CA2218625A1 - Stress treatment and preconditioning against stress - Google Patents

Abstract

Symptoms of stress such as elevated blood pressure in mammals are treated, and mammalian bodies are preconditioned to manifest reduced adverse reactions to subsequently encountered stresses, by injecting into the mammalian patient a small quantity of the patient's own blood which has been previously extracted and subjected extracorporeally to three stressors simultaneously, namely ultraviolet radiation, ozone-oxygen gaseous mixture and mild heating.

Description

CA 0221862~ 1997-10-20 .
..

This invention relates to the field of medicine and medical treatments. In particular, it relates to stress treatment and more specifically to a method and composition for treating m~mm~l S, including humans, in order to provide them with improved reactions and resistance to stress.

The effects of stress on a m~mm~l normally manifest themselves in an increase in body temperature, along with a change in hemodynamic parameters, including an increase in heart rate and an increase in blood pressure. For patients already suffering from elevated blood pressure (hypertension), the effects of stress can therefore be particularly dangerous, since hypertension is a major risk factor for cardiovascular disease.

Stresses to which a m~mm~l may be subjected, and which can result in these effects, can take a wide variety of physical forms. Psychological stresses induced by restraint, confinement, sudden exposure to danger, shock and the like translate into physical stresses affecting one or more organs of the body.
Similarly, physical stress such as exposure to heat or cold, injury including surgical injury, over-exertion and the like, result in abnormal functioning of body organs. Stress is now recognized as a major detrimental factor in many diseases such as cardiovascular disease, cancer, and immunological dysfunction. One common physiological factor which appears to underlie all stress responses is the induction and upregulation of synthesis, in all body cells, of a group of specialized intracellular proteins known as heat stress proteins or heat shock proteins (HSP's). These HSPs function to protect the cells from potential damage caused by whatever form of stress is being applied.

One particular species of physical stress is ischemia, which is the deprivation of blood flow. Ischemia in a body organ, if severe enough, causes the eventual death of cells in the organ, primarily by necrosis. Re-perfusion of the ischemic organ by CA 0221862~ 1997-10-20 :' . ~

resumption of blood flow thereto often results in further injury to the organ, and does not re-invigorate already necrosed cells.
Repeated application of mild ischemic stress to an organ often leads to an increased ability to withstand stress ischemia, an effect thought to be partially related to upregulated synthesis of HSPs. Ischemia may occur as a pathological condition, e.g. as the result of spasm, thrombosis, or other blood vessel obstruction.
Ischemia may be deliberately induced by clamping of blood vessels during surgery.
It is known to precondition the body of a m~mm~l ian patient by subjecting it to controlled stresses, so as better to equip the body for subsequent encounters with uncontrolled stresses of the same type. Physical exercise and training, for example, equips a body for better handling of physical exertion stresses.
Heating a body or a body organ repeatedly under controlled conditions has been shown to provide the body or body organ with preconditioning for the better handling of subsequent heat stresses. Even in respect of ischemia, a body organ such as the heart which has previously suffered mild ischemia is better able to resist the effects of later ischemia, of the type causing myocardial infarction. As stated by Gersh et al., "Preconditioning is an important phenomenon, probably with clinical implications, because repetitive anginal episodes in patients may develop into full fledged infarction. Patients with pre-infarction angina may suffer from a less severe infarct than those thought to undergo sudden coronary occlusion without the opportunity for preconditioning. In contrast, patients with multiple short-lived attacks of ischemia might become tolerant through the development of protective preconditioning, according to animal data."1 Preconditioning by subjection to heat or ischemia is however clearly impractical in respect of most m~mm~l ian bodies and body organs.

CA 0221862~ 1997-10-20 , ' ' U.S. Patent 4,968,483 Mueller et al., describes an apparatus for oxygenating blood, by treating an aliquot of a patient's blood, extracorporeally, with an oxygen/ozone mixture and ultraviolet light, at a controlled temperature. The apparatus is proposed for use in hematological oxidation therapy.

U.S. Patent 5,591,457 Bolton, discloses a method of inhibiting the aggregation of blood platelets in a human, a method of stimulating the immune system and a method of treating peripheral vascular diseases such as Raynaud's disease, by extracting an aliquot of blood from a patient, subjecting it to ozone/oxygen gas mixture and ultraviolet radiation at a temperature in the range of about 37-43~C, and then reinjecting the treated blood into the human patient.
International Patent Application PCT/GB93/00259 Bolton, describes a similar process for increasing the content of nitric oxide in the blood of a m~mm~l ian patient, potentially useful in treating conditions such as high blood pressure in m~mm~l ian patients.

It is an object of the present invention to provide a novel method of treating stress in a m~mm~l ian patient.

It is a further object to provide a process of preconditioning a m~mm~l ian patient to improve the patient's resistance and reaction to subsequently encountered stress.

According to the present invention, there is provided a process of treating a m~mm~l ian patient to counteract the adverse effects of stress and/or to precondition the patient for improved resistance and reaction to subsequently encountered stress, which comprises extracting from the patient an aliquot of blood, subjecting the extracted blood aliquot extracorporeally to at least one stressor selected from an oxidative environment, W radiation CA 0221862~ 1997-10-20 . , .

and elevated temperature, and reinjecting at least a portion of the treated blood aliquot into the patient.

The method of the invention involves the extraction of an aliquot of blood from the patient, the subjection of the blood aliquot extracorporeally to stressors, and the reinjection of the treated blood aliquot into the patient. The treatment counteracts the effects of stress from which the patient is suffering at the time and shortly after the patient receives the treatment. More significantly and importantly, as a result of the treatment, preferably a series of treatments, the patient is better equipped to withstand the adverse effects of subsequently encountered stress. The treatment process according to the present invention causes the m~mm~lian patients, when subsequently stressed, to exhibit decreased stress response as detected by smaller rises in body temperature, smaller increases in heart rate and/or smaller increases in diastolic blood pressure.

The size of the blood aliquot to be treated is, in the case of human patients, generally from about 0.1 ml to about 400 ml, preferably from about 0.1-100 ml and most preferably 5-15 ml, with suitable prorating according to relative body weight for non-human patients. It is preferred to subject the blood aliquot to all three of the aforementioned stressors (blood temperature above body temperature, an oxidative environment such as a mixture of ozone and oxygen introduced into the blood aliquot, and ultraviolet radiation), simultaneously. Care must be taken not to utilize an excessive level of the stressors, to the extent that the cell mem~branes of the white cells of the blood are caused to be disrupted.

The temperature stressor must keep the aliquot in the liquid phase and should not heat it above about 45~C. Any suitable source of heat known in the art may be employed to heat the blood, preferably one or more infrared lamps. The temperature stressor CA 0221862~ 1997-10-20 preferably warms the aliquot being treated to a temperature above normal body temperature, i.e. to about 38-45~C, and most preferably from about 38-43~C, e.g. about 42.5~C. Preferably the temperature of the blood aliquot is maintained at this elevated temperature during the treatment with W and ozone. Alternatively, however, the blood sample can be heated while being subjected to W
radiation, until the blood reaches a predetermined temperature (preferably about 42.5~C), at which point bubbling of ozone gas through the blood is commenced. The concurrent W /ozone treatment is then maintained for a predetermined period of time from about to about 10 minutes, and preferably about 1-5 minutes, most preferably about 3 minutes.

The application of the oxidative stressor preferably involves exposing the aliquot to a mixture of medical grade oxygen and ozone gas, most preferably by bubbling through the aliquot, at the aforementioned temperature range, a stream of medical grade oxygen gas having ozone as a minor component therein. The ozone gas may be provided by any conventional source known in the art.
Suitably the gas stream has an ozone content of from about 1.0-100 ~g/ml, preferably 3-70 ~g/ml and most preferably from about 5-50 ~g/ml. The gas stream is supplied to the aliquot at a rate of from about 0.01-2 litres per minute, preferably 0.05-1.0 litres per minute, and most preferably at about 0.06-0.18 litres per minute (STP).

The ultraviolet radiation stressor is suitably applied by irradiating the aliquot under treatment from an appropriate source of W radiation, while the aliquot is maintained at the aforementioned temperature and while the oxygen/ozone gaseous mixture is being bubbled through the aliquot. The ultraviolet radiation may be provided by any conventional source known in the art, for example by a plurality of low-pressure ultraviolet lamps.
The method of the invention preferably utilizes a standard W-C
source of ultraviolet radiation, namely W lamps emitting primarily CA 0221862~ 1997-10-20 in the C-band wavelengths, i.e. at wavelengths shorter than about 280 nm. Ultraviolet radiation corresponding to standard W-A and W-B sources can also be used. Preferably employed are low-pressure ultraviolet lamps that generate a line spectrum wherein at least 90~ of the radiation has a wavelength of about 253.7 nm. An appropriate dosage of such W radiation, applied simultaneously with the aforementioned temperature and oxidative environment stressors, is obtained from lamps with a power output of from about 15 to about 25 watts, at the chosen W wavelength, arranged to surround the sample container holding the aliquot, each lamp providing an intensity, at a distance of 1 meter, of from about 35 - 75 mW/sq.cm. Several such lamps surrounding the sample bottle, with a combined output at 253.7 nm of 15 - 25 watts, operated at maximum intensity, may advantageously be used. At the incident surface of the blood, the W energy supplied is typically 0.25 Joules per cm2. Such a treatment provides a blood aliquot which is appropriately modified according to the invention ready for re-injection into the patient.

The time for which the aliquot is subjected to the stressors can be from a few seconds to about 60 minutes. It is normally within the time range of from about 0.5 - 60 minutes.
This depends to some extent upon the chosen intensity of the W
irradiation, the temperature and the concentration of and rate at which the oxidizing agent is supplied to the aliquot. Some experimentation to establish optimum times and dosages 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 about 0.5 - 10 minutes, most preferably 2 - 5 minutes, and normally around 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 patient to patient.

CA 0221862~ 1997-10-20 In the practice of the preferred process of the present invention, the blood aliquot (or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, these various leucocyte-containing combinations, along with whole blood, being referred to collectively throughout as the "aliquot") may be treated with the stressors using an apparatus of the type described in U.S. patent 4,968,483 Mueller. The aliquot is placed in a suitable, sterile, W-radiation-transmissive container, which is then fitted into the machine. The temperature of the aliquot is adjusted to the predetermined value, e.g. 42.5~C, by the use of a suitable heat source such as an IR lamp, and the W
lamps are switched on for a fixed period before the gas flow is applied to the aliquot providing the oxidative stress, to allow the output of the UV lamps to stabilize. Then the oxygen/ozone gas mixture, of known composition and controlled flow rate, is applied to the aliquot, for the predetermined duration of 0.5 - 60 minutes, preferably 2-5 minutes and most preferably about 3 minutes as discussed above, so that the aliquot experiences all three stressors simultaneously. In this way, the blood aliquot is appropriately modified according to the present invention sufficient to achieve the desired effects.

The process of the present invention shows utility both in treating a patient's stress symptoms evident at the time the treatment is administered, and in preconditioning a m~mm~l ian patient against the adverse effects of subsequently encountered stress, of any of the aforementioned types. It is not specific to providing tolerance to a specific stress or type of stress, but appears to be of general application. A patient who has undergone a treatment or a series of treatments according to the process of the present invention will exhibit notably reduced adverse reactions to subsequently encountered stress, such as a notably reduced rise in body temperature and/or a reduced increase in heart rate and/or a reduced increase in diastolic blood pressure, in response to stress, as compared with a similar but untreated CA 0221862~ 1997-10-20 , patient. The process is thus particularly useful for patients who are scheduled to undergo stress such as surgery at a predetermined future date. They can precondition their bodies to be ready for surgery by undergoing a treatment or a series of treatments according to the invention prior to surgery, with the result that they will withstand the surgery better and will recover from it more quickly.

Another preferred use of the present invention is in protecting tissues and organs from stress-induced damage, in a manner similar to ischemic preconditioning. As noted previously, repetitive mild ischemic (anginal) episodes can render tissues and organs less susceptible to stress-induced damage, by ischemic preconditioning, although application of ischemic preconditioning by current methods is largely impractical. The process of the present invention can take the place of ischemic preconditioning, ischemia being a species of physical stress. Accordingly, the process of the present invention offers potential for treatment of unstable angina and decrease of infarct size, a treatment not effectively addressed by available therapies.

Similarly, the process of the present invention is applicable in the protection of body organs destined for transplantation. Treatment of the donor body by the process of the present invention serves to protect body organs against damage resulting from the inevitable ischemia which the organ will suffer on removal from the donor body, transportation and subsequent surgical introduction into the recipient body. The treatment according to the invention extends the useful life of the transplant organ between its removal from the donor body and its surgical introduction into the recipient body, thereby reducing losses of viable transplant organs due to transportation delays.

A further, specific clinical application of the process of the invention is in treatment of patients suffering from CA 0221862~ 1997-10-20 ' ' transient ischemic attacks (TIA's, pre-strokes), which are due to temporary obstruction of blood flow to certain areas of the brain.
They commonly indicate the likelihood of suffering a major stroke in the near future. Subjection of such patients to treatment according to the process of the invention, at the onset of TIA's, will precondition the brain to avoid or at least to lessen the severity of the effects of the forthcoming major stroke.

The beneficial effects of the present invention have been ~emo~strated in vivo by clinical experiments on juvenile and adult rats, specifically rats of an inbred strain of genetically hypertensive rats (SHR's). Genetically hypertensive rats (SHR's) are the most widely used animal model for hypertension research, and are well known and readily available to researchers in this field. SHR's have several genetic defects, one of the most important being failure to produce appropriate amounts of HSPs when subjected to stress. SHR's develop hypertension rapidly and exhibit exaggerated increase in heart rate, blood pressure and body temperature in response to stress. They represent a model of hypersensitivity to stress. The results obtained using them provide reliable indications of potential results obtainable with human patients.

In the accompanying drawings:
Figure 1 is a graphical representation of the results obtained according to specific Example 1 described below;

Figure 2 is a graphical presentation of the body temperature results obtained according to Example 2 described below;

Figure 3 is a graphical presentation of the heart rate results obtained according to Example 2 described below;

CA 0221862~ 1997-10-20 :'' Figure 4 is a graphical presentation of the diastolic blood pressure results obtained according to Example 2 described below;

Figure 5 is a graphical presentation of the body temperature results obtained according to Example 3 described below;

Figure 6 is a graphical presentation of the body temperature results obtained according to Example 4 described below;

Figure 7 is a graphical presentation of the body temperature results obtained according to Example 5 described below; and Figure 8 is a graphical presentation of the heart rate results obtained according to Example 5 described below.

Blood from sacrificed SHR's of the same strain as the test animals was collected, treated with sodium citrate anticoagulant and pooled. A portion of the blood was then placed in a sterile container, and subjected simultaneously to the W
radiation, ozone/oxygen gas oxidative environment and elevated temperature stressors, in an apparatus as generally described in the aforementioned Mueller Patent U.S. 4,968,483. More specifically, the blood sample in the sterile, W-transparent container was heated using infrared lamps to 42.5~C, and whilst being maintained at that temperature, it was subjected to W
radiation of wavelength 253.7 nm under the preferred conditions previously described. Simultaneously, a gaseous mixture of medical grade oxygen and ozone, of ozone content 13.5-15.5 ~g/ml, was CA 0221862~ 1997-10-20 bubbled through the blood sample at a rate of flow over a range from 60 mls/min increasing eventually to about 180 mls/min. The time of simultaneous W exposure and gas mixture feed was 3 minutes.

A further portion of the pooled blood, to act as a control, was similarly placed in a sterile container and placed in the aforementioned apparatus, but was not heated, nor subjected to W radiation nor subjected to application of any ozone/oxygen gas mixture. In addition, to provide a further control, a sterile aqueous physiological saline solution was prepared.

A total of 44 seven week old SHR's were selected and divided into three groups, group A containing 15 animals, group B
containing 15 animals, and group C containing 14 animals. For a period of 10 days (at 7-9 weeks of age), each animal of group A
received a daily intragluteal injection of 150 ~l of the W, heat and ozone treated blood. Each animal of group B received at the same time a similar daily injection of the untreated blood. Each animal of group C received at the same time a similar injection of physiological saline.

At the age of 9 weeks, 4 days after completion of the injections the animals were anaesthetized and, a telemetry probe was inserted surgically into the femoral artery of each animal.
The telemetry probe (trade-mark DATAQUEST LABPRO, available from Data Sciences International) is a commercially available probe equipped with a radio transmitter, to permit heartbeat, systolic blood pressure, diastolic blood pressure and other signals to be received and recorded without further handling of the animals, which might induce further, uncontrolled stress reactions. An additional probe was surgically inserted into the peritoneal cavity of each animal, to measure body temperature.

CA 0221862~ 1997-10-20 Continuous daily recordings of body temperature, blood pressure and heart rate were made from each ~n;m~l, during the 10 day period following the surgery, readings being taken during the inactive, at-rest portion of the animals' daily cycle, i.e. the daylight ("light-on") portion, and during the activity time (night). Group A animals which received injections of blood treated according to the process of the invention demonstrated a significantly more rapid recovery of normal body temperature following surgery (6 days vs. 10 days), as compared with group B
10 ~n;m~l S which received untreated blood and group C animals which received saline injections, as shown especially by the readings taken during the resting periods. The differences are less evident from night-time, activity phase readings, suggesting that the higher cortisol levels present during activity may have an influence on the results. This demonstrates a significant effect of the treatment of the present invention on lessening the m~mm~l ian body's response to the stress of surgery.

The results of these experiments are presented graphically on Figure 1, a plot of measured body temperatures against days after surgery, each plotted value being the mean of values obtained from the whole group during the at-rest periods.
Curve A is derived from group A animals, curve B from group B
animals and curve C from group C animals.

The 44 animals treated as described in Example 1, namely the Group A of 15 animals which had received injections of blood treated according to the process of the invention, Group B of 15 ~n;m~l S which had received injection of untreated blood, and Group C which had received injection of saline, 10 days after the probe implantation surgery described in Example 1, were subjected to psychological stress through standard immobilization stress test, CA 0221862~ 1997-10-20 by placing them in small restraint cages for a period of 30 minutes (age of animals - 11 weeks). During this immobilization period, readings of body temperature, blood pressure and heart rate, at one minute intervals, were recorded.

Accompanying Fig. 2 of the drawings is a graphical presentation of the results of the body temperature measurements of the three groups, namely a plot of time as ordinate against body temperature as abscissa over the 30 minute duration of the immobilization stress test. As Fig. 2 shows, curve 2A derived from experimental Group A is consistently and significantly below curve 2B obtained from control Group B and curve 2C obtained from control Group C. Statistical analysis of all the data obtained confirms the high significance of the differences in the figures obtained from experimental Group A.

Figure 3 of the accompanying drawings presents graphically the results of heart rate measurements on the three groups, with heart rate (beats per minute, bpm) plotted as ordinate against time of the stress test. Again, the results (averaged over the ~nim~l s in each group) show that the group which received the injections of blood treated according to the invention, Group A, had a lower increase in heart rate, as compared with the other two groups, over substantially the entire duration of the test. The differences between the respective groups are statistically significant.

Figure 4 of the accompanying drawings presents graphically the results of the measurements of diastolic blood pressure of each of the three test groups, with diastolic blood pressure (mm/Hg) plotted as ordinate against time of the stress test. Again, the values from Group A (averaged), the group which had received injections of blood treated according to the invention, are consistently and significantly lower than those from the other two groups.

CA 022l862~ l997-l0-20 The Group C animals from Example 2, i.e. the control group which had, at age 7-9 weeks, received injections of physiological saline, were divided into three sub-groups Ca, Cb, and Cc. Each group was given a course of 10 daily injections of, respectively, 150 ~l of the treated blood, 150 ~l of the untreated blood, and 150 ~l of physiological saline. The course of injection started when the animals were 12 weeks old, i.e. fully matured adults. The telemetry probes remained in place. The same 30 minute immobilization stress test was performed on each animal, at age 16 weeks, and measurement of heart rate, blood pressure and body temperature were taken.

In body temperature response, the group Ca injected with blood treated according to the process of the invention showed a significantly more blunted increase during the stress period. This is illustrated in Fig. 5, a graphical presentation of the results similar to Fig. 2. It can be seen that curves Ca, derived from the Group Ca animals, is consistently lower than curve Cb derived from Group Cb animals and consistently lower than curve Cc derived from Group Cc ~n;m~l S .

The Group A experimentally treated animals from Example 2 which had received injections of blood treated according to the invention at age 7-9 weeks were divided into three sub-groups, labelled Aa, Ab and Ac, five animals in each group. They were then subjected to a second series of 10 daily injections of 150 ~l of, respectively, the treated blood, the untreated blood and the physiological saline. The course of injections started when the ~nlm~l S were 12 weeks old. The telemetry probes were left in place from Example 1, so that the surgery did not need to be repeated.

CA 0221862~ 1997-10-20 The animals were then subjected again, at 16 weeks of age, to the same immobilization stress test as described in Example 2 for 30 minutes, and measurements taken at 1 minute intervals of heart rate, blood pressure and body temperature.

The differences between the groups with regard to body temperature rise were very significant. They are illustrated on Fig. 6, a plot of body temperature against time, based on averages, similar to Fig. 2. After about the 12th minute of the test, group Aa which had received 2 courses of injection with the blood treated according to the invention exhibited consistently and significantly the lowest rise in body temperature. Curve Aa derived from Group Aa is consistently below curve Ab derived from Group Ab and curve Ac derived from Group Ac. It will be observed that the values on curve Aa are also lower than those on curve A of Fig. 2, indicating that a second treatment according to the invention has additional benefits on conditioning the animals for tolerance of stress. In contrast, the values on curve Ac are higher than the values on curve A of Fig. 2, and lower than the values on curve C of Fig. 2, indicating that the effects of the treatment tend to be lost after about 24 days from the conclusion of the course of treatment, absent a second "booster" treatment according to the invention.

The stress responses of animals from Example 1 which had been given two courses of injection with the same fluid (saline followed by saline, untreated blood followed by untreated blood, and treated blood followed by treated blood), at 7 weeks of age and 12 weeks of age, were measured during a second immobilization stress test, conducted as previously described, on animals aged 16 weeks. The results obtained from body temperature measurements are shown on Fig. 7, a graph of body temperature against time during the 30-minute stress test, similar to Fig. 2. Curve AA is derived CA 0221862~ 1997-10-20 from the measurements (average of 5 animals) of animals which had received two courses of ten daily injections of blood treated according to the invention. Curve BB is derived from the measurements (average of 5 animals) of animals which received two such courses of injections of untreated blood. Curve CC is derived from the measurements (average of 5 animals) of animals which received two such courses of saline injections. As the Figure shows, the values obtained from ~n;m~l S treated according to the process of the present invention are consistently and significantly lower than those derived from the other two groups.

Fig. 8 of the accompanying drawings similarly presents the heart rate measurements for the three groups, during the stress test. Again, Curve AA derived from animals which had received two courses of injection of blood treated according to the invention is significantly lower than the other two curves.

Claims (10)

1. Use in treating symptoms of stress and/or in preconditioning against the adverse effects of stress to be encountered subsequently, in a mammalian patient, of an aliquot of compatible mammalian blood which has been subjected extracorporeally to at least one stressor selected from an oxidative environment, UV radiation and elevated temperature up to about 45°C.

2. Use according to claim 1 wherein the aliquot of blood is the patient's own blood.

3. Use according to claim 2 wherein the blood aliquot is of volume about 0.1 ml to 400 ml.

4. Use according to claim 3 wherein the blood aliquot is subjected to all three said stressors simultaneously.

5. Use according to claim 4 wherein the oxidative environment stressor to which the blood aliquot is subjected is a mixture of medical grade oxygen and ozone, with an ozone content of from about 0.1-100 µg/ml.

6. Use according to claim 3, claim 4 or claim 5 wherein the ultraviolet radiation stressors is ultraviolet radiation from UV lamps emitting primarily at wavelengths of 280 nm or shorter.

7. Use according to claim 3, claim 4, claim 5 or claim 6 wherein the elevated temperature stressor is a temperature in the range from about 38-43°C.

8. Use according to claim 4, claim 5, claim 6 or claim 7 wherein each said stressor is applied to the blood aliquot for a period of time in the range 0.5-60 minutes.

9. Use in treatment of unstable angina and in decreasing infarct size, in a mammalian patient, of an aliquot of compatible mammalian blood which has been subjected extracorporeally to at least one stressor selected from an oxidative environment equivalent to that generated by passing through the blood aliquot a medical grade oxygen/ozone gas mixture containing 0.1-100 µg/ml ozone, UV radiation, and elevated temperature up to about 45°C.

10. Use in protecting mammalian donor organs destined for transplantation from a mammalian donor to a mammalian recipient, of an aliquot of the donor's blood which has been subjected extracorporeally to at least one stressor selected from an oxidative environment, UV radiation and elevated temperature up to about 45°C.