WO1999029329A1 - Immunotherapy using ie water - Google Patents

Abstract

Disclosed are methods for the induction and regulation of cytokines in mammalian cells. Also disclosed are methods for potentiating immunity and regulating tumorigenesis in animals, and particularly humans through the use of IE water compositions. Disclosed are methods for increasing synthesis and secretion of cytokines and other mediators from peripheral blood monocytes.

Description

DESCRIPTION

IMMUNOTHERAPY USING I_E WATER

1.0 BACKGROUND OF THE INVENTION

1.1 FIELD OF THE INVENTION

The present invention relates generally to the fields of immunology and oncology. Disclosed are methods for the induction and regulation of cytokines in mammalian cells. Also disclosed are methods for potentiating immunity and regulating tumorigenesis in .animals, and particularly humans, through the use of I_E water compositions.

1.2 DESCRIPTION OF RELATED ART 1.2.1 CYTOKINES

It has become clear to scientists in recent years that antigen-specific immune response is largely dependent on preferential activation by agents that affect particular subsets of CD4⁺ T helper cells. These cells secrete defined patterns of cytokines that trigger strikingly different effector mechanisms. Two subpopulations of helper T cells, termed type 1 (Thl) and type 2 (Th2) cells show distinct and mutually exclusive patterns of cytokine secretion. For instance, Thl, but not Th2 cells usually secrete IL-2, IFN-γ, IL-12 and TNF-β while Th2, cells produce IL-4, IL- 5, IL-6 and IL-10. Other cytokines such as IL-3, TNF-α, GM-CSF are produced by both Thl and Th2 cells. The different cytokine patterns of the two types of Th cells lead to very distinct effector functions. In general, Th2 cells provide excellent helper functions for immunoglobulin synthesis whereas Thl cells are involved in the induction of delayed type hypersensitivity (DTH) responses. Studies in mouse models have suggested that immune responses to some infectious agents may be associated with preferential activation of antigen specific Thl or Th2 cells. These T-helper cell populations comprise functionally distinct subsets that are characterized by the patterns of lymphokines they produce following activation (Paul and Seder, 1994). Although these distinct subsets were first identified by in vitro .analysis of murine T-cell clones, strong evidence now exists for similar subsets in vivo in mice, rats and humans. In mice and man, at least three CD4 subsets exist: Thl, Th2 and ThO. Thl secrete IL-2, IFN-γ, and TNF-β and supports macrophage activation, delayed type hypersensitivity (DTH) responses, and immunoglobulin (Ig) isotype switching to IgG2a. Th2 cells secrete IL-4, IL-5, IL-6, IL-10, and IL-13, which activate B cells sensitive to IgG and IgE isotypes, as well as antibody production. ThO cells are characterized by production of cytokines of both the Thl and Th2 types, and are thought to be obligatory precursors of Thl and Th2 cells (Table 1).

TABLE 1 CYTOKINE SECRETION PROFILE OF HUMAN CD4 T-CELL SUBSETS TM Th2 ThO

IL-2 ++ - ++

IFN-γ -H- - ++

TNF-β ++ - +

IL-4 - ++ +

IL-5 - ++ +

IL-3 ± ++ +

IL-6 ± ++ +

GM- CSF ± ++ +

TNF-α ++ + ++

IL-10 ± ++ +

± .and + refer to the amount of cytokine produced by the majority of T cell clones.

Several factors, including the dose of antigens, the type of .antigen presenting cells (APC) and the major histocompatibility complex (MHC) class II haplotypes influence the differentiation of naive CD4⁺ T cells into specific Th subsets. However, the best characterized factors affecting the development of Th subsets are cytokines themselves (Paul and Seder, 1994). For example, IFN-γ inhibits the differentiation and effector functions of Th2 cells and may effect to dominant Thl responses. The APC-derived cytokine IL-12 strongly drives the differentiation of Thl cells in vitro .and in vivo, partly through its potent induction of IFN-γ products. Conversely, IL-4 strongly directs the development of Th2 cells, both in vitro and in vivo, for example, mice in which the IL-4 gene has been disrupted have an impaired ability to generate Th2 responses. Furthermore, IL-4, IL-10 and IL-13 inhibit Thl proliferation, and oppose the effects of IFN-γ or macrophages. Therefore, reciprocal regulation occurs, between the Thl and Th2 cell subsets. The in vivo relevance of the functional division of Th into subsets has been extensively studied in systems involving strong or persistent antigenic stimulation. For example, strains of mice that are genetically proven to mount a Thl type response against the parasite Leishmania major (and in particular, strains such as C57B1/6, B10-D2, and C3H/HeN) resist infection, while mice generating a Th2-type response (Balb/c) cannot control the infection (Shen and Coffman, 1992). Moreover, the use of cytokines or cytokine antibodies at the time of primary infection has been shown to alter the type of two Th subset generated, thereby affecting the disease outcome. Polarized Th2 responses have been implicated in several other pathological situations such as parasite infections (Shen and Coffman, 1992), atopic disease, infection with HIV and systemic autoimmune diseases (Goldman et al, 1991 ).

Strongly polarized human Thl -type and Th2-type responses not only play different roles in protection, they can also promote different immunopathological reactions. Thl -type responses appear to be involved in organ specific autoimmunity, in contact dermatitis, and in some chronic inflammatory disorders of unknown etiology. In contrast, in genetically predisposed hosts, Th2-type responses against common environmental allergens are responsible for triggering of allergic atopic disorders. Altered profiles of lymphokine production may account for immune dysfunction in some primary or acquired immunodeficiency syndromes (Romagnani, 1994). It is now clear that the nature of the human specific immune response against offending agents is determined by the set of lymphokines produced by T-cells. The human T-cell response is heterogeneous, but under some in vitro, and probably in vivo conditions, T-cell stimulation can result in the development of a restricted Thl type or Th2 type pattern of lymphokine production. Obviously, genetic differences at the individual level contribute to modulate the nature of the specific immune responses as suggested by the following observations: (i) all subjects are exposed to common environmental allergens, but these latter preferentially activate and/or expand Th2-like cells only in predisposed (atopic) populations and (ii) the early response to some infectious agents that is protective to the majority of people, in a few individuals may result in a subsequent shift to a less protective phenotype, whereas in others it may evolve to acute or chronic immunopathology.

Cytokines represent important compositions in a variety of medical methods. For example, TNF-α, IL-2, GM-CSF represent import.ant compositions for immunotherapy against cancer. Cytokines such as IL-12 are known to boost anti-infection response, while IL-10 has been implicated in the prevention of autoimmune diseases.

1.2.2 I_E WATER

Recent studies by Lo (1996) and Lo et al. (1996) have reported on the physical properties of water with I_E structures. These structures exhibit a variety of distinct physical and chemical properties as compared to ordinary control water preparations. It was suggested that stable rigid structures (or water clusters) could be formed from water molecules at room temperature and normal pressure using an electric field. The authors determined that this I_E water had different properties from pure water, including dielectric constants, electromotive force, resistivity, fluorescence, and temperature stability. Samples of this I_E water (obtainable from

American Technologies Group, Inc., Monrovia, CA) typically had a fluorescence peak at 298 nm, a thermal response indicative of water containing approximately 3% water clusters in solution, and was stable under conditions of both elevated pressure and temperature. 2.0 SUMMARY OF THE INVENTION

The present invention describes for the first time the use of I_E water compositions in the regulation and alteration of biological and cellular activity, and presents for the first time, novel methods for using compositions comprising this I_E crystal water in the treatment of a variety of diseases and physiological abnormalities in a mammal. The inventors' surprising data reveal for the first time that I_E water has utility in a variety of therapeutic and pharmaceutical applications, and can produce dramatic effects in mammalian cells, including the alteration of cytokine activity, and the secretion of cytokines by mammalian cells, and may be used to treat autoimmune disorders and various forms of susceptible cancers and other carcinomas in an animal.

In a first embodiment the invention provides a method of altering cytokine activity in an animal cell. The method generally comprises providing to an animal cell an amount of an I_E water composition effective to change or alter the cytokine activity in the cell. Preferably, the animal cell is mammalian, with human cells being particularly preferred. The altering of activity is preferably an increase in cytokine activity within the cell, or .an increase in the secretion of one or more cytokines from the cell. However, in certain circumstances, the I_E water composition may be provided to a cell for the purpose of reducing or decreasing the cytokine activity within a cell, or decreasing the secretion of one or more cytokines from the cell, or decreasing the rate of production of the cytokine or decreasing its rate of secretion from the cell. Although any cytokine produced by an animal cell may be altered by a composition that comprises I_E water, the preferred cytokines regulated or affected by the compositions are preferably selected from the group consisting of IFN-γ, IL-2, IL- 3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TNF-α, TNF-β, IL-15, IL-18, GM-CSF, CSF, SCF, Fas-R, Fas-L, TRAIL, TRAIL receptors, soluble receptors of such cytokines, and the like.

The inventors contemplate that one or more compositions comprising I_E water may be administered to the cell directly, or indirectly by administering the composition to the animal in which the cell is located. Thus, both in vitro and in vivo uses for these compositions represent important aspects of the invention. The I_E water composition may be added by itself, or in combination with one or more pharmaceutical agents, excipients, drugs, immunomodulating agents, immunosuppressive agents, cytokine inducing agents, cytokine regulating agents, anticancer agents, or other therapeutics as may be desirable.

Exemplary immunomodulating agents which may be combined with, added to, or formulated in the I_E water include, but are not limited to, mitogens (e.g.,

LPS, PHA, ConA), cytokines, including those listed above, antigens (e.g., synthetic, microbial, fungal, viral), drugs (including chemotherapeutic drugs e.g., CDDP, VP- 16, Act D, ADR, 5-FU, etc.), inhibitors or activators of cell signaling pathways, or agents (including polypeptides, polynucleotides, ribozymes, antisense RNAs and the like) that regulate, alter, or affect the transcription of one or more cytokine receptor- encoding polynucleotide sequences within the cell, or that regulate, alter, or affect the translation of one or more cytokine polypeptides. Exemplary immunosuppressive agents which may be combined with the I_E water include, but are not limited to, chemotherapeutic drugs like cytoxan, CDDP, VP-16, ADR, 5-FU, CPT-11, Act D, and antibodies directed against certain cell types like T helper cells. Indeed, a number of immunosuppressive agents are well-known to those of the medical arts, and the formulation of such and regimens for their administration are well-known. In fact, because the I_E water itself is an aqueous medium, it is highly possible to formulate various pharmaceutical formulations to comprise the I_E water merely by using the I_E water in place of "regular" or non- I_E water as the diluent, carrier, or excipient itself.

Exemplary anticancer agents that may be combined with the I_E water compositions include, but are not limited to, cisplatin, adriamycin, doxorubicin, etoposide, camptothecin, Actinomycin D, cyclophosphamide, 5-fluorouracil (5-FU), anti-DNA compounds, protein synthesis inhibitors, and the like. A number of anticancer agents including these and others are well-known to those in the medical arts, and particularly to those in the fields of oncology, and as such, may be readily utilized in combined therapy with the I_E water itself.

A further aspect of the invention concerns a method of altering an immune response in an animal. The method generally involves administering to an animal an amount of an I_E water composition effective to alter the immune response in an animal. In certain embodiments it may be desirable to increase an immune response, or alternatively, to decrease or even prevent an immune response in an animal.

The invention also provides a method for stimulating a blood cell to produce a mediator, such as a cytokine. The method generally involves administering to a blood cell, such as a monocyte, an amount of an I_E water composition effective to stimulate the cell to produce the mediator. The stimulation may cause the cell to initiate or to increase the synthesis of one or more of such mediators, or alternatively, cause the cell to begin or to increase the secretion of such mediators from the cell.

A method of increasing cytokine secretion from a mammalian cell, such as a peripheral blood monocyte, is also provided by the invention. The method involves contacting such a cell with an I_E water composition, in an amount effective to increase or permit cytokine secretion from such a cell.

Another aspect of the invention is a method of inducing differentiation in a cell. The method typically comprises administering to a cell an amount of an I_E water composition effective to induce differentiation in the cell. Exemplary cells may include virtually any type of mammalian cells, including such types as bone marrow pluripotent stem cells, blood progenitor cells, brain cells, aging cells, tissues, transplants, etc., and also terminal differentiation of tumor cells into normal cells.

A method of treating an autoimmune disease in an animal is also provided by the invention. This method involves identifying an animal suspected of having an autoimmune disease; and administering to the animal an amount of an I_E water composition sufficient to treat or reduce the symptoms of the autoimmune disease in the animal. Exemplary autoimmune diseases include psoriasis, lupus, Sjogren's syndrome, rheumatoid arthritis, ulcerative colitis, Crohn's disease, sympathetic ophthalmia, myasthenia gravis, multiple sclerosis, orchitis, and osteomyelitis. The method may also involve administering to the animal one or more immunosuppressive or immunomodulatory compositions as described above.

Also provided by the invention is a method of treating cancer in an animal which involves administering to an animal having one or more types of cancer an amount of an I_E water composition effective to treat, ameliorate, lessen, or ablate the cancer. The inventors contemplate the methods are useful in the treatment and/or amelioration of cancers than can differentiate from malignancy into normal cells like leukemia and lymphomas, as well as for cancers that can respond to cytokines/factors by either inhibiting cell proliferation or are killed like leukemias, sarcomas, melanomas, glioblastomas, prostate cancer, ovarian cancer, lung cancer, and colon cancer. I_E water compositions can stimulate such factors from the host blood/tissues and/or from the tumor cells. The mechanism by which I_E water compositions can alter cancer is contemplated by the inventors to involve the induction of one or more cytotoxic cytokines like TNF- , or by the activation of one or more host immune cells by various cytokines and activation of innate immunity (like NK cells, macrophages, etc.) or by activating adaptive immunity for the activation of cell-mediated immunity and antibody response against the cancer. As such, any type of cancer, or any disease state or abnormality in an animal that can be lessened, ameliorated, or treated by the presence of an increased level of one or more cytokines in the cells of the affected mammal, is contemplated to be responsive to treatment with one or more results I_E water compositions.

Another aspect of the invention involves a therapeutic kit that comprises, in a suitable container means, a therapeutically-effective amount of one or more I_E water compositions and one or more pharmaceutically acceptable excipients. Such a kit may also comprise a single container means, or the I_E water composition and the excipient may be present within distinct container means. Preferably, the I_E water composition is suitable for parenteral, intramuscular, or intravenous administration, or alternatively, formulated for oral or topical administration. The kit may also contain one or more immunomodulating agents, immunosuppressive agents, cytokine inducing agents, anticancer agents, or other therapeutic reagents as described above, if desired. The therapeutic reagent may comprise a polynucleotide, a polypeptide, a carbohydrate, a lipid, a lipid complex, a ribozyme, a nanocapsule, or a liposome, or other suitable pharmaceutical formulation of the I_E water composition as described hereinbelow. Likewise, as stated above, because the I_E water itself is aqueous and has many of the properties of regular water, the composition may be formulated as an excipient, or as a carrier for one or more drugs already used in conventional therapies or methods currently used for altering cytokine activity in a cell. Alternatively, the kit may comprises one or more I_E water compositions in combination with instructions, regimens, or indications for their use in the treatment of a particular disease or physiological abnormality.

2.1 IMMUNITY

I_E water compositions may provide a defense against intracellular microorganisms, such as viruses and some types of bacteria and protozoans. Response to microorganismal infection tends to be dominated by cell-mediated forms of immunity, which is characterized by cellular cytolytic activity and the production of cytokines (e.g., IFN-γ, IL-2, TNF-α). Resistance to extracellular forms of pathogens; for example, helminths, is often associated with humoral antibody responses. These are characterized by induction of high levels of pathogen-specific antibodies (immunoglobulins) that neutralize the foreign organisms. One may use the disclosed I_E water compositions by either the oral route or by intravenous, intramuscular, or other such suitable administration routes. Although the formulation of pharmaceutically-acceptable compositions for administration to an animal is well- known to one of skill in the medical arts, a discussion of pharmaceutical formulations and preparations is included hereinbelow to assist one's preparation of the I_E water compositions for administration to the target cell, or selected animal.. While an I_E water composition may be administered to a selected cell or animal alone, it may also in certain embodiment be provided to the cell or the animal in combination with one or more vaccines (including microbial antigen vaccines) or such like, to provide a specific adjuvant effect for a host anti-pathogen immune response.

3.0 BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIG. 1 shows the synergistic response of TNF-a production by human monocytes to LPS stimulation. (DX means concentrated I_E water).

FIG. 2 shows the kinetics of TNF-α secretion by human monocytes in the presence and absence of I_E water compositions. FIG. 3 shows the induction of genes encoding cytokines by I_E water compositions.

FIG. 4 shows the absence of endotoxin in I_E water compositions.

FIG. 5 shows the regulation of cytokines by I_E water compositions.

FIG. 6 shows the effect of I_E water compositions on IL-10 and IL-12 secretion by PBMC. Induction of IL-10 and IL-12 by human peripheral blood monocytes activated by I_E water (diluted 1 :2 and 1 :4) and compared to induction of IL-10 and IL-12 by two concentrations of LPS (100 ug/ml and 10 ng/ml).

FIG. 7 shows that polymyxin B inhibits LPS activity. This figure shows that whereas polymyxin B inhibits significantly IL-10 production by LPS activated human monocytes, polymyxin B has no effect on IL-10 production by I_E water

(diluted 1 :2 and 1 :4) activated human monocytes. These findings demonstrate that I_E water is devoid of LPS-like contaminants.

FIG. 8 shows the effect of polymyxin B on I_E water-stimulated IL-12 secretion by PBMC. Polymyxin inhibits LPS activity. This figure shows that whereas polymyxin B inhibits significantly IL-12 production by LPS activated human monocytes, polymyxin B has no effect on IL-12 production by I_E water (diluted 1:2 and 1 :4) activated human monocytes. These findings demonstrate that I_E water is devoid of LPS-like contaminants.

FIG. 9 shows the effect of I_E water compositions on CD69 expression by PBMC. Human peripheral blood mononuclear cells were treated with I_E water (two dilutions 1 :2 and 1 :4) overnight and the cells were examined for the expression of the cell surface activation marker CD69 by flow cytometry. The findings reveal that I_E water activates the cells and upregulates the expression of CD69. For comparison, the CD69 expression is upregulated by LPS. FIG. 10 shows the effect of I_E water compositions on CD 14 expression by PBMC. Shown is the expression of LPS receptor by CD 14 antigen. I_E was determined as in FIG. 9 by flow cytometry.

4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In a general and overall sense, the present invention provides methods for modulating the activity of blood cells, and increasing the level and secretion of cytokines from monocytes, and in particular, cells of human origins. The invention also discloses methods for modulating or potentiating an immune response in a mammal, and particularly, in a human by the administration of I_E water compositions.

Methods are also provided for preparation of vaccine compositions based on I_E water compositions to reduce, ameliorate, or prevent infection from a number of etiological agents including bacterial and other microbial infections in an animal.

4.1 SOME ADVANTAGES OF THE INVENTION

Studies by the inventors have conclusively demonstrated that I_E water compositions exhibit highly significant activities on normal human peripheral blood and purified cell subsets. The inventors have discovered that, unlike control water preparations, I_E preparations can stimulate blood cells to respond in an antigen-like fashion and secrete various mediators, such as cytokines, that are crucial to generate and regulate immune responses to infection and cancer. The data also indicate that I_E water preparations can also induce differentiation of cells and induce changes of cell characteristics and behavior. This represents the first scientific demonstration that certain forms of water, such as I_E water have biological effects on tissues and in some aspects, mimic the effects seen with various stimuli (physiologic, chemical, microbial, antigenic, etc.). These findings have far reaching implications in the general field of cell biology and medicine. For example, these water preparations, depending on their preparations, may have selective advantages in the preparation of non-toxic vaccines and the selective regulation of immunity against various diseases. One can design and custom-tailor I_E water preparations that perform different functions .and treatments. Also, these preparations may be instrumental for use in maintaining immunologically-balanced health, particularly in older populations. The inventors also contemplate that such I_E water preparations may influence behavioral responses and alter chronic diseases and stress-related symptoms. Provided herein are studies investigating the effect of I_E water in a biological system in order to establish a physiological role for I_E water. The inventors investigated the induction and regulation of synthesis of several cytokines by human peripheral blood-derived leukocytes and purified subsets. Cytokine secretion was determined by a sensitive and specific ELISA and transcriptional regulation of mRNA by RT-PCR™. The inventors have demonstrated that I_E water preparations possess potent immunomodulatory activities in the absence and presence of suboptimal concentrations of T and B cell mitogens. The effects were specific for I_E water as no effect was seen with control water preparations from ATG or laboratory derived water.

The following findings were reproducibly obtained with I_E water preparations. (1) There was significant stimulation of several cytokines in human peripheral blood, namely tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-10, IL-12, and interferon-γ (IFN-γ). The amount of cytokine secretion induced by I_E water was higher or similar to optimal concentrations of mitogen-induced stimulation of cytokines. I_E water also stimulated cytokine secretion in purified subpopulations of lymphocytes and monocytes. (2) The induction of cytokine secretion by I_E water was a function of both the final concentration of the I_E water and the time of incubation. There was a dose-dependent titration of cytokines with different dilutions of I_E. Further, the inventors observed a very rapid kinetics of induction of cytokines by I_E water as early as 2 h following stimulation (FIG. 2). (3) In the presence of suboptimal concentrations of the B cell mitogen (LPS) and the T cell mitogen (PHA), the inventors observed synergistic activity with I_E water but not with control water preparations (FIG. 1). (4) The levels of cytokine induction differed from one individual to another but the pattern remained the same for all individuals. (5) In several instances, the inventors observed a specific pattern of cytokine induction by THl and TH2 subsets. These two subsets regulate the outcome of several human diseases. To demonstrate that the I_E water was not contaminated with bacterial products, like the lipopolysaccharide (LPS), which is mitogenic and stimulates high levels of cytokines by peripheral blood, the inventors examined the effect of a specific inhibitor of LPS, polymyxin B, in the cytokine response by human peripheral blood to both I_E water and LPS. The findings demonstrate that whereas LPS-induced cytokines are completely inhibited by polymyxin B, the cytokine response by I_E water was not affected at all by polymyxin B. These findings rule out LPS as a potential contaminant in the I_E water preparation.

Taken together, these findings demonstrate that I_E water preparations exert potent and selective immunomodulatory activities on human peripheral blood.

Additionally, I_E water potentiates and synergizes antigenic stimulation and thus may have an important role both as an adjuvant and/or a regulator of specific immune responses. Since different water preparations can be prepared with different I_E crystals, the inventors contemplate that each might have a distinct and selective pattern of cytokine induction and immunostimulatory activities.

4.2 TH1 TH2 MEDIATED RESPONSES

T helper lymphocytes can be divided into two distinct subsets of effector cells based on their functional capabilities and the profile of cytokines they produce. The THl subset of CD4⁺T cells secrete cytokines associated with inflammation, such as IFN-γ, TNF-β, and IL-2 and induce cell-mediated immune responses. The TH2 subset produces cytokines such as IL-4, IL-5, IL-6, IL-10, and IL-13 that help B cells to proliferate and differentiate and is associated with humoral type immune responses. A THO subset contains a mixture of THl and TH2 populations.

4.3 DIFFERENTIATION

In the THl family, IFN-γ and IL-12 promote THl differentiation; IFN-γ prevents growth of TH2; and IL-12 directly augments THl differentiation. Within the

TH2 family, IL-4 drives THl differentiation, while IL-10 suppresses THl development. In vivo, cytokine environment influences the differentiation of TH into

THl or TH2. 4.4 MECHANISMS OF I_E WATER ACTIVITY ON CELLULAR PROCESSES

At least three mechanisms have been implicated by which I_E water compositions may trigger cells: (a) changes in cell-membrane potential resulting in signaling the cell and mimicking a ligand-receptor interaction; (b) activation of cell-enzymatic activity; and (c) may complex with nutrients, transcription factors, etc. The results described herein suggest that different dilutions of I_E water preparations may selectively trigger different cytokines, and different preparations of I_E water may trigger selectively targeted cytokines. Likewise, I_E water compositions may be useful in boosting immunity, priming the immune system for a secondary more effective response, overcoming immunosuppression by drugs, restoring immunity to infections, diseases, aging, immunodeficiency syndromes, and cancer, protecting against autoimmune diseases, and maintaining body-mind equilibrium.

4.5 PHARMACEUTICAL COMPOSITIONS

The I_E water compositions disclosed herein may find important utility in a variety of pharmacological embodiments. As such, they may be formulated for oral administration, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.

For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

The active compounds may also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for ex.ample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

For oral administration, the I_E water-containing compositions may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouth wash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's

Solution). Alternatively, the active ingredient may be incorporated into a solution containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include binders, abrasives, flavoring agents, foaming agents, humectants, and other solvents. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

The composition can be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the .art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to

1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

Because of the non-toxicity of I_E water compositions, the inventors contemplate that in its simplest form of administration, such compositions may be suitable for direct ingestion. Alternatively, sterile preparation of I_E water may be directly injected or used as a diluent or carrier for the administration of other pharmaceuticals.

4.6 LIPOSOMES AND NANOCAPSULES In certain embodiments, the inventors also contemplate the use of one or more liposome and/or nanocapsule formulations for the introduction into a host cell or animal the particular pharmaceutical formulations that comprise I_E water. Such formulations may be preferred for the introduction of pharmaceutically-acceptable formulations of one or more nucleic acids, peptides, and/or antibodies in combination with an I_E water composition as disclosed herein. Such compositions may be used to regulate cytokine synthesis in a host cell, or to modulate or otherwise regulate or control cellular activity, immune response, or to treat a particular condition in an animal, such as an infection or in certain instances, tumorigenesis or other cancers.

The formation and use of liposomes is generally known to those of skill in the art (see for example, Couvreur et al, 1977 which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy of intracellular bacterial infections and diseases). More recently, liposomes were developed with improved serum stability and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen .and Choun, 1987). In one instance, the disclosed composition may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. The term "liposome" is intended to mean a composition arising spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures .and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991).

Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al, 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyano- acrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made, as described (Couvreur et al, 1977; 1988). Methods of preparing polyalkyl-cyano-acrylate nanoparticles containing biologically active substances and their use are described in U. S. Patent

4,329,332, U. S. Patent 4,489,055, and U. S. Patent 4,913,908.

Pharmaceutical compositions containing nanocapsules for the oral delivery of active agents are described in U. S. Patent 5,500,224 and U. S. Patent 5,620,708 (each specifically incorporated herein by reference in its entirety). U. S. Patent 5,500,224 describes a pharmaceutical composition in the form of a colloidal suspension of nanocapsules comprising an oily phase consisting essentially of an oil containing dissolved therein a surfactant and suspended therein a plurality of nanocapsules having a diameter of less than 500 nanometers. U. S. Patent 5,620,708 describes compositions and methods for the oral administration of drugs and other active agents. The compositions comprise an active agent carrier particle attached to a binding moiety which binds specifically to a target molecule present on the surface of a mammalian enterocyte. The binding moiety binds to the target molecule with a binding affinity or avidity sufficient to initiate endocytosis or phagocytosis of the particulate active agent carrier so that the carrier will be absorbed by the enterocyte. The active agent will then be released from the carrier to the host's systemic circulation. In this way, degradation of degradation-sensitive drugs, such as polypeptides, in the intestines can be avoided while absorption of proteins and polypeptides form the intestinal tract is increased.

U. S. Patent 5,641,515 and U. S. Patent 5,698,515 (each specifically incorporated herein by reference in its entirety) describe the use of nanocapsules for the oral administration of a polypeptide, specifically, insulin and are incorporated herein by reference. U. S. Patent 5,698,515 described insulin containing nanocapsules intended for oral administration of insulin which comprises a hydrophilic polymer modified with an inhibitor of proteolytic enzyme, insulin and water, wherein the inhibitor of proteolytic enzymes is ovomucoid isolated from duck or turkey egg whites. U. S. Patent 5,556,617 (specifically incorporated herein by reference in its entirety) describes the use of nanoparticles as pharmaceutical treatment of the upper epidermal layers by topical application on the skin.

Poly(alkyl cyanoacrylate) nanocapsules have been used as biodegradable polymeric drug carriers for subcutaneous and peroral delivery of octreotide, a long- acting somatostatin analogue. These nanocapsules were prepared by interfacial emulsion polymerization of isobutyl cyanoacrylate, and gave a uniform particle size of 216 nm in diameter (Damge et al, 1997).

The negative surface charge of nanocapsules makes them particularly susceptible to lysozyme (LZM), a positively-charged enzyme that is highly concentrated in mucosas. This interaction causes destabilization of the nanocapsule by LZM; however, it was observed that the destabilizing effects caused by the adsorption of LZM onto the nanocapsules can be prevented by previous adsorption of the cationic poly(amino acid) poly-L-lysine (Calvo et al. , 1997).

Calvo et al, 1996 describe the use of poly-epsilon-caprolactone (PECL) microparticles for the ocular bioavailability of drugs. Their study showed that PECL nanoparticles and nanocapsules as well as submicron emulsions are shown to be novel corneal drug carriers, and represent a useful approach for increasing the ocular bioavailability of drugs.

An exhaustive review of nanoparticles and nanocapsular carriers is provided by Arshady 1996. Arshady notes that one of the major obstacles to the targeted delivery of colloidal carriers, or nanocapsules, is the body's own defense mechanism in capturing foreign particles by the reticuloendothelial system (RES). This means that following intravenous administration, practically all nanometer size particles are captured by the RES (mainly the liver). The review describes recent initiatives on the design of macromolecular homing devices which seem to disguise nanoparticles from the RES and, hence, are of potential interest to the targeted delivery of nanocapsular carriers. The idea is based on a graft copolymer model embodying a link site for attachment to the carrier, a floating pad for maintaining the particles afloat in the blood stream, an affinity ligand for site-specific delivery and a structural tune for balancing the overall structure of the homing device. Yu and Chang (1996) describe the use of nanocapsules containing hemoglobin as potential blood substitutes. They use different polymers including polylactic acid and polyisobutyl-cyanoacrylate .and modify the surface of the nanocapsules with polyethylene glycol (PEG) or with PEG 2000 PE. The surface modified nanocapsules containing hemoglobin survive longer in the circulation. U. S. Patent 5,451,410 (specifically incorporated herein by reference) describes the use of modified amino acid for the encapsulation of active agents. Modified amino acids and methods for the preparation and used as oral delivery systems for pharmaceutical agents are described. The modified amino acids are preparable by reacting single amino acids or mixtures of two or more kinds of amino acids with an amino modifying agent such as benzene sulfonyl chloride, benzoyl chloride, and hippuryl chloride. The modified amino acids form encapsulating microspheres in the presence of the active agent under sphere-forming conditions. Alternatively, the modified amino acids may be used as a carrier by simply mixing the amino acids with the active agent. The modified amino acids are particularly useful in delivering peptides, e.g., insulin or calmodulin, or other agents which are sensitive to the denaturing conditions of the gastrointestinal tract.

4.7 THERAPEUTIC KITS COMPRISING I_E WATER COMPOSITIONS

In certain embodiments, the inventors contemplate that the methods disclosed herein may be accomplished through the introduction of a pharmaceutical composition comprising an I_E water preparation into a host cell, and particularly, into an animal such as a mammal either for diagnostic or treatment purposes. As such, one embodiment of the invention concerns the compositions disclosed herein provided in either a therapeutic or diagnostic kit. Such kits typically comprise, in one or more suitable container means, one or more I_E water preparation(s) of the present invention in a pharmaceutically acceptable formulation. The kit may comprise a single container means that contains the I_E water composition(s). The container means may, if desired, contain a pharmaceutically acceptable sterile excipient, having associated with it, the I_E water composition(s) and, optionally, a detectable label or imaging agent. The formulation may be in the form of a gelatinous composition (e.g., a collagenous composition), a powder, solution, matrix, lyophilized reagent, or any other such suitable means. In certain cases, the container means may itself be a syringe, pipette, or other such like apparatus, from which the composition(s) may be applied to a tissue site, skin lesion, or wound area. However, the single container means may contain a dry, or lyophilized, mixture of one or more I_E water composition(s), which may or may not require pre-wetting before use.

Alternatively, the kits of the invention may comprise distinct container means for each component. In such cases, one or more containers would contain each of the I_E water preparation(s), either as sterile solutions, powders, lyophilized forms, etc., and the other container(s) would include a matrix, solution, or other suitable delivery device for applying or administering the composition to the body, bloodstream, or to a tissue site, skin lesion, wound area, tumor, vasculature or other sites. Such delivery device may or may not itself contain a sterile solution, diluent, gelatinous matrix, carrier or other pharmaceutically-acceptable components. The kits may also comprise a second or third container means for containing a sterile, pharmaceutically acceptable buffer, diluent or solvent. Such a solution may be required to formulate the I_E water component into a more suitable form for application to the body, e.g., as a topical preparation, or alternatively, in oral, parenteral, or intravenous forms. It should be noted, however, that all components of a kit could be supplied in a dry form (lyophilized), which would allow for "wetting" upon contact with body fluids. Thus, the presence of any type of pharmaceutically acceptable buffer or solvent is not a requirement for the kits of the invention. The kits may also comprise a second or third container means for containing a pharmaceutically acceptable detectable imaging agent or composition. The container means will generally be a container such as a vial, test tube, flask, bottle, syringe or other container means, into which the components of the kit may placed. The matrix and I_E water compositions may also be aliquotted into smaller containers, should this be desired. The kits of the present invention may also include a means for containing the individual containers in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials or syringes are retained.

Irrespective of the number of containers, the kits of the invention may also comprise, or be packaged with, an instrument for assisting with the placement of the ultimate matrix- I_E water compositions within the body of an animal. Such an instrument may be a syringe, pipette, forceps, or any such medically approved delivery vehicle.

5.0 EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

5.1 EXAMPLE 1 - EFFECT OF I_E WATER ON THE SELECTIVE INDUCTION AND REGULATION OF CYTOKINE SYNTHESIS AND SECRETION BY HUMAN PERIPHERAL BLOOD LYMPHOCYTES This example describes the effects of I_E water preparations on the regulation of the immune system. The inventors show that I_E water preparations may preferentially regulate Thl or Th2 or both cells through the cytokines secreted and therefore affect both the innate and adaptive immune responses to various clinical manifestations (Gan et al, 1992; Gan et al, 1994; Jewett and Bonavida, 1994; Jewett and Bonavida, 1995; Jewett and Bonavida, 1996; Jewett et al., 1996). It is essential to understand the mechanism by which I_E water (American Technologies Group, Inc., Monrovia, CA) regulates the Thl-Th2 type responses and also their effect in individuals with different genetic backgrounds and/or with different predisposition to clinical manifestations. The inventors suggest that I_E water, through their regulation of Thl-Th2 type cytokine responses, may have clinical implications. For instance, the knowledge that the cytokine IL-12 is a critical component in the development of Thl -type responses can be directly applied to vaccine development against disease controlled by DTH, as well as to the introduction of new forms of specific immunotherapy in allergic disorders. On the other hand, a better definition of factors responsible for the induction of lymphokine (such as IL-4 and IL-10) which inhibit both release of pro-inflammatory cytokines and Thl cells differentiation can favor the development of new therapeutic approaches for control of harmful immunopathological reactions in chronic inflammatory disorders and in some autoimmune diseases. Further, current strategies to treat diseases like HIV and tumors contemplate the use of cytokines or genetic approaches to alter Thl-Th2 responses.

Therefore, it is possible that I_E water may exert its effect in vivo by altering the Thl- Th2 cytokine profiles and thus reducing and alleviating different clinical manifestations.

To examine the effect of I_E water on the regulation of Thl or Th2 type responses in vitro, studies were done with unfractionated human peripheral blood mononuclear cells (PBMC) and selected stimulants (PHA, ConA, anti-CD3).

Proliferation and cytokine release was assessed. The studies have also been refined by the use of purified CD4 T lymphocytes and short term T cell clones (see FIG. 1).

To investigate the effect of I_E water preparations on the regulation of various cell surface receptors, the inventors have examined the expression of cytokine receptors by flow cytometry and RT-PCR™ and measured the receptor affinity by ligand binding.

In addition, the Thl and Th2 responses are initiated by their interaction with the antigen-presenting cells (APC). APC process antigens and export peptides on the cell surface via the MHC antigens. It is possible that homeopathic preparations regulate either and/or antigen processing and export and upregulation of surface MHC molecules. The effects of I_E water on cytokine production in whole blood are illustrated in Table 2.

TABLE 2 I WATER-INDUCED CYTOKINE PRODUCTION BY WHOLE BLOOD

Water Cytokine (pg/ml) Preparation TNF-α IL-6 IL-12

Laboratory 260 0 8

ATG Control 260 0 9

I_E#1 12190 3906 1041

I_E#2 10735 4797 1190

The effects of I_E water on LPS-induced cytokine production in whole blood are shown in Table 3. The effects of I_E water on PHA-induced cytokine production in whole blood are shown in Table 4.

TABLE 3

EFFECT OF I_E WATER ON LPS-INDUCED CYTOKINE PRODUCTION

BY HUMAN WHOLE BLOOD

Water Cytokine (pg/ml) Preparation TNF-α IL-6 IL-12

Laboratory 8065 4014 1008

ATG Control 7211 4153 907

I_E#1 13222 4331 1040

I_E#2 12933 4902 1239 TABLE 4

EFFECT OF I_E WATER ON PHA-INDUCED CYTOKINE PRODUCTION

BY HUMAN WHOLE BLOOD

Water Cytokine (pg/ml)

Preparation TNF-α IL-6 IL-12 IFN-γ

Lab CH 131 19 40 703

ATG CH 117 19 41 703

I_E #1 13197 2414 1228 1043

I_E #2 10204 2657 1026 2230

Table 5 illustrates the effects of titration of I_E water on cytokine production in whole blood.

TABLE 5

EFFECT OF TITRATION OF I_E WATER ON CYTOKINE PRODUCTION

BY HUMAN WHOLE BLOOD

I_E Water Dilution

1 :2 1 :6 1 :18 Non I_F CH

TNF-α 14176 4649 1593 430 IL-12 2405 1893 529 39

The synergy of I_E water on LPS in PHA-induced cytokine secretion by whole blood is demonstrated in Table 6.

TABLE 6 SYNERGISTIC EFFECT OF I_E WATER ON LPS IN PHA-INDUCED CYTOKINE SECRETION

BY HUMAN WHOLE BLOOD

Water TNF-α IL-6 IL-12 IFN-γ

Preparation Medium LPS Medium LPS Medium LPS Medium LPS

Laboratory Control 131 2344 19 1424 40 166 703 3351

ATG Control 1 17 2816 19 1476 40 92 700 5569

I_E Water 10204 58975 2657 2886 1026 694 2230 34311

The induction of cytokine production in human peripheral blood monocytes is demonstrated in Table 7. Table 8 shows the kinetics of I_E water-induced cytokine production.

TABLE 7 EFFECT OF I_E WATER-INDUCED CYTOKINE PRODUCTION BY HUMAN PERIPHERAL BLOOD MONOCYTES Water Cytokine (pg/ml)

Preparation TNF-α IL-6 IL-12

Laboratory Control Water 1359 168 11

ATG Control Water 979 169 6

I_E Water Preparation # 1 16740 2579 76

I_E Water Preparation #2 22851 2534 215

TABLE 8 TIME KINETICS OF I_E-INDUCED CYTOKINE SECRETION BY HUMAN PERIPHERAL BLOOD MONOCYTES

TNF-α IL-6 IL-10 IL-12

Control Control Control Control h 668 5305 57 82 4 4 13 14 h 2220 19274 188 1997 4 4 14 14 h 2245 24999 1689 2835 4 13 18 50 h 610 9465 2372 2775 4 190 34 139 h 241 992 3125 3125 5 109 37 155 days 4072 819 2741 2741 5 41 41 78

The inventors have also determined the effects of I_E water on cytokine secretion by human monocytes. These data are illustrated in FIG. 2 and Table 9.

TABLE 9

EFFECT OF I_E WATER ON CYTOKINE SECRETION

BY NONADHERENT HUMAN PERIPHERAL BLOOD MONOCYTES

Water Cytokine (pg/ml)

Preparation TNF-α IL-6 IL-12

Control Water 13,000 2016 120 I_E Water 38,765 2119 720

The absence of endotoxins in I_E water preparations was confirmed to ensure contaminants were not responsible for the activities observed (FIG. 4 and Table 10).

TABLE 10

ABSENCE OF ENDOTOXIN IN I_E WATER PREPARATIONS

Polymyxin B LPS (ng/ml) IE Water

10 μg/ml 10 3 1 Control 1:1 1:3 1:10 Medium

TNF-α 15229 8381 1860 430 14176 4649 1593

+ 9897 481 341 273 23870 9127 1395

% Inhibition (35%) (94%) (82%) (36%) none none (12%)

IL-12 2268 425 39 2405 1893 529

+ 25 6.3 5.0 2470 2061 371

% Inhibition (0.7%) (99%) (99%) (87%) none none (29.9%)

5.2 EXAMPLE 2 - IMMUNOSTIMULATION IN VIVO IN MICE DRINKING I_E

WATER COMPOSITIONS

In vitro studies have demonstrated that I_E H₂0 compositions stimulate peripheral blood to become activated and secrete key cytokines that mediate the immune response to foreign pathogens. Based on these findings, the inventors hypothesized that mice drinking I_E crystal water would exhibit the following: (1) the baseline immunological activity would be augmented; (2) mice immunized with a well-defined antigen would mount a more vigorous antibody and cell-mediated immune responses as compared to control mice; (3) mice infected with a microbial pathogen would be cured or reject a lethal dose as compared to control mice; and (4) genetically immune deficient mice would increase their basal immunity compared to controls. These studies were performed to assess these hypotheses.

5.2.1 EXPERIMENTAL DESIGN 5.2.1.1 AUGMENTATION OF BASELINE IMMUNE FUNCTION BY I_E WATER

Two highly inbred strains of mice (Balb/c and C57BC/6) were used. Groups (10 each) were fed either I_E water (American Technologies Group, Inc., Monrovia, CA [ATG]) or control water after weaning. The mice were kept until they reach the age of 6 weeks. Appearance, body weight, and size were recorded at weekly intervals. Two mice from each group were sacrificed at 6, 8, 10, and 12 weeks of age, and blood, spleen, and lymph nodes were collected. The peripheral blood was assessed for the number of circulating white and red blood cells, the levels of circulating albumin, transferin, immunoglobulin, complement, etc. Also, circulating cytokines were determined. The spleen and lymph node are analyzed for cell constitution (cell number of leukocytes and subset) and tested for in vitro immune response .and production of cytokines. The cells are stimulated with mitogens (T cell mitogen and B cell mitogen) and immune parameters are determined (proliferation, cytotoxicity, surface membrane receptor expression, cytokine synthesis, .and survival). 5.2.1.2 IN VIVO IMMUNE RESPONSE TO A WELL-DEFINED ANTIGEN

As described above, the two inbred strains of mice, at age 8 weeks old, were immunized with two agents: soluble antigen (diphtheria toxoid) and particulate alloantigen (allogeneic tumor: P815-x2 for C57BC/6 mice and EL-4 for Balb/c mice). The mice (3 in each group) were then sacrificed and bled, spleen and lymph nodes removed. The blood was then separated to obtain serum, and the serum was tested for circulating antibody specific against the antigens used. The spleen and lymph nodes were tested for T helper response in vitro and for cytotoxic T cell response. Cytokines produced were also determined in both the supernatants and cultures.

5.2.1.3 IN VIVO CONTROL OF A BACTERIAL INFECTION IN MICE

The same two groups of mice were also inoculated with different doses of S αureus (a Gram-positive organism) and S. typhimurium (a Gram-negative bacterium). The mice were monitored for survival and the sub-lethal dose of bacteria was established. Groups of mice (neonatally under I_E water and control water) were inoculated with 3 doses of bacteria: suboptimal, lethal dose, and superlethal. The mice were then monitored for (1) survival, (2) immunity against the bacteria (antibody and cell-mediated), (3) immunological memory, and (4) cytokine profiles.

5.2.1.4 DEVELOPMENT OF IMMUNITY IN IMMUNODEFICIENT MICE

The innate immune response was also tested in genetically immunosuppressed mice (nude mice, with no T or B lymphocytes) and SCID (severe combined immunodeficient mice). Mice were treated with sublethal doses of bacteria and then monitored for immunity by circulating NK cells, monocytes, and neutrophils. As above, in vitro cell-mediated immunity and cytokine production were also determined. 5.3 EXAMPLE 3 - EFFECT OF I_E CRYSTAL WATER ON THE MATURATION

AND DIFFERENTIATION OF HUMAN MONOCYTES AND MONOCYTIC LEUKEMIAS

The inventors have demonstrated that human peripheral blood monocytes cultured in I_E water undergo maturation and differentiation. The inventors hypothesize that the cells have been triggered externally and/or internally to initiate signaling events leading to differentiation. This example describes the investigation of intracellular and genetic molecular events which occur as a result of I_E water administration.

5.3.1 METHODS

To investigate the molecular events by which the monocytes undergo moφhological and biochemical changes, one may compare the molecular intracellular signaling pathways mediated by monocyte differentiation factors with the signaling mediated by I_E water compositions. This permits the delineation of the role of I_E water in the terminal differentiation of human monocyte leukemia. Such studies are also important in the investigation of malignancy of the differentiated cells in nude/SCID mice.

5.4 EXAMPLE 4 - EFFECT OF I_E WATER ON ENDOTHELIAL CELLS AND ITS

ROLE AS BARRIER FOR CELL INFILTRATION AT SITE OF INFECTION OR CANCER

Based on experimental studies, I_E water appears to modify the normal endothelial cell layer lining blood vessels. These modifications include changes in the surface expression of receptors (adhesion molecules, cytokine receptors) and the induction of cytokines and chemokines that will attract leukocytes. The permeability of the endothelial cells in the presence of I_E water apparently changes, to allow the crossing of leukocytes to site of inflammation. The increase in permeability and chemoattraction of leukocytes is important in fighting infections and cancer. The infiltrating cells become activated to destroy infection and cancer tissues. 5.4.1 METHODS

This study is performed in an in vitro model system using endothelial cells monolayers and double chambers. The top chamber consists of the endothelial monolayer and the bottom chamber consists of media. Cell penetration is determined by examining the presence and number of cells in the bottom of the filter separating the chambers and/or in the bottom chamber. Endothelial cells cultured in I_E water are examined for cell morphology changes, expression of surface receptors (by flow cytometry and by PCR™), expression of adhesion molecules and cytokine synthesis of cytokines and chemokines. Cells used for penetration consist of human peripheral blood leukocytes (separate monocytes, lymphocytes, and neutrophils). In addition, the effects of I_E water (drinking water or t.v. administered water) on the infiltration of leukocytes at sites of infection or implanted cancer may be examined in an in vivo animal model system.

5.5 EXAMPLE 5 - EFFECT OF I_E WATER ON THI AND TH2 CYTOKINES

The effect of water preparations on the secretion of THl and TH2 type cytokines by peripheral blood derived mononuclear cells (PBMC) have been investigated. In this study, the inventors used unfractionated PBMC from normal individuals, which are activated by PHA, LPS, superantigens (bacterial exotoxins) and cultured for various periods of time. Cell proliferation was determined by HTdR incoφoration, and the supernatants derived from the cultures were tested for the presence of the cytokines listed in Table 1 using conventional ELISA. Similar studies may also be performed as above except that purified subpopulations of lymphocytes {CD4 , CDS , T, NK) and macrophages may be used. Likewise, the regulation by I_E water of various cell surface markers (e.g.,

MHC class I and II, CD3, CD4, CD8, CD25, CD28, CD40, CD69 and others) for T lymphocytes and some additional markers for NK cells (CD 16, CD56, CD57, NKRPl) also represents important steps in identifying the uses for I_E water in biological systems. Since various cytokines interact with cell surface receptors, the expression of such receptors e.g., TNF-R1 (I and II), IL-2R, IFN-γR, IL-4R, IL-10R,

IL-12R .and IL-15R may also be examined in the presence and absence of I_E water. These markers are conveniently determined by flow cytometry. In some applications, binding studies using radioactive ligands may be performed to establish the affinity of binding by Scatchard analysis. Such a study may be performed in 2-3 blood cell purified subpopulations.

5.6 EXAMPLE 6 - RESPONSE OF TUMOR CELLS TO VARIOUS CYTOTOXIC DRUGS AND COMBINATION WITH I_E WATER COMPOSITIONS

The inventors have tested the sensitivity of tumor cell lines to various cytotoxic drugs (ActD, CDDP, ADR, VP-16, 5-FU) used alone or in combination with cytokines (TNF-α, IFN-α, IFN-γ) or cytotoxic antibodies (anti-FasR) (Borsellino et al, 1995; Mizutani et al, 1995; Mizutani et al, 1994; Mori et al, 1996; Morimoto et al, 1993; Safrit et al, 1992; Safrit et al, 1993a; Safrit et al, 1993b; Uslu and Bonavida, 1996).

Tumor cells resistance can be regulated at multiple levels including the expression of (a) drug resistant genes like MDR-1 and MRP; (b) the upregulation of anti-oxidant enzymes i.e. glutathione transferase; (c) the induction of factors/cytokines (TGF-β, IL-6, IL-10); (d) the modulation of surface receptors (IL- 6R, EGF-R, TGF-βR). These factors may be analyzed at the level of the protein and also at the level of gene transcription by RT-PCR™ for the effect of I_E water preparations used alone or in the presence of small concentrations of single or combination of drugs. Such studies are useful in determining the effects of I_E water compositions on the properties of tumor cells. The effects of I_E water on modulation of genes involved in various genes, both .anti-apoptotic (Bcl-2, FAP-1, RIP, c-myc, p53, etc.) and apoptotic genes (Bax, c-myc, p53) as well as transcription factors (e.g., NFKB), also represent important areas for exploitation of I_E water compositions.

5.7 EXAMPLE 7 - STUDIES TO RULE OUT THE EFFECT OF LPS-LIKE CONTAMINANTS IN I_E WATER

I_E water can stimulate cytokine production by peripheral blood mononuclear cells (PBMC). Also, cytokine production by I_E regulates THl and TH2 cytokine profiles. Production of IL-12 (THl) and IL-10 (TH2) by PBMC in medium containing laboratory water (water), I_E water (I_E 1 :2, 1 :4) has been examined and compared to the strong mitogenic stimulation by LPS (lipopolysaccharide at 2 concentrations 100 ng/ml and 10 ng/ml). Little secretion of IL-10 and IL-12 is detected by ELISA when medium is prepared with laboratory water (FIG. 6). In contrast, there was moderate secretion of IL-10 and significant secretions of IL-12 by medium containing I_E water. As expected, LPS stimulated both IL-10 and IL-12 by PBMC.

In order to determine that the I_E medium-stimulated IL-12 and IL-10 was not due to a lipopolysaccharide- (LPS) like contaminant, polymyxin B, an inhibitor of LPS, was used and the data were compared. The addition of polymyxin B inhibited

IL-10 secretion and inhibition was more pronounced with LPS concentrations of 10 ng/ml (FIG. 7). However, there was little inhibition by I_E medium. Likewise, the secretion of IL-12 by LPS was inhibited by polymyxin B. In contrast, the secretion of IL-12 by I_E water was not affected (FIG. 8). These results demonstrated that the stimulation of cytokine secretion by I_E water is not due to the presence of an LPS-endotoxin like contaminant. Had LPS contaminant been present in the water preparations, it would have been expected to be responsible in part of the I_E.mediated effects. These findings, however, indicate that the I_E-mediated effects were due to the physical properties of I_E water and/or other factors, and not upon the present of some contaminant in the water composition.

5.8 EXAMPLE 8 - EFFECT OF I_E WATER ON THE REGULATION OF CELL

MEMBRANE EXPRESSION OF RECEPTORS INVOLVED IN SIGNALING PHYSIOLOGICAL RESPONSES All cells in the body express on the cell surface a large number of molecules/receptors each of which is endowed with specific functions when triggered. Further, different types of tissues express selectively specific receptors that provide them with tissue specificity and selective types of responses when triggered. The homeostatic mechanisms and in particular the hormonal, neuronal, and immunological responses are all governed by the expression of specific receptors on the corresponding tissues. Thus, any regulation of the expression of such receptors on the cell surface will result in significant biological and molecular and physiological responses that will affect the whole homeostatic response. For example, the regulation of receptors like the major histocompatibility complex (MHC) antigen receptors on the surface can directly affect the overall immune response. The inventors examined whether I_E water-prepared medium can affect the expression of cell surface receptors on human peripheral blood-derived mononuclear cells. The expression of such receptors was determined by analysis of flow cytometry.

5.8.1 UPREGULATION OF THE EXPRESSION OF THE ACTIVATION MARKERS

CD69

CD69 is a dimeric surface membrane glycoprotein known as activation- inducer molecule. This activation antigen is the earliest appearing cell-surface glycoprotein after immune cells activation. It is expressed mainly on activated T, NK and B lymphocytes and activated macrophages and absent in resting cells. It marks immune activation.

The inventors analyzed the expression of CD69 and compared it to the positive control of upregulation of CD69 by LPS. As shown in Table 11, CD69 is upregulated by LPS and upregulation is dependent on the concentration of LPS used. The upregulation by LPS was shown by increase on the percentage of cells that are positive for CD69 (% Fluo) compared to laboratory medium control. Also, the mean fluorescence intensity and the degree of fluorescence intensity was upregulated (FIG. 9). In comparison, I_E medium also upregulated modestly the expression of CD69 as determined by all 3 parameters.

TABLE 11 EFFECT OF I_E WATER ON UPREGULATION OF CD69 EXPRESSION³

Control +I_E Water +I_E Water +LPS +LPS

Medium (1:4) (1:2) (10 ng/ml) (100 ng/ml)

% Fluor.⁰ 19.8 19.0 22.5 25.5 31.9

Me.an Fluor.^c 7.31 7.81 7.38 8.09 8.45

Fluor. Inten.^d 10.3 11.5 12.3 11.1 14.0

Flow cytometry at the single cell level.

Percent fluorescent cells. c Mean fluorescence. Fluorescence intensity.

These findings indicate that I_E water affects the cell membrane and regulate the expression of the activation marker CD69.

5.8.2 UPREGULATION OF THE LPS RECEPTOR TO CD14 BY I_E MEDIUM

CD 14 surface antigen is a single chain surface molecule membrane protein, expressed on monocytes and macrophages. It functions as a high affinity receptor for the complex lipopolysaccharide (LPS) and LPS-binding protein (LBP). It functions to stimulate the phagocytic system for microbial destruction.

The expression of CD 14 on PBMC by I_E water was examined and compared to the strong stimulation LPS (Table 12). Clearly shown in FIG. 10, the expression of CD 14 was significantly upregulated by I_E medium compared to laboratory control medium. The upregulation was significantly induced by I_E compared to the strong stimulus LPS.

TABLE 12 EFFECT OF I_E WATER ON UPREGULATION OF CD14^a

Control +I_E Water +I_E Water +LPS +LPS

Medium (1:4) (1:2) (10 ng/ml) (100 ng/ml)

% Fluor.⁰ 27.0 29.2 33.8 28.1 33.8

Mean Fluor.^c 45.8 64.0 65.5 80.2 83.1

Fluor. Inten. 60.3 89.9 93.5 108.6 1 15.7

Flow cytometry at the single cell level. Percent fluorescent cells. Mean fluorescence. Fluorescence intensity.

These findings are noteworthy as the receptor for LPS is important for regulating the interaction of bacteπa with phagocytic cells that result in their elimination That is, response to bacterial infection is in large part dependent on

CD 14 and therefore, the potentiation of CD 14 by I_E medium has important implications on host defense mechanisms against microbial infection

5.8.3 UPREGULATION OF CD8 ON CYTOTOXIC T LYMPHOCYTES BY I_E WATER CD8 antigen is a dimenc surface membrane protein expressed on a subset of T cells, pπmaπly the cytotoxic T cells. CD8 antigen acts in concert with the T cell receptor for activation by Class I MHC and foreign peptides. It activates the killing mechanisms of the lymphocytes.

The cell surface receptor CD8 is expressed on a subset of T lymphocytes, particularly those involved in destroying virally infected, bacterial infection, and cancerous cells.

As shown in Table 13, the I_E medium upregulated the expression of CD8 on T lymphocytes and LPS, which only activates B cells and monocytes, had no effect. These findings demonstrate that I_E medium can regulate the functional properties of cytotoxic CD8⁺ lymphocytes for killing of microorganisms and neoplastic cells TABLE 13 EFFECT OF I_Γ WATER ON UPREGULATION OF CD8 RECEPTOR EXPRESSION³

Control +I_E Water +I_E Water +LPS

Medium (1:4) (1:2) (50 ng/ml)

CD8 %^ϋ 20.6 19.6 19.0 20.8

Mean^c 30.8 35.0 37.6 34.9

Intensity 81.8 94.5 96.5 91.0 ^aFlow cytometry at the single cell level Percent fluorescent cells. ^cMean fluorescence.

Fluorescence intensity

5.8.4 FAILURE OF I_E WATER TO UPREGLLATE OTHER CELL SURFACE

RECEPTORS Human leukocyte antigen (HLA) Class I is a cell surface protein expressed on the surface of all human cell types HLA Class I represents one of the most important cell surface proteins which distinguishes one person from another It is responsible for immune activation of killer cells

Human Leukocyte Antigen (HCL) Class II is a cell surface protein complex expressed by immune competent cells, particularly the antigen-presenting cells. It is important in presenting foreign antigens to the immune system

Some receptors are not upregulated by I_E medium For instance, the expression of Class I MHC. Class II MHC, C16 and CD3 were not affected by I_E medium (Table 14) These findings indicate that I_E medium is not a non-specific modulation of cell surface expression of receptors but retain selective effects These data also suggest that I_F mediated-effects regulate gene transcription in a very specific way. TABLE 14 FAILURE OF I_E WATER TO REGULATE CD16, CD3, CLASS I AND II MHC

Control +I_E Water +I_E Water +LPS Medium (1:4) (1:2) (50 ng/ml)

CD16 %^a 6.0 5.3 4.5 5.2

Mean ^b 9.8 9.7 9.7 8.8

Intensity⁰ 13.0 12.1 12.4 12.4

CD3 % 79 81 81 83

Mean 51.8 53.0 55.8 47.9

Intensity 68.8 68.5 74.0 59.3

MHC II % 67.1 52.3 58.9

Mean 27.1 26.6 27.0

Intensity 39.4 38.0 43.7

MHC I % 99.8 99.2 99.3

Mean 50.4 47.7 43.9

Intensity 62.3 66.4 63.4

Percent fluorescent cells. Mean fluorescence. ^cFluorescence intensity.

5.8.5 UPREGULATION OF CHEMOKINES SECRETION BY I_E WATER

Chemokines are molecules involved in attracting various types of immune cells to sites of action. MCP-1 (monocyte chemotactic protein- 1), MIP-1 alpha (macrophage inflammatory protein- 1 alpha), and RANTES (regulated upon activation, normal T cells expressed and secreted). These chemokines are chemotactic for monocytes, lymphocytes, dendritic cells, eosinophils, and basophils. They are secreted by various types of cells. Involved in the regulation of THl and TH2 types of immune responses. The inventors examined, based on the demonstration that I_E water triggers cytokine secretion by PBMC, whether it also affects the secretion of other family mediators involved in cell-cell sites. Representative chemokines were studies such as MCP-1, MlP-α, and RANTES. As clearly shown in Table 15, all the chemokines are significantly induced by I_E water, over 50 fold background levels with laboratory control water. Noteworthy, the stimulation was comparable to the most potent activation of chemokine products by LPS.

These findings emphasize the role of I_E water in the positive regulation of host immune defense mechanisms.

TABLE 15 STIMULATION OF CHEMOKINES BY I_E WATER

Sample MCP-1 MlP-α RANTES (pg/ml)^a

Control Medium 63 0 1800

+ I_E Water (1 :2) 4,000 1 ,100 2,300

+LPS (10 ng/ml) 3,700 2,460 2,200 ^aChemokines measured by ELISA.

The stimulation of chemokines by I_E water was determined by incubating human peripheral blood mononuclear cells in the presence of I_E water and control water. Following overnight incubation, the supernatants were harvested and tested for chemokines by ELISA.

5.9 EXAMPLE 9 - EFFECT OF I_E WATER ON THE REGULATION OF

INTERACTION BETWEEN LYMPHOCYTES AND TUMOR TARGETS The inventors examined whether I_E water, which regulates the expression of various receptors on cell surface membranes, could also influence the adhesion of lymphocytes to corresponding targets (infected cells, tumor cells, etc.). The inventors examined the interaction of lymphocytes to tumor cells by examining, at the single cell level by flow cytometry, the frequency of lymphocytes-target conjugates (Bonavida et al., 1993; Lebow et al., 1990). As shown in Table 16, there is a clear augmentation of the frequency of the conjugates in I_E water-containing medium compared to laboratory (or control) water-containing medium.

TABLE 16 FREQUENCY OF LYMPHOCYTE-TUMOR TARGET CONJUGATES

Control H^O I_E Water (1:2) +LPS (10 μg/ml)

Study 1

Conjugates 687/7944 807/7700 (10.48) 689/7946 (8.67) Changes +21.16% +0.23% Study 2

Conjugates 552/14003 (3.942) 620/14095 (4.40) 453/13973 (3.243) Changes + 1 1.68% -17.77%

These findings indicate that I_E water influences the interaction between lymphocytes and tumor targets and thus increasing the ability of the lymphocytes to kill the tumor cells. The above findings reveal new and highly important properties of I_E water in modulating the expression of cell surface receptors on various types of cells and tissues. These activities are significant for all aspects of cell biology and have significant implications in host responses to internal and external factors and also in the regulation of many homeostatic mechanisms. The peripheral blood mononuclear cells were treated with I_E water overnight. Thereafter, the blood cells were mixed with tumor target cells (effectoπtarget 1 :2) for 30 minutes and the frequency of lymphocyte-tumor target doublets (conjugates) were enumerated by flow cytometry.

6.0 REFERENCES

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All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

CLAIMS:

1. A method of altering cytokine activity in an animal cell, comprising providing to said cell an amount of an I_E water composition effective to alter cytokine activity in said cell.

2. The method of claim 1, wherein said animal cell is a mammalian cell.

3. The method of claim 2, wherein said mammalian cell is a human cell.

4. The method of claim 3, wherein said human cell is selected from the group consisting of a bone marrow cell, a pluripotent stem cell, a blood progenitor cell, a blood cell, a brain cell, an aging cell, a cancer cell, and a tumor cell.

5. The method of claim 1, wherein said cytokine is selected from the group consisting of IFN-╬│, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TNF-╬▒, TNF-╬▓, IL-15, IL-18, GM-CSF, CSF, SCF, Fas-R, Fas-L, TRAIL, and TRAIL receptor.

The method of claim 5, wherein said cytokine is selected from the group consisting of IFN-╬│, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TNF-╬▒, TNF-╬▓, IL-15, and IL-18.

7. The method of claim 1, wherein said composition further comprises an immunomodulating agent.

8. The method of claim 7, wherein said immunomodulating agent is selected from the group consisting of a mitogen, a cytokine, an antigen, a chemotherapeutic agent, a polypeptide, a polynucleotide, and an antibody.

9. A method of increasing the concentration of a cytokine in an animal cell, comprising providing to said cell an amount of an I_E water composition effective to increase the concentration of said cytokine in said cell.

10. A method of increasing the amount of a cytokine secreted from an animal cell, comprising contacting said cell with an I_E water composition in an amount effective to increase the amount of said cytokine secreted from said cell.

11. The method of claim 10, wherein said cell is a peripheral blood monocyte.

12. A method of increasing the rate of secretion of a cytokine from an animal cell, comprising contacting said cell with an I_E water composition in an amount effective to increase the rate of secretion of said cytokine from said cell.

13. The method of claim 12, wherein said cell is a peripheral blood monocyte.

14. A method of stimulating a mammalian blood cell to produce a cytokine, comprising contacting said cell with an amount of an I_E water composition effective to stimulate said cell to produce said cytokine.

15. The method of claim 14, wherein said cytokine is selected from the group consisting of IFN-╬│, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TNF-╬▒, TNF-╬▓, IL-15, IL-18, GM-CSF, CSF, SCF, Fas-R, Fas-L, TRAIL, and TRAIL receptor.

16. The method of claim 4, wherein said cell is comprised with an animal and said cell is contacted with said composition by administering said composition to said animal.

17. The method of claim 16, wherein said composition is administered to said animal orally, subcutaneously, intramuscularly, or intravenously.

18. A method of producing an immune response in an animal, comprising administering to said animal an amount of an I_E water composition effective to produce said response in said animal.

19. The method of claim 18, wherein said I_E water composition further comprises a polynucleotide or a polypeptide.

20. A method of inducing differentiation in an animal cell, comprising administering to said cell an amount of an I_E water composition effective to induce differentiation in said cell.

21. A method of treating an autoimmune disease in an animal, comprising administering to an animal suspected of having an autoimmune disease; an amount of an I_E water composition sufficient to treat said autoimmune disease in said animal.

22. The method of claim 21, further comprising administering to said animal an immunomodulator or an immunosuppressant.

23. The method of claim 22, wherein said autoimmune disease is selected from the group consisting of psoriasis, lupus, Sjogren's syndrome, rheumatoid arthritis, ulcerative colitis, Crohn's disease, sympathetic ophthalmia, myasthenia gravis, multiple sclerosis, orchitis, and osteomyelitis.

24. A method of treating cancer in a mammal, comprising administering to an animal suspected of having a cancer an amount of an I_E water composition effective to treat said cancer.

25. The method of claim 24, wherein said cancer is selected from the group consisting of leukemia, sarcoma, melanoma, glioblastoma, prostate cancer, ovarian cancer, lung cancer, and colon cancer.

26. The method of claim 24, wherein said composition further comprises an anticancer agent.

27. The method of claim 26, wherein said anticancer agent is selected from the group consisting of cisplatin, adriamycin, doxorubicin, etoposide, camptothecin, Actinomycin D, cyclophosphamide, and 5-fluorouracil.

28. A therapeutic kit comprising, in suitable container means, a therapeutically- effective amount of an I_E water composition.

29. The kit of claim 28, further comprising a pharmaceutically-acceptable excipient.

30. The kit of claim 28, further comprising an immunomodulating agent.

31. The kit of claim 28, further comprising an immunosuppressive agent.

32. The kit of claim 28, further comprising a cytokine.

33. The kit of claim 28, further comprising an anticancer agent.

34. The kit of claim 28, further comprising a polynucleotide, a polypeptide, a carbohydrate, or a lipid.

35. The kit of claim 28, further comprising a liposome or a nanocapsule.