A non-limiting list of viral infections treatable by modified particles of the present invention include herpevirus infections, hepatitis B virus infections, West Nile virus infections, flavivirus infections, influenza species, rhinovirus infections, papillomavirus infections, paromixovirus infections, parainfluenza infections, and retroviral infections . Preferred viruses are those that infect a subject's central nervous system. Most preferred viruses are those that cause encephalitis or meningitis.

[0094] A non-limiting list of bacterial infections treatable with the modified particles of the present invention include staphylococcal infections, streptococcal infections, mycobacterial infections, rod infections, Salmonella infections, Vibrio infections, spirochetal infections and Neisseria infections. Bacteria that infect a subject's central nervous system are preferred. Most preferred are bacteria that cause encephalitis or meningitis.

[0095] In some embodiments, the present invention relates to the use of the compositions of the present invention before the onset of the disease. In other embodiments, the present invention relates to the use of the compositions of the present invention for inhibiting an ongoing disease. In some embodiments, the present invention relates to the eradication of a disease in a subject. By eliminating a disease in a subject is meant treating, preventing or suppressing a disease in a subject.

[0096] In some embodiments, the present invention relates to the prevention of relapse of the disease. For example, an undesired immune response may occur in a specific region of a peptide (such as an antigenic determinant). A relapse of the disease associated with an undesired immune response can occur when the immune response attacks various regions of the peptide. Since the carboxylated particles of the present invention are free of attached peptides or antigenic fragments, the particles will be effective against several epitopes. T-cell responses in certain disorders of the immune response, including MS and other TH1 / 17-mediated autoimmune diseases, can be dynamic and develop during a relapsing-remitting and / or chronic progressive disease. The dynamic nature of the T cell repertoire is important for the treatment of certain diseases, as the goal may change as the disease progresses. Previously existing knowledge of response patterns was necessary to predict disease progression. The present invention provides compositions that can prevent the effect of a dynamically changing disease, an “epitope scattering” function. A known relapse model is the immune response to proteolipid protein (PLP) as a model of multiple sclerosis (MS). An initial immune response may occur in response to PLP _139-151 . Subsequent onset of the disease can occur by a recurring immune response to PLP151-171.

[0097] Some embodiments of the present invention relate to the treatment of pathological conditions associated with undesirable hypersensitivity. Hypersensitivity may be of any of types I, II, III and IV. Immediate hypersensitivity (type I). The frequency of administration, as a rule, will correspond to the time of exposure to the allergen. Suitable animal models are known in the art (e.g., Gundel et al., Am. Rev. Respir. Dis. 146: 369, 1992, Wada et al., J. Med. Chem. 39, 2055, 1996; and WO 96 / 35418).

[0098] Other embodiments of the present invention relate to transplantation. This refers to a transplant of a tissue or graft sample from a donor to a recipient and is often performed for people who need tissue in order to restore the physiological functions provided by the tissue. Transplanted tissues include (but are not limited to) whole organs, such as a kidney, liver, heart, lungs, parts of organs, such as skin grafts and cornea; and cell suspensions, such as bone marrow cells and cell cultures, selected and cultured from bone marrow or peripheral blood, and whole blood transfusions.

[0099] A serious potential complication of any transplant results from antigenic differences between the recipient and the transplanted tissue. Depending on the nature and extent of the difference, there may be a risk of an immunological attack on the graft by the host or graft on the host, or both can occur. The degree of risk is determined by monitoring the response pattern in the population of subjects receiving the same treatment and having the same phenotype, with a correlation of various possible factors in accordance with generally accepted clinical procedures. An immunological attack may be the result of an existing immune response (e.g., a preformed antibody) or a response initiated by transplantation (e.g., generation of TH cells). An antibody, T helper (TH) cells, or cytotoxic T (Tc) cells can be involved in any combination with each other and with various effector molecules and cells. However, the antigens involved in the immune response are generally unknown, thereby creating difficulties in developing antigen-specific treatments or inducing antigen-specific tolerance. The modified particles of the present invention are particularly useful in the prevention of organ rejection, since it is not necessary to conjugate attached peptides or antigens with modified particles in order for the particles to be effective in inducing tolerance or eliminating the inflammatory immune response.

[00100] Some embodiments of the present invention relate to reducing the risk of a graft-versus-host reaction leading to rejection of a tissue graft by the recipient. Treatment may be performed to prevent or reduce the effects of hyper-acute, acute, or chronic rejection. Treatment is predominantly initiated well in advance of transplantation, so that by the time the graft is introduced, tolerance will already be formed; but where this is not possible, treatment can be started simultaneously with or after transplantation. Regardless of the time of initiation, treatment is usually continued at regular intervals for at least the first month after transplantation. Subsequent doses may not be necessary if a satisfactory graft adaptation is present, but their administration may be resumed if there is any evidence of rejection or inflammation of the graft. Of course, the tolerization procedures of the present invention can be combined with other forms of immunosuppression to achieve an even lower level of risk.

EXAMPLES

[00101] The following examples are intended to further illustrate the advantages and characteristics of the present invention, but do not limit the scope of the present invention.

Materials and methods

Virus cultivation

[00102] As described previously (Getts et al., J Neurochem. 103: 1019, 2007), West Nile virus (Sarafend strain) was obtained from the brains of newborn mice and used to infect monolayers of Vero confluent cells, with a multiplicity of infection of 5 plaque forming units ( PFU) per cell. Cells were incubated with the virus for 40 hours at 37 ° C, after which they were frozen. The flasks were then thawed and the virus-saturated supernatant was clarified by centrifugation, after which aliquots were stored at -70 ° C until use.

Mice and infection

[00103] C57BL / 6 (CD45.2) females aged 8 to 12 weeks and congenic B6.SJL-Ptprc ^a Pep3 ^b / BoyJ (CD45.1) mice were obtained from the Animal Resources Center, Western Australia. C57BL / 6-7.2fms-EGFP transgenic (CD45.2) mice were obtained from the Transgenic Animal Resources Center Queensland, Australia. All procedures were performed with permission from the University of Sydney Animal Ethics Committee. All animals were kept in a class II biohazard in cells with a hepa filter located at the top of the cell. Food and water were provided ad libitum.

[00104] Intranasal infection with high doses was performed as previously described, 6x10 ⁴ PFU of WNV in sterile phosphate buffered saline (PBS; Gibco BRL, California, USA). For infection with a low dose, mice were inoculated with 6x10 ³ PFU WNV. False infection was carried out by inoculating mice with sterile PBS only. (Getts et al., J Neurochem. 103: 1019, 2007, Wacher et al., J Virol. 81: 860, 2007)

[00105] After infection, the mice were weighed twice a day. Mice were sacrificed under anesthesia by cardiac perfusion using 40 ml of ice-cold PBS. For histological examination, mice were additionally perfused with 20 ml of 4% paraformaldehyde (Sigma Aldrich, St. Louis, USA) in PBS.

Plaque assay for determining virus titer

[00106] To determine live virus titers in tissue samples, plaque analysis was performed using virus-responsive BHK cells (kindly provided by Guna Kapuriah, John Curtin Medical School, Canberra, Australia). As described previously (Getts et al., J Neurochem. 103: 1019, 2007), tissue samples were excised from animals and separated by an electronic homogenizer (Tissue Tearor, Biospec, Bartles, Oklahoma, USA). Brief description: Five-fold dilutions of clarified homogenates were prepared in Roswell Park Memorial Institute 1640 medium (RPMI; CSL Biosciences) and used to infect confluent BHK cells in 6-well plates (1 x 10 ⁶ cells seeded overnight in 2 ml of RPMI medium).

[00107] Cells were incubated for 1 hour at 37 ° C, after which the inoculum was removed by aspiration. Wells were coated with 3 ml of 1.5% (v / v) Agarose II fusible (Amresco, Solon, Ohio, USA) in 2X minimal support medium (MEM; GibcoBRL, Grand Island, New York, USA). The cells were incubated for another 3 days at 37 ° C, after which they were fixed with 3 ml of 10% formalin (Co.) For 2 hours before removing the agarose plug. A 3% solution of crystalline violet (Hopkins and Williams, Essex, England) in 20% methanol (Fronine, Riverstone, New South Wales, Australia) was used to stain fixed cells. Plaques were counted using a colony counter (IUL S.A., Barcelona, Spain), and the final PFU per gram (tissue) was determined by factoring the number of plaques, inoculum volume and sample dilution.

Getting chimeric mice

[00108] As previously described (Getts et al., J Neurochem. 103: 1019, 2007), B6.SJL-PtprcaPep3b / BoyJ mice (CD45.1) aged 6 to 8 weeks were irradiated with a single dose of 950 rad. Twelve hours later, mice were reconstructed with 10 ⁷ bone marrow cells from C57BL / 6-7.2fms-EGFP donors. Mice were injected with sulfamethoxazole (Sigma Aldrich) and trimethoprim (Sigma Aldrich) in drinking water for 10 days after exposure. Six weeks after irradiation, mice were infected with WNV as described above. Chimerization was verified by flow cytometry and consistently had 96-99% donor origin, as previously demonstrated (Getts et al., J Neurochem. 103: 1019, 2007).

Intravenous Pellets

[00109] 0.5; 0.05 and 3 μl FITC or naked Bright Blue (BB) Flouresbrite and carboxylated polystyrene granules were obtained from PolyScience, New York, USA. Naked granules of polylactic-co-glycolic acid (PLGA) and carboxylated granules were obtained from Phosphorex, Massachusetts, USA.

[00110] Granules were diluted in sterile PBS, and 300 μl was injected intravenously into the tail vein of infected or mock infected mice. To study the survival rate after infection with a high dose (6x10 ⁴ PFU VZN intranasally), the mice were injected with one dose of granules every day, starting from 6 days after infection. If necessary, mice were given injections every day until the mass was not stabilized. To study the survival rate at infection with a low dose (6x10 ³ PFU VZN intranasally), the mice were injected either with granules from the 6th day after infection, or in the case of recording significant weight loss (4-6% of the total body weight). Mice were injected with 300 μl of granules once a day for 5 days or until the mass was not stabilized. To select tissue, mice infected with a dose of 6x10 ⁴ or 6x10 ³ were injected with 300 μl of granules either on the 6th day after infection, or if significant weight loss was recorded, followed by sorting if necessary. Bodies were selected for immunohistochemical analysis, flow cytometry, plaque analysis and cytokine analysis, as described below.

Immunohistology

[00111] Fluorescence immunohistochemistry (IHC) was performed for the brain, spleen, liver, lung, and kidneys selected from C57BL / 6 and CFMS-EGFP (in B6.SJL-PtprcaPep3b / BoyJ) chimeras. After perfusion, organs were fixed in 4% paraformaldehyde at 4 ° C for 4 hours and then immersed in 30% sucrose overnight before freezing in the Optimum Cutting Temperature Compound (OCT; Tissue-Tek, Tokyo, Japan). Slices of tissue with a thickness of eight microns were obtained on a cryostat microtome and dried in air. Fluorescence IHC was performed as described previously (Getts et al, J Exp Med. 29: 2319, 2007), with the addition of an amplification system on a tiramid (TSA kit; Perkin Elmer, Belgium), used in accordance with the manufacturer's instructions. Tissue sections before imaging were counter-stained with DAPI (Vector).

Microscope and image acquisition

[0100] Images were obtained on an Olympus BX-51 microscope (Olympus, Japan) using a DP-70 camera and DP manager 2.2.1 software (Olympus).

Isolation of white blood cells from the brain and liver

[0101] Leukocytes were obtained, as described previously (Getts et al, J Exp Med. 29: 2319, 2007), from the brain of mice perfused with PBS by splitting the brain material for 60 minutes at 37 ° C in PBS with deoxyribonuclease (0.005 g / ml, Sigma Aldrich) and collagenase IV (0.05 g / ml, Sigma Aldrich). Cleavage was stopped by the addition of 10% FCS and the homogenate was passed through a nylon sieve with a hole diameter of 70 μm (Becton Dickinson, New Jersey, USA). The precipitate obtained after 10 minutes was centrifuged at 340xg, resuspended in 30% Percoll (Amersham, Norway) and layered on 80% Percoll. White blood cells were selected from 30% / 80% of the boundary layer after centrifugation at 1140xg for 25 minutes at room temperature. The same protocol is also used to obtain white blood cells from the liver, and tissue is weighed before treatment.

Isolation of white blood cells from the spleen, blood and bone marrow

[0102] For analysis by flow cytometry, the right femur was excised and bone marrow cells were washed with PBS-filled syringes. At least 4 mice were used to isolate bone marrow precursors. The cell suspension obtained after washing was filtered through a cell filter with a pore size of 70 μm and centrifuged for 5 minutes at 340xg. The red blood cells in the resulting pellet were lysed in NH ₄ Cl erythrocyte lysis buffer (BD Pharm Lyse ^™ ; BD Pharmingen), and then centrifuged for 5 minutes at 340xg. In the case of peripheral blood, blood was collected by cardiac puncture and immediately transferred to citrate buffer (mmol, Sigma Alrich). The resulting suspension was layered on 70% Percoll and centrifuged at 1140xg for 20 minutes at room temperature with disinhibition. The boundary layer was selected, and the cells were washed once in PBS, centrifuged at 340xg. To isolate leukocytes of the spleen, the spleen was passed through a cell sieve with a pore diameter of 7070 μm and centrifuged for 5 minutes at 340xg. The red blood cells in the resulting pellet were lysed in NH ₄ Cl erythrocyte lysis buffer (BD Pharm Lyse ^™ ; BD Pharmingen), and then centrifuged for 5 minutes at 340xg.

Flow cytometry

[0103] Cells selected (as described above) from the brain, liver, blood and bone marrow were washed in PBS and blocked with anti-CD16 / CD32 antibody (BioLegend). Viable cells were counted using the trypan blue exclusion method, which constantly showed> 95% cell viability.

[0104] The expression of the molecule on the cell surface was measured, and the distribution of the cells was carried out on a FACS ARIA (Becton Dickinson) equipped with an argon ion and helium-neon laser. Viable populations were gated by direct and angular scattering, and after that the identified fluorescent populations were determined by predictive gating. Sorting was performed using specific fluorescence and scattering parameters to determine the populations of interest. Sorting accuracy was established to achieve a bone marrow population purity> 98%.

[0105] The acquired FACS data files were analyzed using Flow Jo flow cytometry software (FlowJo, Ashland, Oregon, USA). Quantification of cell populations of interest was carried out based on percent flow cytometry during the assays and the absolute number of cells of each organ.

Adaptive transfer

[0106] Both Ly6C ^hi / cFMS-EGFP ⁺ / CD11b ⁺ populations and Ly6C ^lo / Cfms-EGFP ⁺ / CD11b ⁺ populations were selected from mice intranasally infected with WNV on day 6. Ly6C ^hi BM was labeled with 10 mmol of an orange (CMTMR [5- (and -6) - (((4-chloromethyl) benzoyl) amino) tetramethylrodamine) mixed isomer cell tracker; Invitrogen). Sorted cells were centrifuged and grown in 1 ml PBS containing 10 μmol CMTMR. Cells were stained for 10 min, after which the reaction was stopped by the addition of 10% FCS. Cells were washed in 50 ml of PBS at least three times. Labeled CMTMR Ly6C ^hi BM cells were mixed in a 1: 1 ratio with Ly6C ^lo cells, which, with the addition of CMTMR, was similarly performed for Ly6C ^hi populations. A total of 2 × 10 ⁶ BM cells were intravenously administered to either WNV-infected mice on day 6.5 or mock-infected Ly5.1-C57BL / 6 congenic mice. This was immediately followed by an intravenous injection of 300 μl of BB polystyrene granules. After 12 hours, the brain, spleen, and liver were selected by the method described above. The presence of donor cells was investigated using flow cytometry using both CFMS-EGFP and CMTMR orange cell tracker.

Multiplex ELISA

[0107] Multiplex ELISA on the plates were performed in accordance with the manufacturer's instructions (Quansys Biosciences, Logan, Utah, USA). Brief description: the brain, spleen, liver and tissues were homogenized in PBS, clarified by centrifugation at 1000xg and stored at -20 ° C until analysis. Serum samples were also used. Thawed samples and standards were diluted in the provided buffer, and 30 μl of each was added to each plate containing 16 wells, each of which contained an immobilized antibody for a particular soluble protein. Then the plates were incubated for 1 hour on an orbital shaker at 120 rpm. The plates were washed three times, and 30 μl of detectable antibodies was added to each well and incubated for another hour. After washing three times, streptavidin-HRP was added and incubated for another 15 minutes. The plates were then washed 6 times and a substrate mixture was added. The readings on the CCD sensor were immediately taken from the tablets (Kodak, Rochester New York, USA). Plate images were analyzed using Quansys Q-View software (Quansys Biosciences).

Induction and evaluation of experimental autoimmune encephalitis (EAE)

[0108] C57BL / 6 mice were subcutaneously injected with an emulsion containing 0.1 mg of MOG peptide (MEVGWYRSPFSRVVHLYRNGK; Auspep, Parkville, Victoria, Australia;> 95% purified HPLC) and Freund's complete adjuvant containing 2 mg / ml Mycobacterium tuberculosis (Sig ) Two days later, mice were intraperitoneally injected with 500 μl of pertussis toxin (Sigma Aldrich). Mice were monitored for disease progression and rated on the following scale: 1, flaccid tail and / or weakness in one hind limb; 2, weakness in more than one limb, impaired gait; 3, paralysis of one limb; 4, paralysis of more than one limb, urinary incontinence; 5, dying.

Induction of thioglycolate-induced peritonitis

[0109] Induction of peritonitis was carried out by injection of 1 ml of thioglycolic acid (4% (v / v); Alrich Sigma) dissolved in PBS. Intraperitoneal lavage was performed on mice sacrificed by displacement of the cervical vertebrae. Brief description: 5 ml of peritoneal washing buffer (PLB; PBS containing 0.5 mM EDTA (Fronine) with heparin (Sigma Aldrich) 9.9 u / ml) was injected into the peritoneum. The peritoneum was carefully massaged before the introduction of PLB into a 10 ml syringe. This process has been repeated twice. Then the lavage volume was increased to 50 ml in PLB, and the lavage was centrifuged at 340xg for 5 minutes. Cells were prepared for flow cytometry as described above.

Statistics

[0110] Graphs were constructed, and computerized statistical analysis was performed on GraphPad Prism and InStat, respectively (both software programs are GraphPad, San Diego, California, USA). Depending on the data, an unpaired, two-tailed Student test or one-way analysis of variance followed by the Tukey-Cramer test with P <0.05, which was considered statistically significant, was applied.

To analyze the correlation between parameters such as mass loss, infiltration, and virus titer, nonlinear regression (fit curve) with a second-order polynomial (Y = A + B * X + C * X ^ 2) was used.

Example 1

Characterization of encephalitis models induced by high and low doses of West Nile virus

[0111] Intranasal infection of C57BL / 6 mice with high doses of WNV equal to 6x10 ⁴ PFU of WNV resulted in 100% mortality on day 7 after infection (Figure 1A). The macrophages and immigrating microglia, which we showed earlier, are formed from circulating precursors - Ly6c ^hi monocytes (Getts et al, J Exp Med. 29: 2319, 2007), infiltrate the brain infected with WNV on the 5th day after infection. T cells, NK cells and neutrophils also enter the central nervous system at this time point, with peak infiltration at 7 days after infection (Figure 1F-H, Figure 2A-D). The virus titer in the brain also exponentially increases from 5 days after infection. reaching high levels on day 7 after infection (Figure 1C), and the body weight of infected mice is significantly reduced from 5 days after infection to death (Figure 1B).

[0112] Inoculation of mice with a 10-fold lower dose of the virus, i.e. 6x10 ³ PFU intranasally, gives sublethal results. There is some variation in mortality when comparing independent experiments, which ranges from 40-60% (Figure 1A), but a strong correlation between the percentage of weight loss and the titer of the virus / infiltration (Figure 1 B-H, Figure 2A-E) is successively demonstrated. Daily monitoring of mouse mass has proven to be a reliable way to predict the outcome of infection in humans and suggests that mice require intervention in order to survive after infection (Figure 1 B).

[0113] Mice that do not lose weight after inoculation with a dose of WNV of 6x10 ³ do not die from infection and are immune to subsequent reinfection with a dose of 6x10 ⁴ (Figure 1A-B). These mice did not show any disease symptoms upon initial infection or high-dose reinfection, including weight loss. Conversely, mice losing a significant amount of weight compared to normal fluctuations in the mock-infected control groups (usually> 4% of body weight in a 24-hour period) continue to lose weight, usually within 2-3 days before death. In this model of encephalitis, most mice that die from infection begin to lose weight between 6 days and 11 days after infection, and death occurs before 16 days after infection. These mice showed high virus titers in the brain at TOD, which is comparable to mice infected with a dose of 6x10 ⁴ on day 7 after infection (Figure 1C).

[0114] A comparison of flow cytometry of leukocyte infiltration in the brain of mice infected with a dose of 6x10 ⁴ and 6x10 ³ showed that the percentage of weight loss on day 7 after infection strongly correlates with total leukocyte infiltration (Figure 1F), macrophages (Figure 1H) immigrating microglia (Figure 2A), T cells (Figure 2C) and NK cells (Figure 2E) with some correlation observed between mass loss and neutrophil immigration (2D). Not surprisingly, the number of CD45 ^lo microglia remained relatively unchanged in all mice, as this population mainly contained resident brain microglia (Figure 2B).

[0115] For further study of the low-dose infection model, mice infected with a dose of 6x10 ³ were weighed and killed daily on days 0-14 with a selection of the brain for flow cytometry and plaque analysis. Some correlation was observed between the percentage of weight loss at the time of killing and the titer of the virus (Figure 2F) or leukocyte infiltration (Figure 2G). However, a comparison between the titer of the virus and leukocyte infiltration showed that some mice had high virus titers without significant infiltration (Figure 2H), and while some of these mice also lost a significant amount of mass, others did not (Figure 2F). Because mice needed to be killed to determine the titer of the virus in the brain, it is not clear whether these mice had the virus in the brain without significant weight loss or leukocyte infiltration neutralized the virus without demonstrating mass loss or infiltration, or if a high titer of the virus precedes brain infiltration / weight loss .

Example 2

Treatment with carboxylated granules substantially improves survival in high and low WNV-induced encephalitis models

[0116] Fluorescent granules are often used in the literature to track the migration of subpopulations of monocytes in various in vivo disease models. However, these studies do not take into account the potential effect of these theoretically “inert” granules with possible monocyte function on the outcome of the disease.

[0117] In an attempt to track monocytes during WNV infection, we used both bare and carboxylated polystyrene granules and found unexpected changes in the course of the disease. The following data show that both naked and carboxylated polystyrene granules significantly reduced brain infiltration by cells of the immune system, and also contributed to the long-term survival of infected mice.

[0118] the Administration to mice infected with a dose of 6x10 ⁴ WNV, 4.41x10 ⁹ carboxylated polystyrene granules with a diameter of 0.5 μm in 300 μl of PBS, on the 6th day after intravenous infection gave 10% long-term survival of mice in this lethal disease model (Figure 3A) . To ensure that survival results can be improved, we injected the same concentration of granules into mice infected with a low dose of 6x10 ³ on day 6 after infection. However, this strategy reduced the survival of mice compared to control groups treated with PBS (Figure 3B). These results emphasize that the granules must be administered therapeutically, i.e. when the mice initially show significant mass loss, which, as we have shown, indicates a high titer of the virus and brain infiltration by leukocyte populations (see Figures 1, 2).

[0119] Therefore, we have established a therapeutic approach for introducing particles into a low-dose infection model. Mice were weighed daily (Figure 3E-G), and 4.41 x ^{10 9} carboxylated polystyrene granules were administered in case of significant weight loss detected in a 24-hour period (usually> 4% of total body weight compared to normal fluctuations of mock-infected control groups) . Using this strategy, we were able to increase the survival rate of mice, which usually continue to lose weight and die without intervention by 60% (40-80% in independent experiments) (Figure 3D). As expected, all mice that lost weight and received PBS continued to lose weight and died 2-4 days later (Figure 3E, G). Mice that did not lose a significant amount of mass at any time showed similar stability in mass, as did the control groups to which PBS was administered (Figure 3E-F).

[0120] Mice received carboxylated granules within 5 days of the initial detection of significant weight loss. For some mice, a 5-day treatment of the granules was sufficient, and the mass remained stable after the administration of the granules was stopped, and these mice survived the infection without further intervention (Figure 4A-B). However, some mice began to lose weight again after discontinuation of treatment on day 5, so treatment was resumed until the mass was stabilized, and these mice also showed long-term survival (Figure 4C-D).

[0121] On day 9 after infection, flow cytometry was performed on the brain of mice infected with a WNV dose of 6x10 ³ that received either carboxylated polystyrene granules or PBS. Mice that did not lose weight also received pellets or PBS on day 8 as control groups for the experiment. As shown in Figure 4D-L, mice that did not lose weight on day 8 after infection but received granules (Figure 4H-I) or PBS (Figure 4D-E) showed no brain infiltration by macrophages or immigrated microglia formed from monocytes, T cells, NK cells, or neutrophils, with the main isolated population being CD45 ^{lo / int} CD11b ⁺ resident microglia. In mice that lost weight on day 8 after infection and received PBS, massive brain infiltration was apparent on day 9 after infection (Figure 4F-G), and the main population was CD45 ^hi inflammatory macrophages formed from monocytes (Figure 4L). However, mice that lost weight and received pellets on day 8 after infection still had significant infiltration, but was significantly reduced compared to mice treated with PBS (Figure 4J-L). Flow cytometry should be repeated for mice infected with a 10 ^3- dose treated with granules or PBS with weight loss to demonstrate that a decrease in infiltration occurs when the mice receive granules 6-14 days after infection.

Example 3

Pellet carboxylation is critical to improving substantive survival and changes in leukocyte populations in mice infected with WNV

[0122] At this stage of the study, it was unclear whether carboxylation of the granules was critical in the improvements noted for survival and reduction of white blood cells migrating to the brain. Mice infected with a dose of WNV of 6x10 ³ were injected with 4.41x10 ⁹ bare or carboxylated granules with a diameter of 0.5 μm in 300 μl of PBS or PBS alone when significant weight loss was recorded. Mice injected with carboxylated granules showed a significant improvement in survival of 60%, while mice treated with naked granules showed a much lower improvement of 25% (Figure 5A). Mice that did not lose weight were not treated, and such mice survived the infection without intervention, while all mice that lost weight and received PBS died on day 14 after infection (Figure 5B-D). Weight loss was controlled in these mice up to 20 days after infection; mice that did not lose a significant amount of mass showed a stability pattern similar to the control groups that were injected with PBS (data not shown), while mice that received either carboxylated or bare granules (Figure 5B-C) eventually stabilized and survived, or continued to lose weight, and died after 2-4 days. All mice that lost weight and received PBS (B, D) continued to lose weight and died after 2-5 days.

[0123] Flow cytometry was performed for mice infected with a dose of 6x10 ⁴ PFU VZN to study changes in leukocyte populations and to identify cells that can be detected in association with either carboxylated or bare granules. 4.41x10 ⁹ carboxylated FITC granules or bare FITC granules with a diameter of 0.5 μm in 300 μl of PBS or only PBS on day 6 after infection were injected into intravenous mice. Mice were killed on day 7 after infection, and blood, brain, bone marrow, liver and spleen were selected for flow cytometry.

[0124] "Free" granules, as well as granules in association with CD45 ⁺ leukocytes, could be detected by flow cytometry (Ex / Em Max 441/486 nm). As expected, the granules could not be detected in the blood of the control groups that were injected with PBS (Figure 5E-G), but could be detected in mice treated with naked (Figure 5H-J) and carboxylated (Figure LM) granules. The granules were cleaned more efficiently if they were carboxylated, and of the remaining granules in the bloodstream, many were in association with CD45 ⁺ leukocytes (Figure 5O) compared to bare granules (Figure 5K), which were initially “free”. These data suggest that carboxylation somehow facilitates the uptake of granules by leukocytes from the bloodstream compared to bare granules, some of which still remain “free” in the bloodstream 24 hours after administration.

[0125] Flow cytometry of the brain revealed some significant differences in the decrease in the leukocyte population in the brain of mice receiving either carboxylated or bare granules. Carboxylated or naked granules cannot be detected in the brain on day 7 after infection, 1 day after treatment (Figure 6A-C), suggesting that cells capturing the granules do not penetrate the infected CNS. The total number of leukocytes in the brain of mice treated with bare granules and in the brain of mice treated with carboxylated granules was essentially reduced compared to PBS control groups, as well as the total number of macrophage and microglia populations (Figure 6D) . However, only carboxylated granules reduced the number of T cells and NK cells in the brain of these mice. As Ly6c ^hi, and Ly6c ^{lo / int} population of macrophages were significantly reduced processing plant both types, without noticeable changes in small populations with Ly6 ^- cells. As for CD45 ^int activated microglia reducing in Ly6c ^hi subpopulation were installed only for treatment carboxylated beads, but reduce Ly6c ^{lo / int} and Ly6 with ^- subpopulations of beads were set for both types of treatment. No changes were noted in a small subpopulation of Ly6c ^hi CD45 ^{lo of the} remaining microglia, however, treatment with granules of both types significantly reduced Ly6c ^{lo / int} and Ly6 c ^- subpopulations.

[0126] Flow cytometry of the spleen demonstrated that many granules were captured by leukocytes in this organ with some interesting differences in populations between treatments. As shown in Figure 7A-I, the granules ⁺ cells were mainly CD11b ⁺ , CD11c ⁺ + and Ly6 c ⁺ .

[0127] Further analysis showed that the three main subpopulations of interest captured granules in the spleen — CD11b ⁺ , CD11c ^— monocytes (Figure 7J, M, P), CD11b ⁺ , CD11c ⁺ “myeloid” dendritic cells, primarily Ly6c ^{lo / -} (Figure 7K, N, Q), and CD11b ^- , CD11c ⁺ dendritic cells, primarily Ly6c ^{lo / -} (Figure 7L, O, R).

[0128] Increases in these three cell populations were also apparent in the spleen of mice receiving carboxylated granules. Increases in total amounts of CD11b ^+, CD11c ^- monocytes, as well as the total number Ly6c ^{int / hi,} granules ⁺ Ly6c ^{int / hi,} and granules ^- Ly6c ^{int / hi} subpopulations were detected in mice treated with carboxylated beads (Figure 8A). An increase in the total amount of Ly6c ^{- / lo} , granules ⁺ Ly6c ^{- / lo} and granules ^- Ly6c ^{- / lo were also found} in the treatment with carboxylated granules. These data suggest that monocytes can migrate to the spleen as a result of treatment with carboxylated granules, but not only ⁺ granules are involved, but also granules ^- cells.

[0129] Among the CD11b ⁺ , CD11c ⁺ dendritic cells in the spleen, it was found that the Ly6c ^{- / lo} population captured both types of granules (Figure 8B). However, only carboxylated granules increased the total number of CD11b ⁺ , CD11c ⁺ dendritic cells in the spleen, and of these, Ly6c ^{- / lo} populations. Since there was no increase in granule ^- Ly6c ^{- / lo} dendritic cells, it is obvious that only granules ⁺ Ly6c ^{- / lo} dendritic cells migrate to the spleen after capture of carboxylated granules. It is possible that these cells are formed from Ly6c ^hi inflammatory monocytes that capture granules from the bloodstream, are sent to the spleen, instead of migrating to the brain, where they inhibit Ly6c expression and activate CD11c expression after their differentiation into dendritic cells.

[0130] From populations of dendritic cells CD11b ^- , CD11c ⁺ in the spleen, Ly6c ^{- / lo} cells captured both types of granules (Figure 8C). When treated with carboxylated granules, an increase in the total number of CD11b ^- CD11c ⁺ populations of dendritic cells, in particular Ly6c ^{- / lo} cells, was observed. Increases were observed both in granules ⁺ and granules ^{- in the} cells of this population, suggesting that not only granules ⁺ , but also granules ^- cells increase in the spleen. This population may contain resident DCs of the spleen, non-myeloid DCs of the spleen attracted from the periphery, or may also potentially consist of inflammatory monocytes that captured granules, suppressed CD11b and Ly6c and activated CD11c expression.

[0131] Granules were also found to be associated with a small percentage of B cells in the spleen, however, only carboxylated granules significantly increased the number of B cells in the spleen (Figure 9A-D). The only other significant increase found for the spleen was an increase in CD8 ⁺ T cells, while the number of CD4 ⁺ T cells, NK cells and neutrophils remained unchanged.

[0132] Granules could also be found in the liver, especially in association with CD11b, CD11c and Ly6c expressing cells (Figure 10A-I). As for the spleen, it was found that CD11b ⁺ CD11c ^- monocytes (Figure 10J), CD11b ⁺ CD11c ⁺ (Figure 10K) and CD11b ^- CD11c ⁺ (Figure 10L) dendritic cells capture granules in the liver. However, there was no significant increase or decrease in any liver white blood cell population. A small number of granules were also found in the bone marrow, in a small population of CD11b ⁺ , CD11c ⁺ Ly6 c ⁺ leukocytes (Figure 11A-G). However, no significant increase or decrease was found in any bone marrow leukocyte populations.

Example 4

[0133] Mice infected with a WNV dose of 6x10 ³ were intravenously administered a low dose of 0.1% or a high dose of 0.5% carboxylated polystyrene granules with a diameter of 0.5; 0.05 or 3 μm in 300 μl of PBS or PBS alone with significant weight loss. Mice receiving a low dose of granules with a diameter of 0.5; 0.05 or 3 μl, showed similar improvements in survival, approximately 40-50% (Figure 12A). However, high-dose treatment of mice was detrimental to survival, with much less improvement of 20%. A significant number of mice treated with high doses of granules showed atypical symptoms of the disease, and it was suggested that the presence of such a high dose of granules in vital organs for several days was detrimental to the mouse. This course will be repeated with the potential inclusion of other lower doses of granules. Flow cytometry will also be performed on mice infected with a WNV dose of 6x10 ⁴ , treated 6 days after infection with granules of various sizes.

[0134] These data emphasize the fact that carboxylated polystyrene granules may not be destroyed in vivo, and therefore may have limited therapeutic use. Therefore, we obtained biodegradable polylactic-co-glycolic acid (PLGA) spheres with a diameter of 0.5 μm, which were either carboxylated or bare (however, the bare spheres also contain a small amount of carboxyl groups). Mice infected with a dose of WNV equal to ⁶ × ^{10 3} were injected with 4.41 × ^{10 9} bare or carboxylated polystyrene granules or PLGA spheres with a diameter of 0.5 μm when determining significant weight loss. As can be seen in Figure 12B, to date, PLGA naked and carboxylated spheres have shown similar survival as carboxylated polystyrene beads.

[0135] To check whether carboxylated PLGA spheres introduced on day 6 after infection reduced brain infiltration on day 7 in mice infected with a WNV dose of 6x10 ⁴ , we compared the treatment of PBS mice with carboxylated granules with a diameter of 0.5 μm, carboxylated PLGA spheres with a diameter of 0.5 μm and 0.5 μm carboxylated diamond particles at a dose of 4.41x10 ⁹ administered intravenously. The brain, blood, spleen, and liver were processed for flow cytometry. Analysis of this experiment is ongoing, however, a preliminary analysis of the brain of these mice showed that all three particles successfully reduce brain infiltration by immune cells (Figure 12C). There was also an increase in the total number of leukocytes in the spleen, in accordance with the results that we saw in mice treated with carboxylated granules.

Example 5

Pellet treatment of mice with T-cell deficiency

[0136] Wild-type mice C57BL / 6 (DT) and RAG (T cell deficiency) were infected with a dose of 6x10 ⁴ or 6x10 ³ PFU WNV. DT mice infected with a high dose of 6x10 ⁴ showed 100% mortality on day 7 after infection, while RAG mice infected with the same dose began to die 8 days after infection, with some mice living up to 13 days after infection. DT mice infected with a low dose of 6x10 ³ showed 60% mortality on day 8 after infection, but with 40% long-term survival after this point in time. RAG mice infected with the same dose did not die until 14 days after infection, however, by day 16, all RAG mice were dead (Figure 13A-B). These data suggest either a direct or indirect role of T cells in the immunopathology of WNV-induced encephalitis, since mice with T-cell deficiency survive longer than mouse RAGs. However, it also emphasizes that T cells are crucial for controlling viral infection, since all low dose RAG mice died from the infection, while 40% of mouse DT survive immunity.

[0137] Mass loss of these mice was recorded daily, and it was shown that weight loss precedes the death of both RAG mice and DT mice (Figure 13B). However, it was found that mouse DTs live only for a maximum of 2-3 days after primary weight loss, while RAG mice could live up to 5 days after the start of weight loss. An analysis of plaque formation for the brain taken from RAG mice and DT of mice infected with a dose of either 6x10 ⁴ or 6x10 ³ showed that the percentage of weight loss strongly correlates with the titer of the virus (Figure 12C). In general, RAG mice showed greater weight loss at the time of death and higher virus titers than mouse DT. Apparently, this can be explained by the fact that they were able to survive after initial weight loss for a longer period than mouse DT and, thus, the virus continued to grow in the brain and the mice continued to lose weight until death, as opposed to direct the result of the inability to control the virus without T cells. This hypothesis is supported by flow cytometry of the brain (Figure 13D-E), in which the brain was compared on day 7 of DT mice and RAG mice on day 7 / day 8. RAG mice show a significant decrease in brain infiltration by macrophages of microglia, T-cells (because of deficiency) and neutrophils, which does not increase between 7 days and 8 days after infection. This may explain the reason why these animals live longer after primary weight loss than mouse DT, if immunopathology is the main cause of death of these animals.

[0138] RAG and mouse DT were also infected with a dose of 6x10 ³ PFU WNV, and were weighed daily to verify the effectiveness of carboxylated granules in the absence of T cells. With significant weight loss (> 4% within 24 hours), mice were injected intravenously with 4.41 x ^{10 9} carboxylated granules or PBS delivered in a volume of 300 μl. There was no significant improvement in the survival of RAG mice after pellet administration (Figure 12F). RAG mice continued to lose weight after the introduction of granules or PBS and died after 2-5 days (Figure 12G). This experiment must be repeated again to confirm the results.

Example 6

Pellet Treatment with EAE

[0139] C57BL / 6 mice will be primed with MOG and CFA adjuvant. By the time a symptom of the disease develops, which will be determined by changes in gait, posture and other properties, carboxylated particles will be administered intravenously or every 8 hours, every 16 hours, every day or every 48 hours. The severity of the disease will be determined by changes in the indicators on the scale of the disease. The introduction of particles will prevent the migration of monocytes into the brain and the subsequent priming of T cells, which leads to a significant decrease in the disease scale.

Example 7

Treatment with granules of atherosclerosis and neointimal smooth muscle cell proliferation

[0140] APO / E deficient mice will receive a diet rich in fats. From 4 weeks of age, carboxylated particles will be administered intravenously every 8 hours, every 16 hours, every 24 hours, every 48 hours, every 72 hours, once a week or once a month. The severity of the disease will be determined by changes in arterial histology. The introduction of particles will prevent the migration of monocytes into the walls of the artery and prevent subsequent immune complications, which are crucial in triggering proliferation of smooth muscles and the formation of plaques in the intima.

[0141] While specific embodiments of the present invention have been described and illustrated, such embodiments of the present invention should be construed as illustrating the present invention and not limiting the present invention, which is construed in accordance with the appended claims.

[00112] All patents, applications, and other references cited herein are incorporated in their entirety by reference.

Claims (12)

1. A method of reducing the duration or severity of an inflammatory immune response in a subject, comprising administering to said subject a pharmaceutical composition containing carboxylated particles, wherein attached biologically active substances or attached antigenic molecules are absent in said particles, said particles being polylactic-co-glycolic particles acid (PLGA), polystyrene particles or diamond particles, and wherein the diameter of said particles is between about 0.1 μm d about 10 microns. 2. The method according to p. 1, characterized in that the diameter of said carboxylated particles is between about 0.3 microns to about 5 microns, between about 0.5 microns to about 3 microns, between about 0.5 microns to about 1 microns, or approximately 0.5 microns. 3. The method according to p. 1, characterized in that the subject has an autoimmune disorder, such as multiple sclerosis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, systemic lupus erythematosus, Raynaud's syndrome, Sjogren's syndrome, autoimmune uveitis, autoimmune myocarditis or Crohn has ischemic reperfusion injury, atherosclerosis, suffered from myocardial infarction, is a transplant recipient, has psoriasis or dermatitis, or suffers from an allergic disorder such as eczema, asthma, allergic rhinitis or perchuvstvitelnost skin. 4. The method according to p. 1, characterized in that the composition is administered orally, nasally, intravenously, intramuscularly, through the eye, percutaneously or subcutaneously. 5. A method of treating a viral or bacterial infection in a subject, comprising administering to said subject a pharmaceutical composition containing carboxylated particles, wherein there are no attached biologically active substances or attached antigenic molecules in said particles, and wherein said particles are polylactic-co-glycolic acid particles (PLGA), polystyrene particles or diamond particles, and wherein the diameter of said particles is between about 0.1 μm to about 10 μm. 6. The method according to p. 5, characterized in that the diameter of said carboxylated particles is between about 0.3 microns to about 5 microns, between about 0.5 microns to about 3 microns, between about 0.5 microns to about 1 microns, or approximately 0.5 microns. 7. The method according to p. 5, characterized in that the viral infection is selected from the group consisting of West Nile virus infection, herpes virus infection, hepatitis virus infection, flavivirus, influenza infection, rhinovirus infection, retroviral infection, papilloma virus infection, paramyxovirus infection and parainfluenza virus infections. 8. The method according to p. 5, characterized in that the viral infection infects the central nervous system of the specified subject or causes viral encephalitis or viral meningitis. 9. The method according to p. 5, characterized in that the bacterial infection infects the central nervous system of the specified subject or causes bacterial encephalitis or bacterial meningitis. 10. The method according to p. 5, characterized in that the composition is administered orally, nasally, intravenously, intramuscularly, through the eye, percutaneously or subcutaneously. 11. A pharmaceutical composition for use in eliminating an inflammatory immune response in patients in need thereof, comprising carboxylated particles, while there are no attached biologically active substances or attached antigenic molecules in said particles, said particles being polylactic-co-glycolic acid particles ( PLGA), polystyrene particles or diamond particles, and wherein the diameter of said particles is between about 0.1 μm to about 10 μm. 12. The composition according to p. 11, characterized in that the diameter of said carboxylated particles is between about 0.3 microns to about 5 microns, between about 0.5 microns to about 3 microns, between about 0.5 microns to about 1 microns, or approximately 0.5 microns.

Images