WO2000012134A1 - Composition traitee par voie electrique et utilisation d'une telle composition - Google Patents

Composition traitee par voie electrique et utilisation d'une telle composition Download PDF

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Publication number
WO2000012134A1
WO2000012134A1 PCT/DK1999/000460 DK9900460W WO0012134A1 WO 2000012134 A1 WO2000012134 A1 WO 2000012134A1 DK 9900460 W DK9900460 W DK 9900460W WO 0012134 A1 WO0012134 A1 WO 0012134A1
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Prior art keywords
composition according
individual
composition
prophylactically
cell
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PCT/DK1999/000460
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English (en)
Inventor
John F. Wetling
Arsalan Kharazmi
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Wetling John F
Arsalan Kharazmi
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Application filed by Wetling John F, Arsalan Kharazmi filed Critical Wetling John F
Priority to AU54080/99A priority Critical patent/AU5408099A/en
Publication of WO2000012134A1 publication Critical patent/WO2000012134A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0004—Homeopathy; Vitalisation; Resonance; Dynamisation, e.g. esoteric applications; Oxygenation of blood

Definitions

  • the invention relates to an electrically treated or affected composition capable of being used in a method for therapeutic treatment of a human being or an animal.
  • the electrically treated composition is particularly useful in suppressing the secretion of histamine from mast cells and, thus, represents a new form of asthma treatment.
  • a composition such as a liquid optionally comprising positive and/or negative charged ions can be subjected to a current or an electrical treatment by for example contacting the liquid with a positive and a negative electrode and connecting the electrodes to a power source, which connection provides a migration of charged particles, preferably ions, in the said liquid.
  • the electrically treated composition has a number of different uses, for example within the field of cell biology.
  • Mast cells are present in connected tissue and epithelia in particularly lungs, the gut and skin. Mast cells are natural components in the immunity defence of the body along with for example macrophages, neutrofile, eosinofile and basofile leukocytes along with B lymphocytes and T lymphocytes.
  • Mast cells contain a cell nucleus surrounded by various organeiles and exist as single cells in a mammal organism such as a human being or an animal.
  • the mast ceil is the presence of a very large number of small and closely packed granula in the cytoplasm of the cell. When the mast cell is stimulated, these granula are secreted by exocytosis. The influence of the mast cell on the inflammation reactions is thought to be due to their ability to form and secrete a number of biologically active compounds which together are termed mediators.
  • mast cells secrete the mediator histamine under for example an allergic reaction. Histamine provides for example contractions in the airways and lungs under an asthma attack. Histamine is one of the only compounds which are common for all types of mast cells.
  • the mast cell is secreting histamine, the result is among other things an increase in the intracellular pH, a change in the membrane potential and a changes ion transport, such as for example an increase in the Na7K + pump activity.
  • Histamine is synthesised from histidine and is stored in the granula of the mast cell in a complex comprising proteoglycans (Warner & Kroegel, 1994). Histamine exerts its effect via H ⁇ H 2 and H 3 receptors. Stimulation of Hi receptors leads to an increased permeability in veins, contraction of smooth muscles, kemotaxis of neutro- files and eosinofiles as well an increased production of prostaglandines. H 2 receptor-stimulation results in stomach acid secretion, increased vein permeability, increased T-cell suppresser function, inhibition of IgE-mediated basofil histamine se- cretion, and decreasing neutrofil and eosinofil chemotaxis.
  • H 3 receptors are present in the central nerve system and in the lungs, and it is believed that H 3 receptors participate in controlling hi histamine formation and secretion via negative feedback (Rothe et al., 1990).
  • the mast cell In some individuals, the mast cell "over-react" which provide asthma and allergies (Redington et al., 1995).
  • the mast cell is, thus, involved in anaphylactic shock which comprises secretion of substantial amounts of heparin and histamine to the serum. It is known that these and other biological active compounds which are con- tained in the granula of the mast cells to a certain degree is responsible to the symptoms that are observed in allergic reactions.
  • Asthma is used herein as defined as an airway suffering characterised by an increased bronchial activity from a stimulus resulting in spasm and/or inflammation of the bronchial wall and a subsequent reduced access of air to the lungs.
  • Bronchitis is ⁇ used herein as defined as an acute or chronic inflammation of any part of the bronchus or the bronchial airways.
  • Allergy is used herein is defined as any way of reacting towards an allergen, typically hypersensitivity, after antigen exposure.
  • Hypersensitivity comprises a significant increased histamine secretion after exposure to an antigen.
  • Type 1 allergy is characterised by degranulation of the mast cell in response to a cross binding between a suitable allergen and immunoglobulin molecules of the E type (IgE), which are present on receptors on the surface of the mast cell.
  • IgE immunoglobulin molecules of the E type
  • pHi intra-cellular pH
  • Nuclear Magnetic Resonance is another method for measuring intracellular pH.
  • An inorganic phosphate signal is used since it is easy to observe the 31 P spectrum, and because this frequency is particularly sensitive to pH in the neutral area.
  • a further method comprises use of pH sensitive micro electrodes, which penetrates the plasmamembrane, which allows monitoring pH changes over time. By using this method, compartmentalisation of the tracer is not a problem. However, one does risk that the cells become damaged when the electrode is introduces through the plasma membrane. The method is, therefore less well-suited for small cells.
  • Fluorescent compounds with pH dependent fluorescent in the physiological area are very useful for examination of regulatory mechanisms and measuring intracellular pH.
  • One preferred probe is 2',7'-bis(2-carboxyethyl)-5-carboxyfluorescein acetoxymehylester (BCECF-AM).
  • BCECF-AM is introduced in the cell as a liquid soluble ester, which is cleaved by esterases in the cytoplasm (Rink et al., 1982; Moolenaar et al., 1983).
  • the fluorescent can be monitored over time and it is, therefore, possible to monitor pH changes over time.
  • the three most important pHi regulatory mechanisms are i) a Na + /H + transporter, ii) a Na + -dependent CI7HCO 3 " transporter, which are both activated by low pHi and are acid secretion mechanisms under physiological conditions, along with iii) a Na + independent CI7HCO 3 transporter, which is activated by high pH and at physiological ion gradients will lead to a reduction of intracellular pH (Thomas,
  • the electrical treatment or affect of a fluid, water-based composition will result in cell- stabilising affect and in a preferred embodiment provide a cell characterised by having a predetermined resting membrane potential or a predetermined resting pHi o and/or a predetermined steady-state resting membrane potential or a predetermined steady-state resting pHi, or a cell capable of entering a condition characterised by maintaining a characteristic resting membrane potential for the respective cell and/or a resting pHi or a characteristic steady-state resting membrane potential for the respective cell and/or steady-state resting pHi.
  • the properties of the electrically treated or affected fluid composition is achieved when a reaction between the fluid composition and the electrodes with which the composition is contacted sets in.
  • the reaction preferably occurs, however, not limited to occur, when the electrodes are having an electric potential.
  • the electrodes having the electric potential will result in a current of charged particles, preferably ions, migrating in the fluid composition, reducing or increasing the amount of gasses in said composition, or reducing or increasing the solubility of the respective gasses or the amount of dissolved gas in the liquid.
  • concentration of ions or the concentration or presence of certain species of ions in the composition is changed as a consequence of the fluid composition being contacted by a current of charged particles, preferably ions.
  • a fluid composition such as an aqueous composition or any other liquids to change not only the concentration of salt, but also the concentration of a gas in a liquid.
  • Increasing concentrations of CO 2 have for example turned out to inhibit the compound 48/80 mediated secretion of histamine independent of pHi.
  • the same effect is observed if a cell liquid is subjected to a current of about 1.5 ⁇ A and subsequently supplemented with mast cells.
  • Compound 48/80 mediated secretion of histamine from the mast cells is also in this case reduced significantly.
  • the reduction is due to cell stabilising effect that provides cells capable of easier or more rapidly entering a condition characterised by maintenance of a characteristic resting membrane potential for the respective cell and/or a resting pHi or a characteristic steady-state resting membrane potential for the respective cell and/or steady state resting pHi.
  • a composition which has been treated with an electrical current or subjected to a ionisation. It has now very surprisingly and unexpectedly turned out that a composition, such as for example liquid, which has been subjected to an electrical current or a ion treatment, is an effective means for treatment of an individual suffering from for example asthma, bronchitis and/or an allergy.
  • the invention relates to an electrically treated composition for use as a medicament.
  • the invention relates to an electrically treated composition for use in a method for therapeutic or surgical treatment, or a method of diagnostics, of a human being or an animal.
  • an electrically treated composition in the manufacture of a medicament for treating a suffering of a human being or an animal.
  • composition obtainable by this method.
  • the electrically treated composition according to the invention is, thus, capable of i) reducing the amount of secreted histamine and/or ii) reducing the increase in intracellular pH associated with the secretion of histamine and/or iii) reducing the increase in the Na7K + pump activity associated with the secretion of histamine.
  • the composition is, furthermore, capable of counter-acting changes in the membrane potential in a cell during the secretion of histamine.
  • the invention provides an anti microbial means, preferably, but not limited to, an anti bacterial means, effective in eliminating bacteria, preferably, but not limited to, pathogenic bacteria, or capable of inhibiting the growth of such bacteria in an environment wherein they occur.
  • a ground-connection is applied to a suitable part of the body, like the wrists, and the ionized air is directed to the area to be treated, like the soles of the feet.
  • the experimental chamber is a metallic cylindrical container A with an inner radius R.
  • a cylindrical electrode B with the radius r is placed in the axis of A and submerged in the liquid to be treated.
  • the length of B in the liquid is h and it is assumed that the distance from the end of B to the bottom of
  • A is much smaller than h. From a voltage supply V a current I is sent through the liquid causing a current density j x at a distance x from the axis which by a good approxima-tion is given by
  • the total energy dissipated in the liquid per unit time (the power) is given by
  • V(J 2 ) m 3.63-10- 4 A-m ,-- 2 :
  • a method of providing a composition with a predetermined property said property being obtainable by contacting said composition with a charged particle, preferably a ion.
  • the predetermined property is preferably essentially sustainable over time.
  • the composition is transiently contacted by said charged particle. It is also preferred that the predetermined property is essentially sustainable over time, and that the composition is transiently contacted by said charged particle.
  • the predetermined property remains associated with the composition for a period of time, the period is at least essentially the same as the period of time in which the composition is contacted by said charged particle.
  • the predetermined property is essentially sustainable over time irrespective of the period of time in which said composition is contacted by said charged particle.
  • the predetermined property is preferably sustainable for at least one week, such as one month, for example six months, for example one year, such as two years, for example five years, such as ten years.
  • the contacting of said composition by a charged particle results in the pas- sage of a current through said composition
  • said current corresponds to at least about 0.01 ⁇ A and preferably less than about 1.5 A.
  • the current may also result in a charge being provided to said composition, said charge being at least about 0.01 mC and preferably less than 10000 mC.
  • the composition comprising said predetermined property is a medicament.
  • composition according to the invention is obtainable by any of the methods described herein immediately above.
  • the composition in one embodiment is for use as a medicament.
  • a composition for use in a method for treatment of the human body by surgery a composition for use in a method for treatment of the human body by therapy, and a composition for use in a diagnostic method practised on the human or animal body.
  • composition according to the invention is for use in a method of prophylactically or therapeutically treating an allergic condition in an individual.
  • the composition may also be used in a diagnostic method capable of diagnosing an allergic condition in an individual, preferably rhinitis, urticaria, allergic conjunctivitis, and an allergic condition occurring in the airways of said individual, such as asthma.
  • the composition may also be used in a method of prophylactically or therapeutically treating an infection in an individual, and it may be used in a diagnostic method capable of diagnosing an infection in an individual.
  • the infection may reside anywhere, but treatment of infections occurring in the airways of said individual are responding particularly well to treatment.
  • On such form of infection is an infection associated with an obstructive disease of the airways, said disease may be sporadically, periodically or chronically occurring.
  • the infection may, prior to treatment, result in an increased mucous production.
  • One such form of infection is bronchitis.
  • composition according to the invention is also for use in a method of treatment of a human being or an animal, said method being capable of reducing or eliminating the secretion of histamin from mast cells.
  • the composition in one embodiment is capable of controlling the intracellular pH value of a human or animal cell and/or capable of reducing the increase in the intracellular pH-value in a human or animal cell during an allergic attack.
  • composition can also be used in a method of prophylactically treating in a human or an animal body a cell at risk of developing cancer, or for therapeutically treating a cancer cell, as well as prophylactically or therapeutically treating a cardio- vascular disease in an individual.
  • composition is also useful in a method of diagnosing an immunodefficient condition in an individual, in a method of prophylactically or therapeutically treating a rheumatic condition in an individual, a method of prophylactically or therapeutically treating a lesion to the skin including burns in an individual, a method of prophylactically or therapeutically treating a disease to the skin in an individual, a method of prophylactically or therapeutically treating eczema, a fungus infection, a bacterial infection, exanthema, or psoriasis in an individual, as well as being useful in a method of boosting the immune response of a human or animal cell.
  • composition according to the invention in the manufacture of a medicament for the treatment of a condition or illness in a human or animal in need of said treatment.
  • the treatment may comprise prophylactically or therapeutically treating an allergic condition in an individual, diagnosis of an allergic condition in an individual, prophylactically or therapeutically treating an infection in an individual, or diagnosing such an infection in an individual.
  • the use of said medicament may also be directed to prophylactically or therapeutically treating a cardiovascular disease in an individual, aimed at diagnosing an im- munodefficient condition in an individual, prophylactically or therapeutically treating a rheumatic condition in an individual, prophylactically or therapeutically treating a lesion to the skin including burns in an individual, prophylactically or therapeutically treating a disease to the skin in an individual, prophylactically or therapeutically treating eczema, a fungus infection, a bacterial infection, exanthema, or psoriasis in an individual, as well as boosting the immune response of a human or animal cell.
  • compositions according to the invention may take place by contacting the composition with other atmospheric ions or with electrodes having an electrical potential.
  • the composition is provided with properties that are useful for example in connection with a therapeutic treatment of a human being or an animal.
  • the composition may also be a compound in powder form or a solid matter inclusive an amorphous material.
  • the composition comprises at least one chemical compound and a carrier, preferably a physiologically and/or pharmaceutically acceptable carrier. The useful effect and the nature of this is not yet known in all details.
  • the method of treating a composition in order to obtain the useful effects described herein can advantageously be used to show the effect on for example secretion of histamine in mast cells and monitor the development in the intracellular pH value under for example an allergic attack in a human being or an animal.
  • the method according to the invention also relates to a method of manufacturing any composition capable of being subjected to an electrical treatment and subsequently shown to have a useful effect, i.e. an effect capable of reducing the secretion of histamine from mast cells under conditions comparable to an allergic or an asthma attack.
  • the mast cells are provoked to secrete histamine, and it is possible by means of chemically manufactured secretory active compound, called compound 48/80. to simulate the secretory process that take place in the body, when a mast ceil is secreting histamine. By adding different concentrations of compound 48/80. it is possible to affect mast cells from rats to secret histamine in various amounts. This provides an opportunity for examining the connection between the form of treatment and histamine secretion.
  • the parameters indicated herein below are for guidance only and are not a condition for achieving the results described herein.
  • the parameters may vary from for example very small units to very large vessels and tanks.
  • the electrical current, electrical potential and time indicated herein below are not a condition for achieving described results.
  • the electrical current, electrical potential and time may be varied and it is possible to achieve different effects. The person skilled in the art will know how electrical current, electrical potential and time may be varied in consideration of the terms described herein and in the examples.
  • a plastic bowl having an internal diameter of 7.25 cm and a height of 3.75 cm.
  • a copper plate (diameter 7.25 cm) is placed, which is connected to a neutral electrode via a sensitive electrical measuring instrument so that the total electrical current through the liquid can be measured.
  • 32 ml of a composition is applied to the bowl and the content of the bowl is treated with an electrical current or with atmospheric ions.
  • Treatment with atmospheric ions involves providing a ion generator, optionally in combination with a blower, above the liquid, and indicated in Fig. 3.
  • the ion generator is supplied with a neutral connection. Ions generated by the ion generator will hit the surface of the liquid and cause an electrical current in the liquid.
  • the electrical current can be measured by an electrometer.
  • a copper electrode (7.25 cm in diameter) is placed in contact with the surface of the liquid. This electrode is connected to one terminal of a generator providing a constant current. Said second terminal of said generator being connected to neutral, cf. Fig. 4. Also in this case, the electrometer will monitor the electrical current in the composition.
  • the composition such as a liquid, is subjected to an electrical current of about 1.5 ⁇ A in 1 hour, so that a total amount of charge of about 5.4 mC is transferred.
  • the composition is subjected to an electrical current of essentially the same intensity from the ion generator.
  • an electrically treated composition for use as an medicament and a electrically treated composition for use in a method of therapeutic or surgical treatment or a method of diagnostics of the human being or an animal preferably comprises a prophylactic or therapeutic treatment, or diagnostic, of an allergic condition, preferably an allergic condition occurring in the airways, such as for example asthma.
  • the method also comprises prophylactic or therapeutic treatment, or diagnostic, of a condition of infection in the airways, such as an obstructive airways disease.
  • the allergic and/or infective condition may either be sporadically or periodically or chronically occurring and optionally also connected with or may lead to an increased mucoid mucous production. In one particular case, the condition is bronchitis.
  • compositions for use in a method capable of effectively reducing or eliminating secretion of histamine from master cells are, furthermore, effective in controlling the intracellular pH value in a cell and preferably also effective in reducing the increase in the intracellular pH value in a cell during a allergic attack.
  • compositions for use in a method for prophylactic or therapeutic treatment, or for diagnostics, of a cancer cell, of a cardio vascular disease, of an immunodeficient condition, of rheumatic sufferings, muscle sufferings and tendon sufferings and for therapeutic treatment of skin lesions such as burns.
  • compositions for use in a method for prophylactically or therapeutically treating, or for diagnosing, stressed and/or sick cells are also capable of being used in a method for prophylactically or therapeutically treating, or diagnosing, skin disease, and there is also provided a composition for use in a method for prophylactically or therapeutically treating eczema, fungus infection, bacteria infection, a rash or psoriasis.
  • the composition according to the invention can also be used in a method of increasing the ability of a cell to counter act a bacterial or viral infection of the cell.
  • composition according to the invention is, furthermore, in one particular preferred embodiment capable of controlling cellular activities mediated by H ⁇ H 2 and H 3 receptors.
  • the composition is, thus, capable of controlling a H T receptor mediated permeability of veins and/or a Hi receptor mediated contraction of smooth muscles and/or a Hi receptor mediated kemotaxis of neutrofiies and eosinofiies and/or ⁇ receptor mediated production of prostaglandines.
  • the composition is also capable of controlling a H 2 receptor mediated stimulation of secretion of stomach acid and/or a H 2 receptor mediated H 2 receptor permeability of veins and/or a H 2 mediated T-cell suppresser function and/or a H 2 receptor mediated inhibition of IgE mediated basofile histamine secretion and/or a H 2 receptor medi- ated neutrophilic and/or eosinophilic chemotaxis.
  • the composition in one particularly preferred embodiment is also capable of controlling a H 3 receptor mediated formation of histamine and secretion thereof in the airways or lungs.
  • the electrically treated composition is suitable for controlling the pHi homeostasis of a cell.
  • the pHi homeostasis of a cell as used herein is defined as the mobile equilibrium condition in the intracellular pH, which provides the cell with an ability to sustain external changes. According, there exists a connection between intracellular pH and the mechanisms mediating the transport of ions across the cell membrane.
  • Physiological conditions as used herein is defined as the conditions that must be present before a cell can enter homeostasis defined as the movable equilibrium condition of the cell, when it is in a natural environment under maintenance of the for the respective cell characteristic resting membrane potential and/or resting pHi or a for the respective cell characteristic steady-state resting membrane potential and/or steady-state resting pHi.
  • a natural environment as used herein is defined as the environment surrounding the cell when this is present in an intracellular solution or in an extra-cellular solution, preferably an extra-cellular solution selected from the group consisting of an interstitial solution, a plasma solution and a trans-cellular solution. These solutions preferably have a composition as indicated in Mainz (1997): Biokemi (Munksgaard, Copenhagen).
  • the respective cell it is not required that the respective cell always will be able to enter into a condition characterised by maintenance of a for the respective cell characteristic resting membrane potential and/or resting pHi or a for the respective cell characteristic steady-state resting membrane potential and/or steady-state resting pHi.
  • a non-healthy cell such as a cancer cell, or a cell infected by a foreign body, such as a virulent virus, or a hyper-secreting mast cell
  • these cells will in one embodiment of the invention be capable of being examined under physiological conditions as described above without this necessarily leading to entry into the above-mentioned characteristic resting membrane potential and/or resting pHi or a steady-state thereof.
  • a cell thus, enters the condition of homeostasis defined as a movable equilibrium that enables a cell to maintain - under physiological conditions as defined herein above - a for the respective cell characteristic resting membrane potential and/or resting pHi, or a for the respective cell characteristic steady-state resting membrane potential and/or steady-state resting pHi.
  • a cell according to the invention will be a cell free from disease or a healthy cell.
  • the composition it is possible to administer the composition to an organism, preferably a human being or an animal, and subsequently promote the formation of or the maintenance of cells free from disease or healthy cells as defined herein above.
  • ceils free from disease or healthy cells are formed or maintained by control of a ion transport mechanism, which the composition according to the invention is capable of exerting.
  • Ion transport as used herein will preferably be understood as the transport of a ion over a biological membrane, preferably a cell membrane, such as for example a plasma membrane.
  • composition according to the invention capable of controlling a Na7H + transporter without exerting any influence on the membrane potential of the cell, said control resulting in the maintenance of and/or re-establishment of the pHi homeostasis of a cell.
  • a composition according to the invention capable of controlling a Na7H + transporter and a bicarbonate dependent transport mechanism, said control resulting in the maintenance of and/or re-establishment of the pHi homeostasis of a cell.
  • composition according to the invention capable of controlling a Na + independent CI7HCO 3 transporter, said control resulting in maintenance and/or re-establishment of the pHi homeostasis of a cell.
  • composition according to the invention capable of controlling a Na + dependent CI7HCO 3 transporter, said control resulting in maintenance and/or re-establishment of the pHi homeostasis of a cell.
  • composition according to the invention capable of controlling a Na7(HCO 3 " ) 3 cotransporter, said control resulting in maintenance and/or re-establishment of the pHi homeostasis of a cell.
  • the invention is not limited to regulation of the herein above mentioned ion transport mechanisms.
  • Other ion transport mechanisms which does not directly transport acid or base, but are involved in a secondary way in the regulation of pHi by adding or removing ions, and in doing so, are capable of altering ion gradients, are also comprised by the invention.
  • a composition capable of controlling a Na + /H + transporter without affecting the cell membrane potential and/or a Na7H + transporter in combination with a bicarbonate dependent transport mechanism and/or a Na + independent CI7HCO 3 transporter, and/or a Na + dependent CI7HCO 3 transporter, and/or a Na7(HCO 3 " ) 3 cotransporter, said control resulting in maintenance and/or re-establishment of the pHi homeostasis of a cell.
  • the use of the composition according to the invention will result in the maintenance or re- establishment of the pHi homeostasis of a cell, which result in naturally occurring cells in a human being or an animal can remain essentially healthy under conditions, such as for example a pathogen infection, which would otherwise have resulted in the conversion of naturally occurring healthy cells or cells free from disease to unhealthy cells.
  • the composition according to the invention will in one preferred embodiment result in a number of naturally occurring healthy cells in a human being or an animal, being maintained essentially unchanged or increased for example an infection with a virus or a pathogen organism.
  • the pathogen infection according to the invention is preferably an infection caused by a micro-organism, preferably a micro-organism selected from the group consisting of a filamentous fungus, a yeast, a bacteria, and a virus.
  • a micro-organism selected from the group consisting of a filamentous fungus, a yeast, a bacteria, and a virus.
  • the unchanged or increased amount of naturally occurring healthy cells will contribute considerably to the ability of the organism to resist infection or cell changes.
  • a composition according to the invention for use an anti-microbial means and/or an anti-microbial means comprising an electrically treated composition said composition being provided by a method comprising the steps of contacting the composition with ions generated by a ion generator or by an electrical current of charged particles, preferably ions, generated by electrodes having an electrical potential and in contact with a power source.
  • a method of eliminating unwanted micro-organisms, such as bacteria in a medium said method comprising treating the medium with charged particles, preferably ions, such as an electrical current of charged particles, for example ions.
  • the composition is preferably a fluid composition, such as for example a liquid or an aqueous solution, preferably, however, not limited to, water, such as corporation water or ordinary tapped water, an aqueous salt solution, such as for example physiological selin (0.9% NaCI), demineralised water, Millipore water, ion exchanged water or water filtrated though a filter of for example active charcoal.
  • the charged particles, preferably ions, such as for example an electrical current preferably provides the fluid composition with a charge of from about 5.4 mC to about. 23.8 mC per about 32 ml of the fluid composition.
  • the anti-microbial means is in one embodiment effective as an anti-bacterial means, since the means eliminate bacteria or inhibit the growth of bacteria, such as bacteria capable of causing disease, including pathogen bacteria in a certain environment, for example a human being or an animal, or an eatable or drinkable product for human or animal consumption, said product optionally being processed or conditioned prior to intake.
  • the product is a meat product, such as for example meat from an ox, a calf, a pig, a chicken, a duck, a turkey, an ostrich, a poultry product such as an egg, or a dairy product such as milk.
  • the milk is treated or affected with a current of charged particles, preferably ions, generated for example by electrodes having an electrical potential, said treatment or affection having an anti-microbial effect including an anti-bacterial effect and improves the shelf-life and storage-stability of milk treated in accordance with the invention.
  • This invention is particularly useful for eliminating or inhibiting the growth of unwanted bacteria in milk and dairy products.
  • the method according to the invention is in one embodiment useful in the production of canned milk and may be used together with a traditional treatment, such as a heat treatment of raw-milk, condensed milk or milk powder, particularly a heat treatment comprising one or more treatments, such as low-pasteurisation, high- pasteurisation, UHT-treatment, sterilisation or evaporation.
  • the method according to the invention is particular useful in the manufacturing of dry milk or milk powder.
  • Pathogen bacteria as used herein are defined as bacteria capable of expressing a pathogen determinant, such as a secondary metabolite such as for example a toxin or a gene-product which does not occur naturally in environment, for example the gut in the human organism or an animal where the pathogen determinant is produced an optionally secreted to the surrounding by the pathogen bacteria.
  • a pathogen determinant such as a secondary metabolite such as for example a toxin or a gene-product which does not occur naturally in environment, for example the gut in the human organism or an animal where the pathogen determinant is produced an optionally secreted to the surrounding by the pathogen bacteria.
  • the fluid composition according to the invention is inhibiting or eliminating the production of the pathogen determinant.
  • the growth of the pathogen bacteria is inhibited or eliminated when it is contacted with the composition according to the invention. It is also possible that both growth and pathogen determinant production is inhibited or eliminated at the same time.
  • the anti-bacterial means is particularly useful in eliminating or inhibiting the growth of bacteria selected from the group of bacteria consisting of Achromobacter xylosoxidans, Acinetobacter calcoaceticus, preferably A. anitratus, A. haemolyticus, A. alcaligenes, and A. Iwoffii, Actinomyces israelii, Aeromonas hydrophilia,
  • Alcaligenes species preferably A. faecalis, A. odorans and A. denitrificans, Arizona hinshawii, Bacillus anthracis, Bacillus cereus, Bacteroides fragilis, Bacteroides melaninogenicus, Bordetella pertussis, Borrelia recurrentis, Brucella species, preferably B. abortus, B. suis, B. melitensis and B. canis, Calymmatobacterium granulomatis, Campylobacter fetus ssp. intestinalis, Campylobacter fetus ssp. jejuni, Chlamydia species, preferably C. psittaci and C.
  • trachomatis Chromobacterium vioiaceum, Citrobacter species, preferably C. freundii and C. diversus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium, preferably C. ulcerans, C. haemoiyticum and C. pseudotuberculosis, Coxiella bumetii, Edwardsiella tarda,
  • Eikenella corrodens, Enterobacter preferably E. cloacae, E. aerogenes, E. hafniae (also named Hafnia alvei) and E. agglomerans, Erysipelothrix rhusiopathiae, Escherichia coli, Flavobacterium meningosepticum, Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter species, Klebsiella species, preferably K. pneumoniae, K. ozaenae og K.
  • rhinoscleromatis Legionella species, Leptospira interrogans, Listeria monocytogenes, Moraxella species, preferably M. lacunata and M. osloensis, Mycobacterioum bovis, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma species, preferably M. pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia species, preferably N. asteroides and
  • Proteus species preferably P. mirabilis, P. vulgaris, P. rettgeri and P. morganii (also named Providencia rettgeri and Morganella morganii respectively), Providencia species, preferably P. alcalifaciens, P. stuartii and P. rettgeri (also named Proteus rettgeri), Pseudomonas aeruginosa, Pseudomonas mallei,
  • Serratia species preferably S. marcescens, Shigella dysenteriae, S. flexneri, S. boydii and S. sonnei, Spirillum minor, Staphylococcus aureus, Staphylococcus epidermidis, St
  • lower eu- caryots comprising for example yeast and fungide, preferably lower eucaryots selected from the group consisting of phycomycetes, ascomycets, basidiomycets, deuteromycet and fungi imperfecti.
  • Pathogenicity of lower eucaryots is defined as for bacteria herein above.
  • a means for eliminating a virus preferably a virus selected from the group consisting of Poxvirus, Herpesvirus, Adenovirus, Papovavirus, Par- vovirus, Picornavirus, Togavirus, Myxovirus, Paramyxovirus, Reovirus, Rhabdovirus, Retrovirus, particularly Human Immunodeficient Virus (HIV) and Arenavirus.
  • a virus preferably a virus selected from the group consisting of Poxvirus, Herpesvirus, Adenovirus, Papovavirus, Par- vovirus, Picornavirus, Togavirus, Myxovirus, Paramyxovirus, Reovirus, Rhabdovirus, Retrovirus, particularly Human Immunodeficient Virus (HIV) and Arenavirus.
  • HIV Human Immunodeficient Virus
  • the treatment comprises contacting the respective microbial organisms with a com- positioin according to the invention, preferably in the form of a fluid composition.
  • the fluid composition is administered to a human being or an animal infected by a microbial organism, such as for example a pathogen bacteria or a lower eucaryot, such as for example a yeast or a fungi.
  • compositions in the form of a ant- microbial means to prophylactically treat and/or alleviate and/or treat and/or cure diseases in a human being or an animal, said diseases being selected from the group consisting of Actinomycosis, Adenovirus-infections, Antrax, Bacterial dysentery, Botulisme, Brucellosis (Bang's disease), preferably caused by B. melitensis and B.
  • a fluid composition according to the invention obtained by a method comprising the step of contacting water or a aqueous solution with a charged particle, preferably a ion, or an electrical current of charged particles, preferably ions, said charged particles or electrical current of charged particles being provided by a ion generator or electrodes having an electrical potential and being connected to a power source.
  • the fluid composition in one preferred embodiment is contacted by charged particles in the form of an electrical current of from about 1.5 ⁇ A to about. 6.6 ⁇ A in about 1 hour.
  • the composition is obtainable in a preferred embodiment by provision of a total charge of from about 5.4 mC to about. 23.8 mC per about 32 mi of the fluid composition.
  • the current may in one embodiment be varied from about 0.01 ⁇ A to about 150 ⁇ A, such as from about 0.05 ⁇ A to about 100 ⁇ A, for example from about 0.1 ⁇ A to about 50 ⁇ A, such as from about 0.2 ⁇ A to about 25 ⁇ A, for example from about 0.3 ⁇ A to about 20 ⁇ A, such as from about 0.4 ⁇ A to about 15 ⁇ A, for example from about 0.5 ⁇ A to about 12 ⁇ A, such as from about 0.6 ⁇ A to about 10 ⁇ A, for example from about 0.7 ⁇ A to about 8.0 ⁇ A, such as from about 0.8 ⁇ A to about 6.0 ⁇ A, for example from about 0.9 ⁇ A to about 4.0 ⁇ A, such as from about 1.0 ⁇ A to about 2.5 ⁇ A, for example from about 1.1 ⁇ A to about 2.2 ⁇ A, such as from about 1.2 ⁇ A to about 2.0 ⁇ A, such as from about 1.3 ⁇ A to about 1.8 ⁇ A, for example from
  • the current in another embodiment may be varied from about 0.01 mA to about 150 mA, such as from about 0.05 mA to about 100 mA, for example from about 0.1 mA to about 50 mA, such as from about 0.2 mA to about 25 mA, for example from about
  • 0.3 mA to about 20 mA such as from about 0.4 mA to about 15 mA, for example from about 0.5 mA to about 12 mA, such as from about 0.6 mA to about 10 mA, for example from about 0.7 mA to about 8.0 mA, such as from about 0.8 mA to about 6.0 mA, for example from about 0.9 mA to about 4.0 mA, such as from about 1.0 mA to about 2.5 mA, for example from about 1.1 mA to about 2.2 mA, such as from about 1.2 mA to about 2.0 mA, such as from about 1.3 mA to about 1.8 mA, for example from about 1.4 mA to about 1.6 mA, such as about 1.5 mA.
  • the current may be varied from about 0.01 A to about 150 A, such as from about 0.05 A to about 100 A, for example from about 0.1 A to about 50 A, such as from about 0.2 A to about 25 A, for example from about 0.3 A to about 20 A, such as from about 0.4 A to about 15 A, for example from about 0.5 A to about 12 A, such as from about 0.6 A to about 10 A, for example from about 0.7 A to about 8.0 A, such as from about 0.8 A to about 6.0 A, for example from about 0.9 A to about 4.0 A, such as from about 1.0 A to about 2.5 A, for example from about 1.1 A to about 2.2 A, such as from about 1.2 A to about 2.0 A, such as from about 1.3 A to about 1.8 A, for example from about 1.4 A to about 1.6 A, such as 1.5 A.
  • the current may be provided in a period of hour from about 0.01 hour to about 5 hours, for example from about 0.05 hour to about 4.5 hours, such as from about 0.1 hour to about 4.0 hours, for example from about 0.2 hour to about 3.5 hours, such as from about 0.4 hour to about 3.0 hours, for example from about 0.5 hour to about
  • 2.5 hours such as from about 0.6 hour to about 2.0 hours, for example from about 0.7 hour to about 1.8 hours, such as from about 0.8 hour to about 1.6 hours, for ex- ample from about 0.85 hour to about 1.4 hours, such as from about 0.9 hour to about 1.2 hours, for example from about 0.95 hour to about 1.1 hours, such as about 1.0 hour.
  • the overall charge being provided to the composition according to the invention is from about 0.01 mC to about 10000 mC, such as from about 0.05 mC to about 8000 mC, for example from about 0.1 mC to about 4000 mC, such as from about 0.2 mC to about 2000 mC, for example from about 0.4 mC to about 1000 mC, such as from about 0.6 mC to about 800 mC, for example from about 0.8 mC to about 600 mC, such as from about 1.0 mC to about 300 mC, for example from about 1.2 mC to about 200 mC, such as from about 1.4 mC to about 100 mC, for example from about
  • 1.6 mC to about 80 mC such as from about 1.8 mC to about 60 mC, for example from about 2.0 mC to about 40 mC, such as from about 2.2 mC to about 30 mC, for example from about 2.4 mC to about 25 mC, such as from about 2.6 mC to about 20 mC, for example from about 2.8 mC to about 18 mC, such as from about 3.0 mC to about 16 mC, for example from about 3.2 mC to about 14 mC, such as from about 3.4 mC to about 12 mC, for example from about 3.6 mC to about 10 mC, such as from about 3.8 mC to about 8 mC, for example from about 4 mC to about 7 mC, pa fra ca.
  • 4.2 mC to about 6.5 mC for example from about 4.4 mC to about 6 mC, such as from about 4.6 mC to about 5.8 mC, for example from about 4.8 mC to about 5.7 mC, such as from about 5.0 mC to about 5.6 mC, for example from about 5.2 mC to about 5.5 mC, such as about 5.4 mC.
  • the fluid composition in a particularly preferred embodiment is provided according to the invention for use as a medicament and/or for use in a preventative, therapeutic or curative treatment of a condition in a human being or an animal.
  • the composition is used as medicament, it is preferably used in a pharmaceutically effective amount, which depends on the condition to be treated and the form of administration which is preferred for this treatment.
  • an electrically treated composition in the manufacture of a medicament for treating a suffering in a human being or an animal.
  • the treatment comprises using the medicament in a method for therapy or surgery, or a method of diagnostics and a particularly preferred embodiment, a method of prophylactically or therapeutically treating an allergic condition, preferably a treatment of the condition, when it is occurring in the airways, such as asthma.
  • compositions in the manufacture of a medicament for prophylactically or therapeutically treating a condition of infection in the airways such as an obstructive airways disease, which as the asthmatic condition described herein above, may occur sporadically or periodically or chronically.
  • the condition may lead to an increased mucoid mucus production, such as it is the case in bronchitis.
  • an electrically treated composition in the manufacture of a medicament, said medicament being effective in reducing or eliminating secretion of histamine from mast cells.
  • the manufactured medicament is, furthermore, effective in controlling the intracellular pH value in a cell and preferably furthermore effective in reducing the increase in the intracellular pH value in a cell during an allergic attack.
  • an electrically treated composition in the manufacture of a medicament which is effective in prophylactically or therapeutically treating, or in diagnosing, a cancer cell, a cardiovascular disease, an Immunodeficient condition, stressed and/or sick cells as well as prophylactically or therapeutically treating a skin disease, such as eczema, fungus, a bacteria infection or psoriasis.
  • the use is also aimed at manufacturing a medicament that is effective in increasing the immune response of a cell during a bacterial or viral infection of said cell.
  • a method for treating a suffering in a human being or an animal comprising contacting the suffering with the electrically treated composition according to the invention.
  • the method comprises treating by prophylaksis, therapy or surgery and/or a method for diagnostics, such as a method for prophylactically or therapeutically treating an allergic condition, preferably a condition occurring in the airways such as asthma.
  • the method according to the invention is also aimed at a treatment comprising a method of prophylactically or therapeutically treating a condition of infection in the airways, preferably a condition comprising an obstructive airways disease, which just as the asthmatic condition is sporadically or periodically or chronically occurring.
  • a condition of infection in the airways preferably a condition comprising an obstructive airways disease, which just as the asthmatic condition is sporadically or periodically or chronically occurring.
  • the condition may lead to an increased mucoid mucous production and is in preferred embodiment bronchitis.
  • the method of treatment according to the invention is effective in reducing or eliminating secretion of histamine from mast cells, effective in controlling the intracellular pH value of a cell, and effective in reducing the increased value of the intracellular pH in a cell during an allergic attack.
  • the method of treatment is also effective in prophylactically or therapeutically treating, or in diagnosing, a cancer cell, a cardiovascular disease, an
  • Immunodeficient condition, stressed and/or sick ceils as well as prophylactically or therapeutically treating a skin disease, such as eczema, fungus, a bacteria infection or psoriasis.
  • the method of treatment is also useful in increasing the immune response of a cell during a bacterial or viral infection of said cell.
  • compositions according to the invention for use in methods - and the use of compositions in the methods - comprising for example washing of organ transplants and/or transplantation of skin or hair.
  • a composition for use in oral or subcutaneous such as intravenous administration of for example a medicament or for use as a dialysis solution.
  • composition by transferring said composition to the skin of a human being or an animal suffering from internal bleeding, such as a hematome, is effective in treating the internal bleeding and effective in improving a curative process.
  • the composition is also assumed to be effective in treatment of insect bites and snake bites.
  • composition will according to one presently preferred hypothesis also be effective in treating an infection condition in a human being or an animal, as well as effective in treating skin being burned following scalding or sun burning.
  • the compositions according to the invention are useful in a mouth hygienic treatment, said treatment comprising either a liquid mouth hygienic solution, contacting the teeth or the oral cavity, or by brushing the teeth. It is also possible to use the composition according to the invention in connection with bathing or washing a human being or an animal, said bathing or washing having a health improving effect on the human being or the animal.
  • composition according to the invention in order to promote the growth and health of plants.
  • the composition is effective in promoting the organoleptic qualities in beer, wine and alcoholic beverages in general. It is, thus, possible to provide the wine with a property that results in the contents of histamine in said wine being reduced, or that the taste of the wine or for example the colour of the wine is affected as well as the bouquet of said wine being improved. According to yet another presently preferred hypothesis, it is possible to use the composition according to the invention in an improved treatment of waste water. In yet another embodiment, there is provided a method of providing sea water with such properties that are useful for fish-farming or useful in improving the quality of fish and the health of fish.
  • a powdery material in a solution such as for example water and treat said dissolved powdery material by a current of for example atmospheric ions or a current provided by electrodes having an electrically potential and being connected to a power source.
  • the treatment will provide the powdery material with properties that are useful in the production of for example chewing gum or lozenges, and it is possible to provide these products with properties that are promoting a healthy mouth hygiene.
  • the treated powdery material can also be brought into contact with for example the skin of a human being or an animal and provide a curative effect or a pain alleviating effect.
  • an powdery material being treated in accordance with invention, said material being brought into contact with crops and plants in a field and said contact providing a growth promoting effect or a prophylactically effect, an effect capable of reducing the occurrence of diseases and an effect of eliminating said diseases.
  • water containing emulsion such as for example butter, margarine, mayonnaise, gels or paint and by doing so obtain an increased shelf-life and durability.
  • water containing emulsion such as for example butter, margarine, mayonnaise, gels or paint
  • the self-life of milk can be improved by treating the milk with the composition according to the invention.
  • Figure 1 illustrates a current passsing through two plane electrodes.
  • Figure 2 illustrates a current passsing through two cylindrical electrodes.
  • Figure 3 illustrates an experimental design for treating a composition with atmospheric ions, optionally in combination with a ion generator which preferably is connected to a neutral electrode. Ions generated by the ion generator will be brought into contact with the surface of the liquid and result in an electrical current in the liquid. This electrical current is measurable by means of an electrometer.
  • Figure 4 shows an experimental design for treating a composition with an electrical current.
  • a copper electrode (7.25 cm in diameter) is brought in contact with the surface of the solution.
  • the electrode is connected to one terminal of a generator providing a constant current and the second terminal is neutral.
  • the electrometer monitors the electrical current in the composition.
  • Figure 5 shows the result of sending a current through the cell liquid in two different ways and, subsequently, transfering mast cells to the treated cell liquid.
  • the cell liquid must first be treated with an electrical current of about 1.5 ⁇ A in 1 hour, corresponding to the transfer of a total charge of about 5.4 mC respectively, and the effect of an electrical current of essentially the same value generated by the current generator and having the same direction, measurements are made without adding compound 48/88 and by adding compound 48/80 in three different concentrations.
  • the figures show the known effect of compound 48/80 on histamine secretion. It is, furthermore, evident that the secretion of histamine is reduced even quite significantly in the case where the liquid has been electrically treated.
  • the effect of negative ions and a direct electrical current from metal electrodes is perceived according to one presently hypothesis to be the same.
  • Figure 6 shows a comparison of the effect on positive and negative ions.
  • the electrical current of both positive and negative ions is kept as close to 1.5 ⁇ A as possible.
  • the time of exposure is also in this case 1 hour. Again, a marked reduction in the histamine secretion is observed.
  • Figure 7 shows the intracellular pH value as function of time determined during the experiments described herein above.
  • T 300 s
  • compound 48/80 is added and it can be seen that the cells present in the liquid, that is treated either with ions (black curve) or directly with electrical current (green curve), is inhibited in their pH response when stimulated with compound 48/89.
  • a control red curve
  • mast cells The following analyses of mast cells makes it possible to gain an insight into the signal mechanisms, which via exocytose leads to secretion of mediators. Histamine secretion is typically a result of an attack of asthma or allergy and gives rise to discomfort and unpleasant side-effects.
  • the peritoneal mast cells of the rat are the cells that contain histamine. These cells can be isolated and purified to a degree of purity of more than 95% and have for many years with success been used in his- tological, biochemical and pharmacological examinations of the cellular changes which takes place under the secretory process. Even though some heterogeneity exists among mast cells in different species and in different parts of the body of single species, certain similarities exist between the steps that lead to secretion via exocytose. Therefore, the mast cell is a good model for analysing secretion by means of exocytos (Diamant, 1990).
  • Spontaneous histamine Indicates if the cells which have been removed from the rat and are now present in a cell liquid have been damaged during the purification process, or if the histamine secretion is generated by the cell liquid, wherein the cells are present. If spontaneous histamine is below 5%, the cells have not been damaged. If, however, the spontaneous histamine is above 5%, the cells are in a form of stress. Spontaneous histamine is a control indicating whether the cells, which are used in the experiments, initially appears to behave as normal, healthy cells.
  • Histamine secretion is a term indicating that the ceil is secreting its granular contents to the surrounding media.
  • Exocytosis The process whereby the mast cell is capable of secreting histamine.
  • Cell suspension Cells and cell liquid.
  • Zero electrode A neutral electrode present in an ion generator, is necessary in order for the current to pass through the cell liquid.
  • Compound 48/80 During an attack of allergy or asthma, the mast cells are provoked to secrete histamine. In the laboratory, it is possible via a chemically produced, secretory active compound, termed compound 48/80. to simulate the secre- tory process taking place in the body, when a mast cell is secreting histamine. Histamine secretion in the body during an asthma or allergy response is thought not to be above 20%. By adding different concentrations of 48/80. it is possible to make the cell secrete different amounts of its content of histamine. On that basis, it is possible to examine the extent to which a treatment may suppress the unwanted secre- tion of histamine from mast cells.
  • a preferred cell liquid, buffer B, according to the in- vention contains , 140 mM NaCI, 1 mM CaCI 2 , 1.2 mM MgSO 4 4.0 mM KCI, 2.46 mM Na 2 HPO 4. 0.615 M KH 2 PO 4. 10 mM HEPES, 5.6 mM glucose, and 0.1% BSA (Bovint Serum Albinum).
  • the cell liquid is preferably comprised so that it corresponds to the composition of the physiological plasma of the rat. Isolation of peritoneal rat mast cells. The rats are anaesthetised with CO 2 gas, whereafter they are decapitated in a guillotine.
  • 10 ml cell liquid (ambient temperature) are containing 50 ⁇ g/ml heparin, to avoid coagulation of any present blood from damaged blood vessels, are injected in the abdominal cavity of the rat to a small hole which has previously been cut.
  • the hole is closed with a peang, whereafter the abdomen is massaged in about 1 min. in this way, the mast cells are released from the surface of the tissue of the abdominal cavity, whereafter a mixed population of peritoneal mast cells is obtained.
  • the extraction of the cells is achieved by carefully cutting the wall of the abdominal cavity and removing the in- ternal organs. The latter is importing since they can easily be perforated when the mast cells are extracted. If this happens, various compounds are released to abdominal cavity, which may cause problems during the further isolation procedure.
  • Abdominal liquid is carefully removed from the abdominal cavity with a syringes and is placed in a 45 ml centrifuge tube, which is already placed on ice (about4°C). The rest of the isolation procedure takes place at about
  • the ceils are centrifuges (230 g in 10 min at 4°C). The supernatant is removed and the cells are resuspended in 0.5 ml cell liquid. The cell suspension is hereafter transferred to an ostonic Percoll (density : 1.017 g/ml), which is centrifuged at 10.000 g in 20 min at 4°C.
  • the top two thirds of the Percoll gradi- ent is removed and the mast cells are transferred to a 45 ml centrifuge tube in which the ceils are washed twice with 40 ml cell liquid each followed by a centrifugation (230 g in 10 min at 4°C). This isolation procedure provides 96-99% mast cells. After the last wash, the supernatant is removed and the cells are resuspended in a know volume so that the cell density is known. The cell suspension is transferred to a in- cubation tubes (10 ml test tubes). 2 x 20 ⁇ l is removed for cell counting and 2 x 40 ⁇ l is removed for histamine determination.
  • the pelloted cells are lysed by boiling for 3 min. in 250 ⁇ l physiological selin (0.9% NaCI). After boiling, the samples are diluted with a 1.75 ml cold buffer (2°C), whereafter they are centrifuged (0 min at 500 g). The supernatant is removed to a new test tube and frozen down. These samples will be indicative of the cellular histamine content. Freezen is optionally, however, preferred since it is easier to freeze the samples and test only a few days during the month instead of testing 4 samples during each day.
  • the determination of the histamine content in the samples is carried out as described by Shore et al. (1959). This method involves condensation of o- phthaldialdehyd (OPT) with histamine at a high pH. In a spectro-fluorometer, the condensed substance will fluoresce at a low pH at 360 nm. This light can be measured at 450 nm. In order to determine the exact content of histamine in the samples, at standard histamine curve is made indicating the quantity of light as a function of various known histamine concentrations.
  • OPT o- phthaldialdehyd
  • the concentration of histamine ( ⁇ g/ml) in these standards are 0.250; 0.200; 0.125; 0.050; 0.025 respectively in a total volume of 1 ml (the standards are mixed in buffer).
  • a histamine free sample of 1 ml buffer is also made. The samples are thawed. A suitable amount of the test volume are transferred to a new test tube and diluted with buffer to a final volume of 1 ml. Thereafter, all the samples are treated the same, irrespective of whether they are samples or standards.
  • 1 ml HCI 0.1 N
  • 0.4 ml NaOH (1 N) was added to the samples.
  • Plastic bowls with a diameter of 7.25 cm and a height of 3.75 cm are applied.
  • the bottom plate of the bowls is made of copper.
  • the Bottom plate is connected via a wire to a measurement means which all the time measures how much current passes through the cell liquid and reaches the bottom plate.
  • cell liquid (32 mi) is treated with a electrical current
  • a copper plate is placed on top of the cell liquid and in contact with cell liquid.
  • the copper plate is also connected to a power source.
  • the mast cells are resuspended in the cell liquid.
  • the mast cells are loaded with the fluorescent indicator BCECF-AM (final concentration: 5 ⁇ M) for 30 min at 37°C in the dark (final volume 400 ⁇ l).
  • BCECF-AM quickly enters the cells across the cell membrane in esterified, uncharged form.
  • BCECF-AM is hydrolysed by the esterases present in the cell. Hydrolysis results in the BCECF being charged and, thus, maintained in the cytoplasm of the cell.
  • BCECF is an indicator that can be used to measure pHi and indicate variations of pHi in the cell.
  • the cells are washed and centrifuged (230 g for 10 min at room temperature) and resuspended in 1.9 ml of the respective assay buffer (37°C), which results in any released dye being removed prior to the experimental observations.
  • 1.8 ml of the cell suspension is transferred to a cuvette which subsequently is placed in a thermo-static cuvette house (37°C) with a stirrer in a spectro-fluorometer.
  • the handling of the spectro-fluormeter and the data collection was carried out online by a computer.
  • the BCECF fluorescent was registered in "ratio mode" by changing the wave length of the excitation from 490 nm and 435 nm in intervals of 0.5 seconds. The emission was measure at 530 nm.
  • the BCECF fluorescent is independent of pH at an excitation wave length of 435 nm (Rink, 1988).
  • a calculated ratio of the two emission signals at 490 and 435 nm excitation respectively gives an indication of the intracellular pH, which is independent of differences in the intracellular BCECF concentration, the number of mast cells, and optionally artefacts related to experimental changes (for example changes in the reflection of light occurring due to changes of the cell volume)(Rink 1998).
  • Calibration of the fluorescent-signal was carried out with nigericin/K + method (Thomas et al. 1979) and the 490/440 ratio was linear between pH 6.4-7.6 results.
  • Figure 5 shows the result of sending a current through the cell liquid in two different ways and, thereafter, adding mast cells to the treated liquid.
  • the current provided by the electrodes was about 1.5 ⁇ A and it was passed through the liquid for 1 hour (white bar indicated by “II”).
  • Atmospheric negative ions were provided with current corresponding to about 1.5 ⁇ A for 1 hour (dark grey bar indicated by “III”).
  • the cells were, thereafter, treated with 0.1 ; 0.2 and 0.3 ⁇ g/ml compound 48/80. respectively.
  • the light gray bar indicatred by "I” is a control of how much histamine the cells secrete when they are merely added untreated cell liquid.
  • a current through the cell liquid of about 1.5 ⁇ A provided by the added electrons is capable of inhibiting the cells in secreting up to 37% histamine which is sufficient to inhibit a physiological response during an asthmatic attack, where the histamine secretion typically is a maximum of about 20%
  • Figure 6 shows an experiment whereby the current is provide by atmospheric negative ions and a current is provided by the atmospheric negative ions to the cell liquid, which are thereafter added to the cells.
  • a current of about 1.5 ⁇ A provided by atmospheric positive ions for 1 hour (white bar indicated by "II") and a current of about 1.5 ⁇ A provided by atmospheric negative ions for 1 hour (dark grey bar indicated by "III") was used respectively.
  • the cells were, thereafter, treated with 0.1 ; 0.2 and 0.3 Gg/ml compound 48/80. respectively.
  • the light gray bar indicated by "I” is again a control of how much histamine the cells secrete when they are merely added untreated cell liquid.
  • FIG. 7 shows that the intracellular pH changes during the secretion of histamine (trace indicated by "B").
  • the observed pH increase is primarily due to an activation of protein kinase C and Na7H + exchange, wherein the Na + is transported in and the H + is transported out of the mast cell in a reaction driven by the electro-chemical gradient for natrium into the cell.
  • Figure 7 shows that the intracellular pH increase during histamine secretion is also reduced, when the cells are added a cell liquid treated with a current provided by electrodes having an electrical potential (about 1.5 ⁇ A for 1 hour) (trace indicated by "C") or provided by atmospheric negative ions (about 1.5 ⁇ A for 1 hour) (trace indicated by "A").
  • a suspension of Staphylococcus aureus 502A was adjusted to an optical density 0.2 at 546 nm equivalent of approximately 1x108 bacteria / ml was prepared. This suspension was diluted to 1 :10 in saline. The bacterial suspensions were then treated for one hour with either ionized air or direct current.
  • CFU Colony forming units
  • a suspension of Staphylococcus aureus 502A was adjusted to an optical density 0.2 at 546 nm equivalent of approximately 1x108 bacteria / ml was prepared. This suspension was diluted to 1 :10 in saline. The bacterial suspensions were then treated for one hour with either ionized air or direct current. 20 ml of the suspension was transferred to a 50 ml sterile tube and then incubated for 24 hrs at 37oC. On day 2 the bacterial suspension was treated again for one hour with either ionized air or direct current. Following incubation 0.1 ml of this suspension and two 0.1 ml aliquotes of subsequent 1 :10 dilutions were plated on blood agar plates. Colony forming units (CFU) were counted after overnight incubation at 37oC. The experiment was performed in triplicates. The data are shown as the mean number of CFU / plate.

Abstract

L'invention se rapporte à une composition traitée ou modifiée par voie électrique, qui peut être utilisée pour le traitement thérapeutique d'un être humain ou d'un animal. Cette composition traitée par voie électrique s'avère particulièrement utile pour supprimer la sécrétion d'histamine par des mastocytes et elle constitue par conséquent une nouvelle forme de traitement de l'asthme.
PCT/DK1999/000460 1998-09-01 1999-09-01 Composition traitee par voie electrique et utilisation d'une telle composition WO2000012134A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
US8377451B2 (en) 2003-03-07 2013-02-19 Wyeth Holdings Corporation Polysaccharide-staphylococcal surface adhesin carrier protein conjugates for immunization against nosocomial infections

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