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| "Composite effects of group drumming music therapy on modulation of neuroendocrine-immune parameters in normal subjects." - Page 4 |
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Laboratory Studies
Peripheral blood mononuclear cells (PBMCs) were isolated from 15 mL of whole blood diluted 1:1 with a wash solution of Hanks balanced salt solution supplemented with 25 mmol/L of HEPES solution and 50 ug/mL of gentamicin using density gra-dient media. The mononuclear cell (PBMC) layer was collected and washed twice with wash solution and once with complete media, which was RPMI-1640 supplemented with 25 mmol/L of HEPES solution, 2 uM L-glutamine, 50 ug/mL gentamicin, and 10% fetal bovine solution. The cells were enumerated using a NOVA Celltrack 2 and adjusted to 2.5 x 10-6/mL.
Natural killer cell sensitive targets, K562 cells obtained from American Type Cell Collection (ATCC), were propagated in RPMI-1640 media, which was supplemented with 10 mmol/L HEPES, 2 uM L-glutamine, 50 ug/mL gentamicin, and 10% fetal bovine solution. On the day of the assay, the targets were labeled with 200 uCi of sterile sodium chromate 51 for 70 minutes in a 200 to 500 uL volume. The targets were washed twice prior to plating (10-4/tissue culture plate wall).
Peripheral blood mononuclear cells were used in a standard NK cell cytotoxicity assay as previously described/26 with minor modifications. Briefly, PBMCs were plated in 96 well plates with complete media, complete media containing human recombinant IL-2 (1 ng/well), or complete media containing human recombinant IFN-y (25 ng/well). After a 45-minute incubation (37°C, 6% carbon dioxide) with media or IL-2, or a 16-hour incubation with media or IFN-y, targets were added to obtain effector-to-target (E:T) ratios in the range of 50:1 down to 6:1. Cytokine incubations were performed at 2 E:T ratios, 25:1 and 12:1. These ratios were chosen to permit the observance of a maximal increase in cytotoxicity of all human specimens when incubated with TH1 activators of NK cell cytotoxicity. Separate controls with media only were assessed in parallel for the 2 cytokines due to differences in incubation times. All data were generated as percent cytotoxicity./22, 27,28
Serum levels of DHEA were measured by standard radioimmunoassay technique. Serum cortisol levels were measured by the cortisol Chiron ACS immunoassay run on the ACS:180 Automated Chemiluminescence Immunoassay Analyzer. Quantitative determination of plasma IFN-y concentrations were performed by the IFN-y Quantikine sandwich enzyme immunoassay. Interleukin 2 quantitation was achieved by the IL-2 Quantikine sandwich enzyme immunoassay.
Statistical Analysis
Statistical analysis of post-pre results for the 6 preliminary supervised groups (61 age-and-sex-matched subjects randomly assigned to groups of 9-11 subjects each) was performed using the binomial test of proportions for the purpose of determining which protocol was most highly correlated with NK cell modulation in a direction opposite to that expected with the classical stress response. For the preliminary groups, post-pre results were calculated as a simple difference between post- and pre-measurements. The composite drumming group was the only preliminary group that demonstrated strong NK cell activity enhancement (P=.055), and therefore was subsequently selected as the treatment model (Figure 1 and Table 1).
Thereafter, pre- and postexperimental results for 60 age-and sex-matched subjects were measured by 18 assays on 30 experimental and 30 control subjects who were randomly assigned to their respective groups. Twelve assays were designated a priori as biological markers and therefore were
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predicted
to be affected by the drumming/visualization intervention (Table 2). The
additional assays were used to assess sample bias, blood volume shift,
blood cell demargination, affective changes, and post hoc exploratory
data analysis. In view of diverse scaling of the measurements, the data
were increased by 1.0 units to eliminate any negative values and allow
a transformation of the data into a normalized measure of change:
log ([postresult +1.0] / [preresult +1.0])
The average normalized change for
the experimental group was compared with that of the control group using
independent t tests for each assay. Initially, however, an omnibus test
of significance across all of the 12 assays was conducted and found to
be significant (Hotelling r2; /><.0005)./29 The
results of multiple independent / tests (univariate analyses) are listed
in Table 2./30 The mean normalized change of the treatment
group is compared with that of the control group in Figure 2 for the 19
tests.
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