Transforming growth factor regulation

The present invention relates to a process of increasing the expression of TGF-&bgr; 1 by cells in a mammalian patient, comprising administering to the patient an effective amount of stressed mammalian blood cells, said stressed cells having been extracorporeally subjected to at least one stressor selected from oxidative stress and ultraviolet radiation. The process of the present invention shows potential in the treatment of ulcers in mammalian patients.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The source of the stressed blood cells for use in the present invention is preferably the patient's own blood, i.e. an aliquot of autologous blood. The terms “aliquot”, “aliquot of blood” or similar terms used herein include whole blood; separated cellular fractions of the blood, including platelets; separated non-cellular fractions of the blood, including plasma; plasma components; and combinations thereof. Preferably, in human patients, the volume of the aliquot is up to about 400 ml, preferably from about 0.1 to about 100 ml, more preferably from about 1 to about 15 ml, even more preferably from about 8 to about 12 ml, and most preferably about 10 ml. The effect of the stressor or the combination of stressors is to modify the blood, and/or the cellular or non-cellular fractions thereof, contained in the aliquot. The modified aliquot is then re-introduced into the patient's body by any suitable method, most preferably intramuscular injection, but also including subcutaneous injection, intraperitoneal injection, intra-arterial injection, intravenous injection and oral administration, following which it causes an increase in the expression of TGF-&bgr; 1 by the patient. According to a preferred process of the present invention, an aliquot of blood is extracted from a mammalian subject, preferably a human, and the aliquot of blood is treated ex vivo, simultaneously or sequentially, with the aforementioned stressors. The blood is then injected back into the same subject. Preferably a combination of both of the aforementioned stressors is used. Preferably, the aliquot of blood is further subjected to mechanical stress. Such mechanical stress is suitably that applied to the aliquot of blood by extraction of the blood aliquot through a conventional blood extraction needle, or a substantially equivalent mechanical stress applied shortly before the other chosen stressors are applied to the blood aliquot. This mechanical stress may be supplemented by the mechanical stress exerted on the blood aliquot by bubbling gases through it, such as ozone/oxygen mixtures, as described below. Optionally also, a temperature stressor may be applied to the blood aliquot, simultaneously or sequentially with the other stressors, i.e. a temperature at, above or below body temperature. The optionally applied temperature stressor either warms the aliquot being treated to a temperature above normal body temperature or cools the aliquot below normal body temperature. The temperature is selected so that the temperature stressor does not cause excessive hemolysis in the blood contained in the aliquot and so that, when the treated aliquot is injected into a subject, the desired effect will be achieved, without development of significant adverse side effects. Preferably, the temperature stressor is applied so that the temperature of all or a part of the aliquot is up to about 55° C., and more preferably in the range of from about −5° C. to about 55° C. In some preferred embodiments of the invention, the temperature of the aliquot is raised above normal body temperature, such that the mean temperature of the aliquot does not exceed a temperature of about 55° C., more preferably from about 40° C to about 50° C, even more preferably from about 40° C. to about 44° C., and most preferably about 42.5±1° C. In other preferred embodiments, the aliquot is cooled below normal body temperature such that the mean temperature of the aliquot is within the range of from about 4° C. to about 36.5° C., more preferably from about 10° C. to about 30° C., and even more preferably from about 15° C. to about 25° C. The oxidative environment stressor can be the application to the aliquot of solid, liquid or gaseous oxidizing agents. Preferably, it involves exposing the aliquot to a mixture of medical grade oxygen and ozone gas, most preferably by applying to the aliquot medical grade oxygen gas having ozone as a component therein. The ozone content of the gas stream and the flow rate of the gas stream are preferably selected such that the amount of ozone introduced to the blood aliquot, either on its own or in combination with one of the other stressors, does not give rise to excessive levels of cell damage, and so that, when the treated aliquot is injected into a subject, the desired effect will be achieved, without development of significant adverse side effects. Suitably, the gas stream has an ozone content of up to about 300 &mgr;g/ml, preferably up to about 100 &mgr;g/ml, more preferably up to about 30 &mgr;g/ml, even more preferably up to about 20 &mgr;g/ml, particularly preferably from about 10 &mgr;g/ml to about 20 &mgr;g/ml, and most preferably about 14.5±1.0 &mgr;g/ml. The gas stream is suitably supplied to the aliquot at a rate of up to about 2.0 litres/min, preferably up to about 0.5 litres/min, more preferably up to about 0.4 litres/min, even more preferably up to about 0.33 litres/min, and most preferably about 0.24±0.024 litres/min. The lower limit of the flow rate of the gas stream is preferably not lower than 0.01 litres/min, more preferably not lower than 0.1 litres/min, and even more preferably not lower than 0.2 litres/min, all rates at STP ( 0 ° C. and 1 atmosphere pressure). The ultraviolet light stressor is suitably applied by irradiating the aliquot under treatment from a source of UV light, i.e. electromagnetic radiation of wavelength from about 180 nm to about 400 nm. Preferred UV sources are UV lamps emitting UV-C band wavelengths, i.e. at wavelengths shorter than about 280 nm. Ultraviolet light corresponding to standard UV-A (i.e., wavelengths from about 315 to about 400 nm) and UV-B (i.e., wavelengths from about 280 to about 315) sources can also be used. As in the case of the oxidative stressor, the UV dose should be selected, on its own or in combination with the other chosen stressor(s), so that excessive amounts of cell damage do not occur, and so that, when the treated aliquot is injected into a subject, the desired effect will be achieved. For example, an appropriate dosage of such UV light, applied simultaneously with the aforementioned temperature and oxidative environment stressor, can be obtained from lamps with a power output of from about 10 to about 30 watts, arranged to surround the sample container holding the aliquot, each lamp providing an intensity, at a distance of 16 mm, of from about 5 to about 20 mW/cm 2 . Up to eight such lamps surrounding the sample bottle, with a combined output at 253.7 nm of 10 to 30 watts, operated at an intensity to deliver a total UV light energy at the surface of the blood of from about 0.025 to about 10 joules/cm 2 , and preferably from about 0.1 to about 3.0 joules/cm 2 , may advantageously be used. Such a treatment provides a modified blood aliquot which is ready for injection into the subject. It is preferred to subject the aliquot to the oxidative environment stressor, the UV light stressor and the temperature stressor simultaneously, following the subjection of the aliquot to the mechanical stress, e.g. by extraction of the blood from the patient. Thus, the aliquot may be maintained at a predetermined temperature above or below body temperature while the oxygen/ozone gas mixture is applied thereto and while it is irradiated with ultraviolet light. The time for which the aliquot is subjected to the stressors is normally within the time range of from about 0.5 minutes up to about 60 minutes. The time depends to some extent upon the chosen combination of stressors. When an UV light is used, the intensity of the UV light may affect the preferred time. The chosen temperature level may also affect the preferred time. When an oxidative environment in the form of a gaseous mixture of oxygen and ozone applied to the aliquot is chosen as one of the two stressors, the concentration of the oxidizing agent and the rate at which it is supplied to the aliquot may affect the preferred temperature. Some experimentation to establish optimum times may be necessary on the part of the operator, once the other stressor levels have been set. Under most stressor conditions, preferred times will be in the approximate range of from about 2 to about 12 minutes, more preferably from about 2 to about 5 minutes, most preferably about 3 minutes. The starting blood temperature, and the rate at which it can be warmed or cooled to a predetermined temperature, tends to vary from subject to subject. Warming is suitably by use of one or more infrared lamps placed adjacent to the aliquot container. Other methods of warming can also be adopted. As noted, it is preferred to subject the aliquot of blood to a mechanical stressor, as well as the chosen stressor(s) discussed above. Extraction of the blood aliquot from the patient through an injection needle constitutes the most convenient way of obtaining the aliquot for further extracorporeal treatment, and this extraction procedure imparts a suitable mechanical stress to the blood aliquot. The mechanical stressor may be supplemented by subsequent processing, for example the additional mechanical shear stress caused by bubbling as the oxidative stressor is applied. In the practice of the preferred process of the present invention, the blood aliquot may be treated with the heat, UV light and oxidative environment stressors using an apparatus of the type described in aforementioned U.S. Pat. No. 4,968,483 to Müller et al. The aliquot is placed in a suitable, sterile container, which is fitted into the machine. A UV-permeable container is used and the UV lamps are switched on for a fixed period before the other stressor(s) is applied, to allow the output of the UV lamps to stabilize. When a temperature stressor is used in combination with UV light stressors, the UV lamps are typically on while the temperature of the aliquot is adjusted to the predetermined value, e.g. 42.5±1° C. Four UV lamps are suitably used by placing around the aliquot containing container. In the preferred method of the invention, a mammalian patient standing to benefit from an increased expression of TGF-&bgr; 1 is given one or more courses of treatments, each course of treatment comprising the administration to the mammalian subject of one or more (e.g., one to six or one to twelve) aliquots of mammalian blood modified as discussed above. For optimum effectiveness of the treatment, it is preferred that no more than one aliquot of modified blood be administered to the subject per day in one or more injection sites, and that the maximum rest period between any two consecutive aliquot administrations during the course of treatment should be no greater than about 21 days. As used herein, the term “rest period” is defined as the number of days between consecutive aliquots or consecutive courses of treatment on which no aliquots of modified blood are administered to the subject. Therefore, except where aliquots are administered to the subject on consecutive days, a rest period of from 1 to 21 days is provided between any two aliquots during the course of treatment. Moreover, at least one of the rest periods during the course of treatment preferably has a length of about 3 to 15 days. Although it may be sufficient to administer only one course of treatment as described above to the subject, it may be preferred in some circumstances to administer more than one course of treatment, or to follow the above-described course of treatment by periodic “booster” treatments, if necessary, to maintain the desired effects of the present invention. For example, it may be preferred to administer booster treatments at intervals of 1 to 4 months following the initial course of treatment, or to administer a second course of treatment to the subject following a rest period of several weeks or months. The invention is further illustrated and described below with reference to Example 1, comprising animal studies conducted in an approved manner, and Example 2, a clinical trial on human patients. The examples are offered for purposes of illustrating the invention and should not be construed as a limitation. 
 EXAMPLE 1 Whole blood was obtained from Balb/c mice, by cardiac puncture through an injection needle, and treated with an anti-coagulant. An aliquot of this anti-coagulated blood was subjected to the process of a preferred embodiment of the invention, to obtain treated blood. The remainder was left untreated, for use in control experiments. Since the Balb/c mice used were genetically identical, the administration of the treated blood to others of the group is equivalent to administration of autologous blood. To obtain treated blood, the selected aliquot, in a sterile, UV-transmissive container, was treated simultaneously with a gaseous oxygen/ozone mixture and ultraviolet light at elevated temperature using an apparatus as generally described in aforementioned U.S. Pat. No. 4,968,483 Müller et.al. Specifically, 12 ml of citrated blood was transferred to a sterile, low density polyethylene vessel (more specifically, a Vasogen VC7002 Blood Container) for ex vivo treatment with stressors according to the invention. Using an apparatus as described in the aforementioned Muiller et al. patent (more specifically, a Vasogen VC7001 apparatus), the blood was heated to 42.5±1° C. and at that temperature irradiated with UV light at a wavelength of 253.7 nm, while oxygen/ozone gas mixture was bubbled through the blood to provide the oxidative environment and to facilitate exposure of the blood to UV. The constitution of the gas mixture was 14.5±1.0 &mgr;g ozone/ml, with the remainder of the mixture comprising medical grade oxygen. The gas mixture was bubbled through the aliquot at a rate of 240±24 ml/min for a period of 3 minutes. There were 4 groups of Balb/c mice. The first, control group A-1 received no treatment. The second, control group B-1, was treated with 50 &mgr;l of physiological saline. The third, control group C-1;, was sham treated with 50 &mgr;l of blood which had been extracted, but not treated with the additional stressors. The fourth, test group D-1, was treated with 50 &mgr;l of blood subjected to stressors as described above. Treatments, each involving injection of 50 &mgr;l of the respective liquid into the gluteal muscle, started on day 1, and were repeated every day for a total of 6 days. The experiment was run in parallel to the test for contact hypersensitivity resistance in the mice, as described in applicants co-pending international patent application PCT/CA00/00433 incorporated herein by reference, so that the various groups had been pre-sensitized with dinitrofluorobenzene (DNFB) and were subsequently challenged on one ear, 24 hours after the last injection, with DNFB as described therein, but this is not a factor in the tests demonstrating the present invention. Each of the animals was sacrificed and the lymph nodes draining the ear that was challenged with DNFB were collected. The expression of the mRNA of the cytokine TGF-&bgr; 1 in the lymph tissue so obtained was analyzed using known RT-PCR techniques, essentially following the procedures described in Kondo et.al., J. Immunology, Vol. 157:4822, 1996. The PCR products were determined by scanning of photonegatives using a laser densitometer, and the densitometric value of the TGF-&bgr; 1 was normalized to that of the housekeeping gene &bgr;-actin. The analyses indicated that animals which had received a course of injection of blood subjected to stressors as described had significantly increased concentrations of TGF-&bgr; 1 in the lymph node, as compared with controls and sham treated animals. The analyses were repeated three times, and the accompanying Figure illustrates the mean of these results. 
 EXAMPLE 2 A total of 20 human patients having moderate to severe psoriasis were randomized into a double blind, placebo controlled clinical trial. Two groups of 10 patients received 2 injections per week intramuscularly, into the gluteal muscle, of treated blood or saline, over a 3 week period. The therapy involved the collection of 10 ml of the patient's venous blood into 2 ml sodium citrate. The blood was transferred to a sterile disposable low-density polyethylene vessel for ex vivo treatment as described in Example 1. Prior to muscular injection, 1 ml of Novocain was injected into the gluteal muscle as a local anesthetic. Skin biopsies were taken at the end of the treatment, fixed in formalin and embedded in paraffin. Histological examination of skin biopsies of patients who had undergone treatment according to the invention was undertaken, by immunohistochemistry using a monoclonal antibody to TGF-&bgr; 1 . Increased production of TGF-&bgr; 1 in the dermis of patients treated according to the invention was seen in slides of tissue ( FIG. 2 a , microphotograph of the biopsied human skin sample after treatment to visualize TGF-&bgr; 1 ) based on increased density of staining in TGF&bgr; 1 producing cells, compared to patients treated with saline ( FIG. 2 b ). This result is indicative of the use of the process of the invention to upregulate TGF-&bgr; 1 and therefore in treating ulcers of the skin. All references cited above are herein incorporated by reference in their entirety.