The impact of C[O.sub.2] dry baths on subpopulations of NK and NK-T lymphocytes, cytotoxic activity, level of inflammation and pain management in elderly with musculosceletal syndromes--a pilot study.
Background--Aim of the study
The effects of carbon dioxide-rich water bathing have been investigated in clinical research; C[O.sub.2] is assumed to exert a direct action on vascular smooth muscles, induce vasodilatation and activate thermoreceptors of the skin. Our overall aim is to test our hypothesis that dry C[O.sub.2] treatment's rehabilitative effect is achieved by using its immune boosting, anti-inflammatory and anti-depressive elements to encage the elderly's pathophysiologic mechanisms capable of supporting a repaired and significantly improved function. In particular, the present study compared the post-therapy effects with pre-therapy baseline of the treatment measuring immune mediated response, the levels of biochemical markers for chronic inflammatory conditions and assessing clinically the short--term effectiveness of pain management in a group of elderly with musculoskeletal syndromes.
Material and Methods
Repeated artificial C[O.sub.2] dry treatments were performed on 37 volunteers with musculoskeletal pain disorders. Group A (intervention) comprised 27 patients (12 men and 15 women) and group B (placebo) tracked 10 patients (5 men and 5 women). Each patient was covered with a specific plastic hypoallergic bag up to neck level and underwent the treatment for 45 min. In group A the bag was filled with C[O.sub.2] gas and in the placebo group B the bag was filled with fresh air only. The DCDB (Dry Carbon Dioxide Bath) treatment was performed for 17 consecutive days. Blood samples were drawn before and after the intervention period. The selected parameters included white blood cells, cytotoxicity, lymphocytes, NK, NK-T cells, [beta]-endorphin, substance P and neurokinin A. Repeated measurements were made in heart frequency, arterial blood pressure and body temperature at the beginning and after all sessions.
In the intervention group, a significant decrease was detected in the diastolic arterial pressure (only in women) and in the heart rate. Significant increase was measured in the white blood cells count, lymphocytes count (in men), NK-T cells count, the cytotoxicity measurements, [beta]-endorphin and substance P levels as well as the neurokinin A levels in men. Interestingly, the intervention group members reported a kind of profound euphoria which remained during the intervention period and lasted for at least one month. The increase of Substance P and neurokinin A in the intervention group was not followed by any increase of pain intensity. In the control group, the parameters related to the arousal of the immune system remained unaffected but the levels of substance P and neurokinin A rose more sharply than in group A.
Dry C[O.sub.2] bath seems to stimulate short-term immune-boosting responses that last during and after the intervention period. The rise in the endorphin levels explains the euphoric sensation reported by the intervention group patients. The effects of dry C[O.sub.2] on the parasympathetic nervous system are induced probably via hemodynamic mechanisms; for the hemodynamic responses triggering, a direct action of dry C[O.sub.2] on specific C[O.sub.2]-receptors in the skin is a possible physiologic pathway.
Keywords: C[O.sub.2], carbon dioxide, dry baths, leukocytes, cytotoxicity, NK cells, [beta]-endorphin, substance P, neurokinin A
Carbon dioxide (Health aspects)
Carbon dioxide (Research)
Cell-mediated cytotoxicity (Health aspects)
Cell-mediated cytotoxicity (Research)
Immune response (Health aspects)
Immune response (Research)
Killer cells (Health aspects)
Killer cells (Research)
|Publication:||Name: Experimental Medicine Publisher: Renaissance Medical Publishing Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 Renaissance Medical Publishing ISSN: 1791-9967|
|Issue:||Date: Jan, 2009 Source Volume: 1 Source Issue: 1|
|Topic:||Event Code: 310 Science & research|
|Product:||Product Code: 2813300 Carbon Dioxide NAICS Code: 32512 Industrial Gas Manufacturing SIC Code: 2813 Industrial gases|
|Geographic:||Geographic Scope: Greece Geographic Code: 4EUGR Greece|
The use of carbonated water in the form of carbon dioxide baths is based on traditional therapeutic methods and is currently limited in the area of "Medical Spa treatments" or "Balneo-therapeutics". C[O.sub.2] treatment can be applied with two different ways; with carbon dioxide enriched water and with dry carbon dioxide bath (DCDB). It has to be mentioned that there are essential differences between the two C[O.sub.2] bath methods, which differentiates them completely. In carbon dioxide bath (CDB), C[O.sub.2] gets in contact with the human body via its dilution in water and in this way it is possible for an individual to inhale the gas. In addition, the immersed body is affected by water buoyancy, hydrostatic pressure and water temperature. In DCDB, the body comes in contact only with C[O.sub.2]. Most studies focus more on the effect of CDB and less of DCDB. DCDB is thought to be effective mainly in the treatment of peripheral vascular diseases, however, the mechanism(s) underlying this traditional therapy remain(s) poorly defined.
In a recent study of 2009, Gapon and Ignatov reported that the therapy with the use of DCDB after acute myocardial infarction, decreases systolic and diastolic arterial pressure and generally reduces cardiovascular complications. (1) Seven years earlier, Abramovich had observed that in elderly patients with hypertension dry carbon dioxide baths rehabilitate skin thermoreactivity and improve microcirculation. (2) Decrease of arterial pressure is mentioned in other studies also. (3,4) The application of DCDB in patients with psoriatic arthritis was directly associated with the positive results in immunological markers. (5) Grigor'eva et al report a 70% clinical improvement with the combined use of dry-air carbon dioxide baths and applications of peloids at low temperatures in the rehabilitation of 106 patients with psoriatic arthritis. (6)
The aim of this study is to investigate the efficiency of treatment with anhydrous C[O.sub.2] for hypothetical immune boosting actions (according to traditional data) in elderly patients suffering from painful musculoskeletal syndromes. Secondary aims of this research are to record in this category of patients biochemical parameters of inflammation, possible haemodynamic responses, pain perception and mood changes.
For the assessment of hemodynamic responses, we examined the arterial pressure, heart rate and body temperature before and after the intervention in every session. To assess immunological responses we measured white blood cells, lymphocytes, natural killer cells (NK), natural killer T cells (NK-T), and cytotoxicity, at the same times. For investigating the pain perception and mood we measured the endogenous opioid peptide, [beta]-endorphin. And for the markers of inflammation we measured tachykinins, substance P and neurokinin A.
Evidence based data on the correlation and interaction between measured variables
Though the impact of substance P and neurokinin A on the immune system remains unclear, many studies indicate that, there may also be exerted profound influence on inflammatory responses, by affecting multiple aspects of immune cell function. (7) This fact establishes a receptor-mediated mechanism by which tachykinin peptides can affect both innate and specific immune responses. (8) Substance P was shown to induce and mediate inflammation, angiogenesis, infections, intestinal mucosal immunity and stress and also activate several immune cells, such as CD4+ and CD8+ T lymphocytes, mast cells, NK cells and macrophages. (9) The functionally active neurokinin-1 receptors can be expressed by human natural killer cells. Substance P might therefore be a novel link between neural structures and innate immunity. (10)
Material and methods
In this research, 37 patients suffering from chronic musculoskeletal syndromes participated. They were randomly divided into two groups. In group A, 27 people participated with an average age of 64,3 [+ or -] 5,67 y (12 men with an average age of 67,7 [+ or -] 5,69 y, 15 women with an average age of 62,9 [+ or -] 5,05 y), and in group B (placebo), 10 people participated with an average age of 65,1 [+ or -] 5,33y (5 men with an average age of 66,4[+ or -]5,23y, 5 women with an average age of 64,4 [+ or -] 5,96y). The participants did not discontinue any pharmaceutical prescription nor did they change it at any point during the experiment. Their diet and routine habits were not altered in any way. During the intervention, one individual was disqualified because he deviated from the prescribed administration of drugs.
Eligibility criteria: age range from 60 to 70 years, Musculoskeletal problem, hypertension, reported generalized weakness, and bad mood (not related to bipolar disorder).
The selected individuals had at least one health problem, from each category listed below:
1. Cardiovascular disease: hypertension, chronic heart failure, coronary heart disease, chronic peripheral vascular disease of the lower extremities.
2. Painful musculoskeletal syndromes: chronic low back pain, lumbar disc herniation, cervical disc herniation, neck syndrome, osteoarthritis, shoulder periarthritis, tendonitis.
3. Osteoporosis with chronic pain (all women)
4. Diabetes mellitus (noticed on one individual).
The anthropometric characteristics were as follows:
Body weight (kg): The mean weight (Kg) of all patients was 82,6 [+ or -] 13,25, for men 92,8 [+ or -] 6,77, for women 74,5 [+ or -] 11,47 with no significance (p) of mean differences between men and women (sex).
Height (m): mean value 1,65 [+ or -] 6,7, for men 1,74 [+ or -] 1, for women 1,61 [+ or -] 3,2 and the significance (p) of mean differences according to sex was p<0.0001. Body Mass Index (BMI): mean value 29,01 [+ or -] 3,63, for men the mean value was 30,07 [+ or -] 2,9, for women mean value was 28,56 [+ or -] 4,01, and there was not significance (p) of mean differences between the two sexes.
C[O.sub.2] dry bath
This method requires a special plastic bag made of hypoallergic material, used to cover each participant's naked body. The plastic bag is sealed with tape on the chest or neck level. Then the individual lies on a bed in a quiet room. Dry C[O.sub.2] is very slowly administered into the bag through a tube, until the bag gets inflated with 180gr of C[O.sub.2]. C[O.sub.2] is heavier than atmospheric air. The time required for this session is 60 minutes. After this, red pigmentation appears on the skin due to the action of C[O.sub.2] on skin capillaries (vasodilatation). The number of sessions were 17 in total.
Design of the study
Participants who volunteered in this project had no food or water intake during their daily therapy sessions. Their height, weight and index of body mass were measured. They all underwent a clinical examination to determine the general condition of their health. This is indicated with a "good health" report sheet. It should be noted that the participants' weight was measured and recorded in the morning by an accurate balance (Electronil, Soehnle, West Germany). Their height was measured using a seca height measurement device (seca 240 Telescopic Height Measure, Germany). Furthermore, their blood pressure was measured prior to and following each DCDB using an Hg manometer (Baum, Baumanometer, Desk Model, USA).
Blood Samples (collection-distribution)
10ml of whole blood was drawn carefully in the morning from the median cubital vein of each participant. For sampling, BD Vacutainer[R] Blood Collection Tubes were used. The blood was distributed equally (5ml) in tubes containing anticoagulant (Heparin) and in others without it. The blood was centrifuged in 2000 rpm for 5 minutes (Biofuge, 17RS, Heraeus, SEPATECH, Gmb, Osterode, Denmark). The serum was collected and stored in -800[degrees]C for 6 weeks.
Methodology of NK cytotoxicity by flow cytometry (FCA)
The basic principle of the quantification of the cytotoxic activity of NK cells with FCA is to discriminate between effector (NK cells) and target (cancer) cell populations. The cell line K562 (cryopreserved) is used as target cells prestained with green fluorescent membrane dye (Beckton-Dickinson, USA). The K562 cell line derived from the blood of a patient suffering from chronic myeloid leukemia in terminal blastic crisis, and represents the most sensitive target cell line for human NK cells. (11) K562 cells lack MHC classes I and II antigens. After incubation of the effector and the target cells, a red fluorescent DNA dye (Sigma, USA) is added to label the target cells permeabilized by NK activity. This dye labels only cells with compromised plasma membranes. In this way, a clear separation between four cell populations can be obtained: live target cells, dead target cells, live effector cells, and dead effector cells. Thus, the actual ratio between effector and target cells (E:T) can be confirmed, but only events that appear to be positive after this analysis (dead and live targets cells) will have to be collected for the determination of the NK cytotoxic activity. This method described by Kane et al (11) included four steps: a) isolation of effector cells includes peripheral blood mononuclear cells (PBMCs) containing the NK cells, b) thawing of K562 target cells, c) cytotoxicity assay, and d) flow cytometric analysis (Figure 1).
Methodology of estimation of the number of NK and NK-T cells
For the estimation of the populations of NK and NK-T cells the technique of flow cytometry (Becton-Dickinson, USA) was used.
The Elisa technique (Phoenix Pharmaceutical, Inc, Germany) was used to identify the substances [beta]-endorphin (ng/ml), substance P (ng/ml) and neurokinin A (ng/ml) in human serum. An Elisa Reader was used to measure the absorption of the above peptides (Expert Plus UV, Microplate Reader, ASYS Hitech GmbH Eugendorf, Austria).
All data was analyzed using SPSS 14.0 for Windows. Appropriate descriptive statistics (mean and standard deviation for all measurable variables) and the parametric method Student's t test (independent-samples t test, 2-tailed) were used for the statistical analysis. The one-sample Kolmogorov-Smirnov test was used to examine whether the data of each of the variables, entering the Student's t-test analysis, were normally distributed. Our data were assessed using the Pearson's Correlation technique. The level of significance was set at 0.05.
In table 1, the measurements of body temperature and cardiac parameters are presented. Men had higher systolic blood pressure (mmHg) than women before (p=0.010) and after (0.002) the intervention. In women the diastolic arterial pressure was decreased (p=0.004) after the intervention. The pulse rate had a significant decrease (p=0.0001) after the DCDB in all participants and no difference was noted between men and women. It should be noticed that the women's heart rate was higher (p=0.007) than the men's in the measurements before the sessions.
Significant increases of [beta]-endorphin levels were observed in all participants after the sessions. Similar increases were found in the Substance P. Neurokinin A levels were increased in the total sample and in men also (Table 2).
The white cells count was increased only in men. The lymphocytes count (%) was increased only in women, who had higher levels before and after the sessions. The levels (%) of NK cells increased in men (p=0.039). The NK-T cells increased in all participants (total, men and women) and presented significant (p=0.002) differences according to sex before the sessions. Improvement of cytotoxicity resulted after the sessions (Table 3).
[FIGURE 1 OMITTED]
In the present study the measured outcomes derived from the action of dry C[O.sub.2] in the human organism, with actions made via the skin and the open body cavities, anus and urethra. Several researchers showed that water immersion, especially in C[O.sub.2]-enriched water, induces bradycardia via vagus nerve stimulation and the absorption of C[O.sub.2] from the skin surface (12), it normalizes the heart rhythm (13) or acts on the subcutaneous microcirculation by increasing parasympathetic and decreasing sympathetic activity. (14,15) In our research the absence of water and high temperature, proves that the considerable reduction of heart rate is due to the direct action of C[O.sub.2] to the parasympathetic system. This is explained by the increase of neuropeptides that suppress the actions of sympathetic system.
The synchronous increase of neuropeptides and NK cells agrees with many studies in the area of neuroimmunomodulation research (16,17) and the finding that there is secretion of opioid peptides by leukocytes. (18-22) In addition, endorphins and substance P increase NK cell cytotoxicity. (23)
The increase of [beta]-endorphin levels explains the pleasant disposal and well-being that was described by all participants. The interactions between leukocyte-derived opioid peptides and peripheral nociceptor endings carrying opioid receptors (24,25) may explain the connection of the pathophysiologic common basis of all the examined variables in our study with the pain mechanisms. The presence of hypodermic receptors of substance P, neurokinin A (26), [beta]-endorphin (26,27) and their stimulation by C[O.sub.2] explains that endogenous opioid analgesia results from neuroimmune interactions that occur in peripheral tissues. (28)
What should also be highlighted is that the level of sensitivity of C[O.sub.2] receptors that have been recognised in mosquitoes, explain how easily these insects can detect low levels of C[O.sub.2] in humans. (29) Finally, because oxygen and carbon dioxide are very important for the function of the human organism, numerous chemosensitive neurons can either obstruct or facilitate openings for stimulations, depending on the varying conditions. (30) Our study incicates that C[O.sub.2] affects the neuropeptides and their receptors which are located on the skin. These skin receptors are as important as other receptors found in the lungs, brain and other vital organs. These receptors rapidly allow the organism to conceptualize environmental alterations in order to adapt. Frequent or continuous contact of C[O.sub.2] to the skin seems to be perceived by the organism as an unpleasant situation.
In conclusion, C[O.sub.2] via the DCDB technique seems to have immune boosting results in elderly patients with chronic musculoskeletal syndromes. Other results of this treatment is decrease of hypertension, pain management and euphoric sensation. Those results are short--term responses that have been measured after the C[O.sub.2] sessions. Further investigation will be needed to determine the long term effects of C[O.sub.2]. Also, the exact participation of the skin receptors in the pathophysiologic mechanisms of the neuroimmune post session response are to be defined with skin biopsies.
We would like to express our thanks to the "Physiokinisi" Company and especially to Ms. Renata Mihailidou for the support.
Conflict of interest: None declared.
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Elias Kallistratos , Ioannis Toliopoulos , Dimitrios Fragkoraptis , Spyridon Gerou , Eleftherios Fragkoraptis 
 Physiotherapy Department, Alexander Technological Educational Institute, Thessaloniki, Greece,
 Konstantinion Research Center of Molecular Medicine and Biotechnology, Thessaloniki, Greece
 Analysis Research and Diagnostic Biomedical Labs, Greece
Elias Kallistratos, Ph.D, Associate Professor Physiotherapy Department, Alexander Technological Institute of Thessaloniki P.O BOX 141, GR-57400 Sindos, Thessaloniki, Greece Tel: +30 2310 791548, Mobile: +30 6972405216 E-mail: email@example.com
Table 1. Measurements of Body temperature and cardiac parameters. Parameters Total Total T-test (P) T.b ([degrees]C) 36.5 [+ or -] 0.03 T.a ([degrees]C) 36.5 [+ or -] 0.06 NS BPs.b (mmHg) 133.0 [+ or -] 12.97 BPs.a (mmHg) 131.4 [+ or -] 12.45 NS BPd.b (mmHg) 82.2 [+ or -] 6.50 BPd.a (mmHg) 80.9 [+ or -] 7.40 NS P.b (BPM) 75.1 [+ or -] 7.80 P.a (BPM) 68.9 [+ or -] 6.26 0.000 Parameters Men Men T-test (P) T.b ([degrees]C) 36.5 [+ or -] 0.02 T.a ([degrees]C) 36.5 [+ or -] 0.01 NS BPs.b (mmHg) 139.9 [+ or -] 4.96 BPs.a (mmHg) 139.1 [+ or -] 5.02 NS BPd.b (mmHg) 81.8 [+ or -] 6.11 BPd.a (mmHg) 82.4 [+ or -] 8.30 NS P.b (BPM) 70.8 [+ or -] 5.92 P.a (BPM) 66.8 [+ or -] 6.22 0.000 Parameters Women Women T-test (P) T.b ([degrees]C) 36.5 [+ or -] 0.03 T.a ([degrees]C) 36.5 [+ or -] 0.08 NS BPs.b (mmHg) 127.5 [+ or -] 14.83 BPs.a (mmHg) 125.2 [+ or -] 13.28 NS BPd.b (mmHg) 82.5 [+ or -] 6.99 BPd.a (mmHg) 79.8 [+ or -] 6.67 0.004 P.b (BPM) 78.5 [+ or -] 7.54 P.a (BPM) 70.6 [+ or -] 5.98 0.000 Parameters Men-Women T-test T.b ([degrees]C) 0.014 T.a ([degrees]C) ND BPs.b (mmHg) 0.010 BPs.a (mmHg) 0.002 BPd.b (mmHg) NS BPd.a (mmHg) NS P.b (BPM) 0.007 P.a (BPM) NS T.b. (Body Temperature before), T.a. (Body Temperature after), BPs.b (systolic blood pressure before), BPs.a (systolic blood pressure after), BPd.b (diastolic blood pressure before), BPd.a (diastolic blood pressure after), P.b. (pulse rate before), P.a. (pulse rate after), NS: no significant difference Table 2. The levels of the peptides before (b) and after (a) the protocol Parameters Total T-test (P) Men ng/ml B-endorphin.b 1.1 [+ or -] 0.57 1.3 [+ or -] 0.65 B-endorphin.o 2.8 [+ or -] 2.26 0.001 3.9 [+ or -] 2.89 Substance P.b 0.7 [+ or -] 0.18 0.7 [+ or -] 0.09 Substance P.a 0.9 [+ or -] 0.33 0.002 0.9 [+ or -] 0.3 Neurokinin A.b 3.1 [+ or -] 0.6 3 [+ or -] 0.48 Neurokinin A.a 3.5 [+ or -] 0.72 0.041 3.50.62 Parameters T-test (P) Women ng/ml B-endorphin.b 1 [+ or -] 0.49 B-endorphin.o 0.024 1.9 [+ or -] 1.03 Substance P.b 0.6 [+ or -] 0.21 Substance P.a 0.005 0.9 [+ or -] 0.36 Neurokinin A.b 3.1 [+ or -] 0.7 Neurokinin A.a 0.023 3.4 [+ or -] 0.81 Parameters T-test (P) T-test ng/ml differences Men-Women B-endorphin.b NS B-endorphin.o 0.000 0.021 Substance P.b NS Substance P.a 0.046 NS Neurokinin A.b NS Neurokinin A.a NS NS NS: no significant difference Table 3. Measurements of immune system markers Parameters Total T-test (P) HCT.b. (%) 41.8 [+ or -] 6.05 HCT.a. 43.2 [+ or -] 5.34 0.000 WBC.6.103/uL 6 [+ or -] 0.9 WBC.a. 5.6 [+ or -] 0.95 0.016 Lymp.b. % 30.7 [+ or -] 7.8 Lymp.a 33.4 [+ or -] 8.99 0.001 NK.b. % 14,1 [+ or -] 4,96 NK.a. 17,4 [+ or -] 5,77 NS NK-T.b. % 11,1 [+ or -] 6,16 NK-T.a. 3,3 [+ or -] 1,23 1 NK-T12.5:1.b. 5,7 [+ or -] 2,18 NK-T12.5:1.a. 20 [+ or -] 12,48 0.000 NK-T25:1.b. 7,3 [+ or -] 1,86 NK-T25:1.a 21,8 [+ or -] 12,01 0.000 NK-T50:1.b. 12,4 [+ or -] 3,89 NK-T50:1.a. 36,4 [+ or -] 13,45 0.000 Parameters Men T-test (P) HCT.b. (%) 44.5 [+ or -] 7.64 HCT.a. 45.9 [+ or -] 6.95 0.002 WBC.6.103/uL 6.1 [+ or -] 0.35 WBC.a. 5.3 [+ or -] 0.51 0.000 Lymp.b. % 25.2 [+ or -] 5.76 Lymp.a 25.4 [+ or -] 5.37 NS NK.b. % 13,2 [+ or -] 2,57 NK.a. 20,2 [+ or -] 5,7 39 NK-T.b. % 7,3 [+ or -] 2,22 NK-T.a. 3,3 [+ or -] 0,47 1 NK-T12.5:1.b. 7,2 [+ or -] 2,2 NK-T12.5:1.a. 24,1 [+ or -] 16,14 24 NK-T25:1.b. 8,4 [+ or -] 1,83 NK-T25:1.a 21,8 [+ or -] 15,8 12 NK-T50:1.b. 13,4 [+ or -] 4,48 NK-T50:1.a. 43,7 [+ or -] 15,15 0.000 Parameters Women T-test (P) HCT.b. (%) 39.6 [+ or -] 3.23 HCT.a. 41.1 [+ or -] 2.03 0.002 WBC.6.103/uL 6 [+ or -] 1.19 WBC.a. 6 [+ or -] 1.12 NS Lymp.b. % 35.2 [+ or -] 6.27 Lymp.a 39.7 [+ or -] 5.46 0.000 NK.b. % 14,7 [+ or -] 6,28 NK.a. 15,2 [+ or -] 5,1 NS NK-T.b. % 14,1 [+ or -] 6,66 NK-T.a. 3,3 [+ or -] 1,64 0.000 NK-T12.5:1.b. 4,6 [+ or -] 1,33 NK-T12.5:1.a. 16,7 [+ or -] 7,69 0.000 NK-T25:1.b. 6,5 [+ or -] 1,41 NK-T25:1.a 21,8 [+ or -] 8,47 0.000 NK-T50:1.b. 11,6 [+ or -] 3,26 NK-T50:1.a. 30,6 [+ or -] 8,61 0.000 Parameters T-test differences Men-Women HCT.b. (%) 0.031 HCT.a. 0.017 WBC.6.103/uL NS WBC.a. NS Lymp.b. % 0.000 Lymp.a 0.000 NK.b. % NS NK.a. NS NK-T.b. % 2 NK-T.a. NS NK-T12.5:1.b. 1 NK-T12.5:1.a. NS NK-T25:1.b. 5 NK-T25:1.a NS NK-T50:1.b. NS NK-T50:1.a. 0,009 b: before, a: after, HCT: Heamatocrit, WBC: White blood cells, Lymp.: lymphocytes, NK NK cells, NK-T: NK-T cells, NK-T 12.5:1, 25:1 & 50:1 cytotoxicity of NK cells in 3 ratios, NS: no significant difference
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