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The Influence of Psychological Intervention upon

Psycho-Neuro-Endocrino-Immune Network

A thesis submitted for the award of


Doctor of Philosophy (Ph.D.)
Imperial College, Faculty of Medicine, London

Akira NAITO M.D.

August 2006

Imperial College London


Faculty of Medicine

Division of Neuroscience and Mental Health


Charing Cross Campus
St. Dunstan’s Road, London W6 8RP
and
Department of Immunology
Chelsea and Westminster Campus
369 Fulham Road, London SW10 9NH

-1-
This thesis is dedicated to my father:

Nagayoshi NAITO
1940 - 2006

My role model and mentor


who taught me the importance of being myself
and passed away at the end of my Ph.D. course.
I thank him for his truthfulness, warm understanding and spiritual support.

Cyclamen
(31 January 2006)
The last piece of work in his private life with my mother, Masako

-2-
Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Abstract
A growing body of evidence suggests that there are cross regulatory influences between
the psychological, neurological, endocrinological and immunological systems. This has led to
the proposal of a psycho-neuro-endocrino-immune network, in which a change in one part has
consequential influences upon other parts of the network. Thus psychological stress can also
affect physical well-being through the network.

This thesis is based on the hypothesis that the detrimental effects of stress upon the
network can be alleviated by psychological intervention. The aim of this project is to examine
whether learning and practising stress-management skills improves psychological and physical
well-being.

An in vivo study using university students confirmed that examination stress provokes
anxiety and increases stress, and that stress perception was associated with NK-cell function.
Immune alterations were observed in vitro when NK-cells and lymphocytes were exposed to
stress hormones. Another in vivo study using HIV-infected patients not receiving anti-retroviral
treatments confirmed that there is a steady decline in CD4 T-lymphocyte counts, and that this
decline was associated with declines in quality-of-life scores.

The psychological interventions used in the project, self-hypnosis and Johrei, aimed to
provide alternative perspectives of, and unique self-help coping strategies for, stressful life
events. These, particularly Johrei, were shown to alleviate or even to reverse the effects of stress
upon the immune system, including a decrease in NK-cells in university students and the
decline of CD4 T-lymphocytes in HIV-infected patients. Neither intervention affected stress
perception significantly, although Johrei appeared to improve the quality-of-life scores.

These findings support the a priori hypothesis that psychological intervention may
counteract the detrimental effects of stress on health and promote well-being, and suggest this
via meaning-focused coping. These warrant the need for further research to explore the inter-
dependent relationships amongst the integrated psycho-neuro-endocrino-immune network, and
the influence of Johrei upon the network.

Ph.D. at University of London -3- Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Contents Page

Abstract 3

List of Figures 6
List of Tables 9
List of Text boxes 14
Abbreviations 15

Acknowledgements 16

Chapter I Introduction
Contents of chapter I 18
1.1 Project overview 19
1.2 Theoretical framework 22
1.3 Approach taken 49

Chapter II Materials and Methods


Contents of chapter II 51
2.1 Participants with regard to outcomes and measurement time points 53
2.2 Psychological intervention 60
2.3 Self-report questionnaires 63
2.4 Materials and methods in vitro 65
2.5 Statistical analyses 77

Chapter III Results


Contents of chapter III 79
3.1 The influence of psychological intervention upon stress-related changes
in university students facing academic examinations 83
3.2 The influence of psychological intervention upon perceived stress and
quality-of-life and various immunological disease-associated
parameters in HIV-infected individuals 102
3.3 In vitro investigation into the effect of exposure to stress hormones
upon Natural Killer cells 128
3.4 In vitro investigation into the effect of exposure to stress hormones
upon T-lymphocytes 151

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Chapter IV Discussion
Contents of chapter IV 171
4.1 The influence of psychological intervention upon sustained stress and
stress-associated changes in university students facing exams and in
patients with HIV-infection 173
4.2 Stress hormone associated changes in vitro in immune cells as an
exemplar of the psycho-neuro-endocrino-immune network interaction 191
4.3 Conclusion 198
4.4 Future directions 199

References 200

Appendices
A-1. Ethics approval, information sheet and consent form 214

A-2. Psychological training intervention protocols 226


i. Self hypnosis training 227
ii. Johrei training 253

A-3. Questionnaires 286


Stress perception (STAI , IES and PSS) 287
Quality of Life and sleep quality (LoC, MCS and PSQI) 290

A-4. Standard operating procedures (SOPs) for laboratory methods 296


3
[ H]-thymidine incorporation using whole blood for proliferation assay 296
Flowcytometry analysis of proliferation response in whole blood assay protocol
298
NK cell cytotoxic activity assay by flow cytometry 300

A-5. Papers 304


i. Peer reviewed publications 304
ii. Presentation and workshop given 305
iii. Supervisor for research students 307

A-6. Additional results 308

A-7. Research proposal for post-doctorial project 316

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

List of Figures
Figure 1: A typical Natural Killer cell
Figure 2: Schema of the neuro-endocrino-immune interaction
Figure 3: Numbers of students recruited and remaining in the study at the Exam and Non-exam
assessment time points. The number of drop-out students is also indicated.
Figure 4: Timing of the Exam and Non-exam assessment time points with respect to recruitment for
individual students in the Cohorts A and B
Figure 5: Timing of training, follow-ups and data-collection sessions (Baseline and Exams assessment
points) and numbers of students at the sessions
Figure 6: Timing of the measurement collection time points (Recruitment and After 4 months) and the
one-month blocks (Baseline, Term one to Term four) for clinical routine blood collections
Figure 7: Timing of the two measurement points (Recruitment and Post-intervention) with regard to the
period of training and practice of the psychological interventions
Figure 8: Time-points (Training time point and the Terms according to the Training). Intervals between
the doted-lines represent one term (one month)
Figure 9: Comparison format with regard to the Training time point (Pre-intervention vs. Post-
intervention). Intervals between dot-lines represent one term (one month) as in Figure 8
Figure 10: FSC vs. SSC dot plot
Figure 11: Individual cortisol levels in the tissue culture medium and in plasma from 34 volunteers
Figure 12: K562 cell growth at starting cell concentrations of 1.5x104, 1.5x105 and 1.0x106 cells per mL
Figure 13: Acquision settings of a flow cytometry method measuring the levels of NK cytotoxic activity
Figure 14: Acquision settings of the flow cytometer in the NCR analyses
Figure 15: Acquision settings of the flow cytometer in the Annexin V- PI analysis
Figure 16: Mean (95% C.I.) PSS scores at the Non-exam and Exam time points
Figure 17: Mean (95% C.I.) State anxiety scores in the STAI at the Non-exam and Exam time points
Figure 18: Mean (95% C.I.) NK-cell percentages in male and female students
Figure 19: Mean (95% C.I.) NKCA (% killing) in the Not-stressed and Stressed subgroups
Figure 20: Mean (95% C.I.) NKCA levels (% killing) in the Not-stressed and Stressed male students
Figure 21: Mean (95% C.I.) per-NK-cell cytotoxic activity (calculated as a ratio: NK cytotoxic activity
(NKCA) / NK-cell percentage (NKC%)) in the Not-stressed and Stressed subgroups
Figure 22: Mean (95% C.I.) per-NK-cell cytotoxic activity (calculated as a ratio: NKCA / NK-cell
percentage) in the Not-stressed and Stressed male students
Figure 23: Mean (95% C.I.) CD4 T-cell levels (%) of male and female students
Figure 24: Mean (95% C.I.) State anxiety scores in the STAI of the three groups at baseline and the
Exam time point
Figure 25: Mean (95% C.I.) levels of NK-cells of the three groups at baseline and the Exam time point
Figure 26: The individual levels of NK-cell percentages in the three groups at baseline and the Exam
time point

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Figure 27: Mean (95% C.I.) levels of CD4 T-cells of the three groups at baseline and the Exam time
point
Figure 28: The individual levels of CD4 T-cell percentages in the three groups at baseline and the Exam
time point
Figure 29: Mean (95% C.I.) levels of CD8 T-cells of the three groups at baseline and the Exam time
point
Figure 30: The individual levels of CD8 T-cell percentages in the three groups at baseline and the Exam
time point
Figure 31: Mean (95% C.I.) PSS levels of the Not-stressed and Stressed subgroups at recruitment and
four months later
Figure 32: Mean (95% C.I.) levels of the State anxiety scores in the STAI of the Not-stressed and Stress
subgroups at recruitment and four months later
Figure 33: Mean (95% C.I.) levels of the IES in the Not-stressed and Stress subgroups at recruitment and
four months later
Figure 34: Mean (95% C.I.) levels of the LoC in the Not-stressed and Stressed subgroups at recruitment
and four months later
Figure 35: Mean (95% C.I.) levels of the MSC in the Not-stressed and Stressed subgroups at recruitment
and four months later
Figure 36: Mean (95% C.I.) levels of the PSQI in the Stress and Not-stressed subgroups at recruitment
and four months later
Figure 37: Mean (95% C.I.) CD4 gradients (cells per l per month) in the Increased control (decreased
scores of the LoC) and Decreased control (increased scores of the LoC) subgroups
Figure 38: Mean (95% C.I.) CD4 gradients (cells per l per month) in the Improved psychological
functioning (Improved QoL: increased scores of the MCS) and Decreased psychological
functioning (Decreased QoL: decreased scores of the MCS) subgroups
Figure 39: Mean (95% C.I.) CD4 gradients (cells per l per month) in the Improved sleep quality
(decreased scores of the PSQI) and Decreased sleep quality (increased scores of the PSQI)
subgroups
Figure 40: Means (95% C.I.) CD4 gradients over the five months study period from the Baseline to the
Post-intervention time point
Figure 41: CD4 gradients of one control subject at the Pre-intervention and Post-intervention periods
Figure 42: CD4 gradients of one Johrei subject at the Pre-intervention and Post-intervention periods
Figure 43: Individual changes in CD4 gradients (cell counts per l per month) in the Self-hypnosis,
Johrei and Database-control groups between the Pre-intervention and Post-intervention
periods
Figure 44: Means (95% C.I.) CD4 gradients (count per l per month) in the Self-hypnosis and Johrei and
Database-control groups in the Pre- and Post- intervention periods
Figure 45: (a) Individual (b) Mean (95% C.I.) NKCA levels at Time 0 and after 24hour incubation

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Figure 46: (a) Individual (b) Mean (95% C.I.) NK-cell percentage in PBMCs at Time 0 and after 24hours
Figure 47: (a) Individual (b) Mean (95% C.I.) NKCA levels with cortisol at 0, 250 and 2500nM
Figure 48: (a) Individual (b) Mean (95% C.I.) NK-cell percentage with cortisol at 0, 250 and 2500nM
Figure 49: Correlation between flow-cytometry method and LDH-releasing method of the NKCA
Figure 50: (a) Individual (b) Mean (95% C.I.) NKCA (50:1) levels (% killing) at Time 0 and after 24hrs
Figure 51: (a) Individual (b) Mean (95% C.I.) NKCA (25:1) levels (% killing) at Time 0 and after 24hrs
Figure 52: Percentages of NK-cell in total and each subset at Time 0 and after 24 hours incubation
Figure 53: (a) Individual (b) Mean (95% C.I.) Nkp46 (m.f.i.) at Time 0 and after 24hrs incubation
Figure 54: (a) Individual (b) Mean (95% C.I.) Nkp30 (m.f.i.) at Time 0 and after 24hrs incubation
Figure 55: (a) Individual (b) Mean (95% C.I.) NKCA (50:1) levels(% killing) of PBMCs incubated with
or without cortisol
Figure 56: (a) Individual (b) Mean (95% C.I.) NKCA (25:1) levels(% killing) of PBMCs incubated with
or without cortisol
Figure 57: Mean (95% C.I.) of the NKCA at Time 0 and after 24hrs incubation of PBMCs with/out
250nM cortisol in the target: effector ratio of 25:1
Figure 58: Correlations between the changes and differences of the NKCA (% killing: between at Time 0
and after 24hrs incubation of PBMCs with/out 250nM cortisol in the ratio of 25:1
Figure 59: Percentages of NK-cell in total and each subset in the PBMCs after 24hours incubation with
and without 250nM cortisol
Figure 60: (a) Individual (b) Mean (95% C.I.) Nkp46 (m.f.i.) after 24hrs incubation of PBMCs with/out
cortisol
Figure 61: (a) Individual (b) Mean (95% C.I.) Nkp30 (m.f.i.) after 24hrs incubation of PBMCs with/out
cortisol
Figure 62: Mean (95% C.I.) and individual expression of Nkp46 (m.f.i.) on the cytotoxic NK-cells
(CD56dimCD16+) at Time 0 and after 24hrs incubation of PBMCs with/out 250nM cortisol
Figure 63: Mean (95% C.I.) and individual level of NKCA at Time 0 and after 24hrs incubation of
PBMCs with/out 250nM cortisol at target: effector ratio of 25:1
Figure 64: Groups defined by tertiary split of the NKCA levels after 24 hours incubation
Figure 65: Mean (95% C.I.) plasma levels of cortisol in the High, intermediate (Mid) and Low NKCA
level groups after 24 hours incubation
Figure 66: Mean (95% C.I.) plasma levels of DHEA-S in the High, intermediate (Mid) and Low NKCA
level groups after 24 hours incubation
Figure 67: Mean (95% C.I.) plasma levels of melatonin in the High, intermediate (Mid) and Low NKCA
level groups after 24 hours incubation
Figure 68: (a) Individual (b) Mean (95% C.I.) background proliferations of PBMCs cultured
Figure 69: (a) Individual (b) Mean (95% C.I.) proliferation response of PBMCs to the PPD antigen
Figure 70: (a) Individual (b) Mean (95% C.I.) proliferation response of PBMCs to the Herpes antigen
Figure 71: (a) Individual (b) Mean (95% C.I.) proliferation response of PBMCs to the SEB at Day 3
with/out cortisol

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Figure 72: (a) Individual (b) Mean (95% C.I.) proliferation response of PBMCs to the PHA at Day 3
with/out cortisol
Figure 73: (a) Individual (b) Mean (95% C.I.) background percentages of CD8 T-cells expressing CD95
Figure 74: (a) Individual (b) Mean (95% C.I.) background percentages of CD4 T-cells expressing CD95
Figure 75: (a) Individual (b) Mean (95% C.I.) percentages of CD8 T-cells expressing CD95 in PHA
stimulated whole blood culture in the absence of cortisol
Figure 76: (a) Individual (b) Mean (95% C.I.) percentages of CD4 T-cells expressing CD95 in PHA
stimulated whole blood culture in the absence of cortisol
Figure 77: (a) Individual (b) Mean (95% C.I.) percentages of CD8 T-cells expressing CD95 in PHA
stimulated whole blood culture in the presence of cortisol
Figure 78: (a) Individual (b) Mean (95% C.I.) percentages of CD4 T-cells expressing CD95 in PHA
stimulated whole blood culture in the presence of cortisol
Figure 79: Mean (95% C.I.) percentages of PHA stimulated CD8 T-cells expressing CD95 after
(a) 24 hours, (b) 48 hours of incubation with and without cortisol
Figure 80: Mean (95% C.I.) percentages of PHA stimulated CD4 T-cells expressing CD95 after
(a) 24 hours, (b) 48 hours of incubation with and without cortisol
Figure 81: Individual percentages of apoptotic (a) CD8 T-cells (b) CD4 T-cells incubated 3 days with
and without cortisol
Figure 82: (a) Individual (b) Mean (95% C.I.) background percentages of CD8 T-cells expressing CD25
Figure 83: (a) Individual (b) Mean (95% C.I.) background percentages of CD4 T-cells expressing CD25
Figure 84: (a) Individual (b) Mean (95% C.I.) percentages of CD8 T-cells expressing CD25 in PHA
stimulated whole blood culture in the absence of cortisol
Figure 85: (a) Individual (b) Mean (95% C.I.) percentages of CD4 T-cells expressing CD25 in PHA
stimulated whole blood culture in the absence of cortisol
Figure 86: (a) Individual (b) Mean (95% C.I.) percentages of CD8 T-cells expressing CD25 in PHA
stimulated whole blood culture in the presence of cortisol
Figure 87: (a) Individual (b) Mean (95% C.I.) percentages of CD4 T-cells expressing CD25 in PHA
stimulated whole blood culture in the presence of cortisol
Figure 88: Mean (95% C.I.) percentages of PHA stimulated CD8 T-cells expressing CD25 after
(a) 24 hours, (b) 48 hours of incubation with and without cortisol
Figure 89: Mean (95% C.I.) percentages of PHA stimulated CD4 T-cells expressing CD25 after
(a) 24 hours, (b) 48 hours of incubation with and without cortisol

List of Tables
Table 1: Classification of the main components of the immune system
Table 2: CD expression by NK-cells and lymphocytes

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Table 3: Definitions of various endogenous molecules


Table 4: Number and percentage of HIV-infected individuals who had CD4 T-cell counts chec at the
monthly time periods from the Recruitment to four months after the recruitment (Term 4)
Table 5: Number and month of subjects who started anti-retroviral medication and dropped from the
study (NB: Term X represents that X months after the Recruitment time point)
Table 6: Mean percentages and counts (standard deviation: SD) of NK-cells, CD4 and CD8 T-cells and
total lymphocyte counts using a single volunteer (9 tubes collected and averaged on three
separate occasions)
Table 7: Mean (95% C.I.) PSS scores at the Non-exam and Exam time points
Table 8: Mean (95% C.I.) State anxiety scores in the STAI at the Non-exam and Exam time points
Table 9: Mean (95% C.I.) NK-cell percentages in male and female students
Table 10: Mean (95% C.I.) levels of NKCA (% killing) in the Not-stressed and Stressed subgroups
Table 11: Mean (95% C.I.) levels of NKCA (% killing) in the Not-stressed and Stressed male students
Table 12: Mean (95% C.I.) ratios of NKCA to NK-cell in the Not-stressed and Stressed students
Table 13: Mean (95% C.I.) ratios of NKCA to NK-cell in the Not-stressed and Stressed male students
Table 14: Mean (95% C.I.) CD4 T-cell levels (%) of male and female
Table 15: The numbers of participant in the three groups (Self-hypnosis, Johrei and Relaxation control)
in the Not-stressed and Stressed subgroups based on the PSS scores at the Exam time point
Table 16: Mean (95% C.I.) State anxiety scores in the Self-hypnosis, Johrei and Relaxation control
groups at baseline and the Exam time point
Table 17: Numbers of participants in the three groups at the Exam time point
Table 18: Mean (95% C.I.) levels of NK-cell (%) in the Self-hypnosis, Johrei and Relaxation control
groups at baseline and the Exam time point
Table 19: Percentages (numbers) of subjects whose NK-cell counts maintained or increased
(Maintained) and decreased (Decreased) in the Self-hypnosis and Johrei and Relaxation
control groups for the Intention-to-treat analysis (missing data were added into the number in
the Decreased group)
Table 20: Mean (95% C.I.) levels of CD4 T-cell (%) in the Self-hypnosis, Johrei and Relaxation control
groups at baseline and the Exam time point
Table 21: Mean (95% C.I.) levels of CD8 T-cell (%) in the Self-hypnosis, Johrei and Relaxation control
groups at baseline and the Exam time point
Table 22: Medians, means and standard deviations of the PSS of the university students at non-exam
and exams and of the HIV-infected individuals at recruitment and four months later
Table 23: Mean (95% C.I.) levels of the PSS in the Not-stressed and Stressed subgroups at the
recruitment and four months later
Table 24: Median, mean and standard deviation of the State anxiety score of the university students at
non-exam and exams and of the HIV-infected individuals at recruitment and four months later
Table 25: Mean (95% C.I.) levels of the State anxiety score of the STAI in the Not-stressed and Stressed
subgroups at the recruitment and four months later

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Table 26: Mean (95% C.I.) levels of the IES in the subgroups of the Not-stressed and Stressed at the
recruitment and four months later
Table 27: Mean (95% C.I.) levels of the LoC in the Not-stressed and Stressed subgroups at the
recruitment and four months later
Table 28: Mean (95% C.I.) levels of the MCS in the Not-stressed and Stressed subgroups at recruitment
and four months later
Table 29: Mean (95% C.I.) levels of the PSQI in the Not-stressed and Stressed subgroups at recruitment
and four months later
Table 30: Correlation between the CD4 gradient (cells per l per month) and the change scores of
perceived quality-of-life scales
Table 31: Mean (95% C.I.) CD4 T-cell change over the five month (cells per l per month) in HIV-
infected individuals of the Increased control and Decreased control subgroups
Table 32: Mean (95% C.I.) CD4 T-cell change over the five month (cells per l per month) in HIV-
infected individuals of the Improved QoL and Decreased QoL subgroups
Table 33: Mean (95% C.I.) CD4 T-cell change over the five month (cells per l per month) in HIV-
infected individuals of the Improved sleep quality and Decreased sleep quality subgroups
Table 34: Mean (95% C.I.) levels of the IES in the HIV-individuals in the Self-hypnosis, Johrei and
Control groups at the Baseline and Post-intervention time points
Table 35: Summary of the numbers (percentages) of the HIV-individuals whose changes in the LoC
between the Baseline and post-intervention either decreased (Gained sense of control) or
increased (Lost sense of control) in the Self-hypnosis, Johrei and Control groups
Table 36: Summary of the numbers (percentages) of the HIV-individuals whose changes in the MCS
between the Baseline and post-intervention either increased (Improved mental quality of life)
or decreased (Decreased mental quality of life) in the Self-hypnosis, Johrei and Control
groups
Table 37: Summary of the numbers (percentages) of the HIV-individuals whose changes in the PSQI
between the Baseline and post-intervention either decreased (Improved sleep quality) or
increased (Decreased sleep quality) in the Self-hypnosis, Johrei and Control groups
Table 38: Mean (95% C.I.) levels of CD4 gradients (counts per l per month) in HIV-infected
individuals in the Self-hypnosis, Johrei and Control groups
Table 39: Percentages (numbers) of participants whose CD4 T-cell counts maintained or decreased over
the five months in the Self-hypnosis and Johrei and Control groups for the Intention-to-treat
analysis (missing data were added into the number in the Decreased group)
Table 40: Mean (95% C.I.) levels of CD4 T-cell (counts per l) in HIV-infected individuals in the
groups of the Self-hypnosis, Johrei and Database-controls at the Training period
Table 41: Mean (95% C.I.) CD4 gradients in HIV-individuals in the Self-hypnosis, Johrei and Database
control groups, and in all the 96 individuals (ALL) at the Pre-intervention period

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Table 42: Percentages of HIV-individuals in the Self-hypnosis, Johrei and Database control groups
whose changes in CD4 gradients between the Pre-intervention and Post-intervention periods
were either increased, stayed the same or decreased with a judgement that a change of four of
less cells per l per month was defined as the same change (Stayed the same subgroup), more
than four increase (Improving subgroup), and more than four decrease (Worsening subgroup)
Table 43: Mean (95% C.I.) CD4 gradients in HIV-individuals in the Self-hypnosis, Johrei and Database
control groups at the Pre-intervention and Post-intervention periods

Appendix 6

Table A-1: Mean (95% C.I.) PSS levels of the Cohorts A and B at the Non-exam and Exam time points
Table A-2: Mean (95% C.I.) State anxiety levels of the Cohorts of A and B at the Non-exam and Exam
time points
Table A-3: Mean (95% C.I.) NK cytotoxic activity in the Cohorts A and B at the Non-exam and Exam
time points
Table A-4: Mean (95% C.I.) NK-cell percentages in the Cohorts A and B at the Non-exam and Exam
time points
Table A-5: Mean (95% C.I.) CD4 T-cell percentages in the Cohorts A and B at the Non-exam and
Exam time points
Table A-6: Mean (95% C.I.) CD8 T-cell percentages in the Cohorts A and B at the Non-exam and
Exam time points
Table A-7: Mean (95% C.I.) NKCA levels at the Non-exam and Exam time points
Table A-8: Mean (95% C.I.) NK-cell and CD4 and CD8 T-cell (%) at the Non-exam and Exam time
points
Table A-9: Number of students in the Not-stressed and Stressed subgroups with regard to gender who
filled in the PSS and had blood collection for lymphocyte and NKCA level analyses
Table A-10: Mean (95% C.I.) NK-cell percentages in the Not-stressed and Stressed subgroups
Table A-11: Mean (95% C.I.) NK-cell percentages in the Not-stressed and Stressed male students
Table A-12: Mean (95% C.I.) NK-cell percentages in the Not-stressed and Stressed female students
Table A-13: Mean (95% C.I.) levels of NKCA (% killing) in the Not-stressed and Stressed female
students
Table A-14: Mean (95% C.I.) levels of NKCA (% killing) of male and female
Table A-15: Mean (95% C.I.) ratios of NKCA to NK-cell in the Not-stressed and Stressed female
students
Table A-16: Mean (95% C.I.) ratios of NKCA to NK-cell in male and female students
Table A-17: Mean (95% C.I.) CD4 and CD8 T-cell levels (%) in the Not-stressed and Stressed
subgroups

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Table A-18: Mean (95% C.I.) CD4 and CD8 T-cell levels (%) in the Not-stressed and Stressed male
students
Table A-19: Mean (95% C.I.) CD4 and CD8 T-cell levels (%) in the Not-stressed and Stressed female
students
Table A-20: Mean (95% C.I.) CD8 T-cell levels (%) of male and female students
Table A-21: Mean (95% C.I.) PSS levels in the three groups at the Exam time point
Table A-22: Mean (95% C.I.) PSS scores in male subjects in the three groups at the Exam time point
Table A-23: Mean (95% C.I.) PSS scores in female subjects in the three groups at the Exam time point
Table A-24: Mean (95% C.I.) State anxiety scores in the male subjects of the Self-hypnosis, Johrei and
Relaxation control groups at baseline and the Exam time point
Table A-25: Mean (95% C.I.) State scores in the female subjects in the Self-hypnosis, Johrei and
Relaxation control group at baseline and the Exam time point
Table A-26: Mean (95% C.I.) NKCA levels (% killing) in the three groups at the Exam time point
Table A-27: Mean (95% C.I.) NKCA levels (% killing) in male subjects in the three groups at the Exam
time point
Table A-28: Mean (95% C.I.) NKCA levels (% killing) in female subjects in the three groups at the
Exam time point
Table A-29: Mean (95% C.I.) levels of NK-cell (%) of male students in the groups of the Self-hypnosis,
Johrei and Controls at baseline and the Exam time point
Table A-30: Mean (95% C.I.) levels of NK-cells (%) in the female subjects in the Self-hypnosis, Johrei
and Relaxation control groups at baseline and the Exam time point
Table A-31: Mean (95% C.I.) levels of CD4 T-cell (%) of male students in the Self-hypnosis, Johrei and
Relaxation control groups at baseline and the Exam time point
Table A-32: Mean (95% C.I.) levels of CD4 T-cells (%) in the female students in the Self-hypnosis,
Johrei and Relaxation control groups at baseline and the Exam time point
Table A-33: Mean (95% C.I.) levels of CD8 T-cell (%) of male students in the Self-hypnosis, Johrei and
Relaxation control groups at baseline and the Exam time point
Table A-34: Mean (95% C.I.) levels of CD8 T-cells (%) in the female students in the Self-hypnosis,
Johrei and Relaxation control groups at baseline and the Exam time point
Table A-35: Correlation between the CD4 gradient (cells per l per month) and the stress perception
scores and the perceived quality-of-life at recruitment time point (pre) and four-months
after the recruitment (post)
Table A-36: Correlations between the CD4 gradient (cells per l per month) and the change scores of
stress perception scales
Table A-37: Number of HIV-infected individuals who had viral load level check at the monthly time
periods from the Recruitment to four months after the recruitment (Term 4)

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Table A-38: Correlation between the viral load level gradients (log-transformed viral copies per l per
month) and the stress perception and the perceived quality-of-life scores at recruitment time
point (pre) and four months later (post)
Table A-39: Correlation between the viral load level gradient (log-transformed copies per l per month)
and the change scores of perceived stress and quality-of-life scores
Table A-40: Correlation between the NK gradient (cells per l per month) and the stress perception
scores and the perceived quality-of-life at recruitment time point (pre) and four-months
after the recruitment (post)
Table A-41: Correlation between the NK gradient (cells per l per month) and the change scores of
perceived stress and quality-of-life scores
Table A-42: Mean (95% C.I.) levels of the State anxiety and PSS scores in the HIV-individuals in the
Self-hypnosis, Johrei and wait-listed control groups at the Baseline and the Post-
intervention time points
Table A-43: Mean (95% C.I.) levels of the LoC, MCS and PSQI in the HIV-individuals in the Self-
hypnosis, Johrei and wait-listed control groups at the Baseline and Post-intervention time
points
Table A-44: Mean (95% C.I.) levels of regression gradients in viral load levels (Log-transformed) in
HIV-infected individuals in the Self-hypnosis, Johrei and wait-listed control groups
Table A-45: Mean (95% C.I.) levels of regression gradients in NK-cell (counts per l per month) in
HIV-infected individuals in the Self-hypnosis, Johrei and wait-listed control groups

List of Text boxes


Text box 1: Project synopsis 21
Text box 2: Appraisal of a stressor 38
Text box 3: Elements of coping with a threat 38
Text box 4: Psychological interventions in this project 43
Text box 5: Summary of hypotheses Re: Stress-related changes of endocrine system which may
contribute to suppress cellular immune responses under sustained stress 48

Ph.D. at University of London - 14 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Abbreviations

ACTH: adrenocorticotrophic hormone NK cell: Natural Killer cell


ANOVA: analysis of variance NKCC: Natural killer cytotoxic activity
AP-1: activation protein 1 PBL: peripheral blood lymphocyte
ART: anti-retroviral treatment PBMCs: peripheral blood mononuclear cells
CBG: cortisol binding globulin PBS: phosphate buffer saline
CD: clusters of differentiation PEI: Personalised Emotional Index
CFSE: carboxyfluorescein succinimidyl ester PFA: paraformaldehyde
cpm: count per minute PHA: phytohaemagglutinin A
CRF: corticotrophin releasing factor / (hormone) PI: propidium iodide
CRP: C-reactive protein PNI: psycho-neuro-immunology
CSF: Colony stimulating factor PPD: purified protein derivative
CTL: (cytotoxic / cytolitic T lymphocyte) PSS: Perceived Stress Scale
CV: coefficient of variation PSQ: Personality Syndrome Questionnaire
DHEA (-S): dehydroepiandrosterone (sulphate) PSQI: Pittsburgh Sleep Quality Inventory
EEG: electroencephalogram QoL: quality of life
E:T ratio: Effector vs. Target ratio RCT: randomised controlled trial
FITC: fluorescein isothiocyanate REM: rapid eye movement
FCS: foetal calf serum SAM: sympathetic adreno-medulla
GABA: gamma aminobutyric acid SAS: Statistical Analysis Software
GCR: glucocorticoid receptor SD: standard deviation
HIV: human immunodeficiency virus SEB: Staphylococcal Enterotoxin B
HPA: hypothalamus pituitary adrenal (axis) SIBS: Spiritual Involvement and Beliefs Scale
HSV: herpes simplex virus SoC: sense of coherence
IES: Impact of Event Scale SOPs: standard operating procedures
IL: Interleukin SPSS: Statistical Package for Social Sciences
IFN: interferon STAI: State and Trait Anxiety Inventory
ITAM / ITIM: immunoreceptor tyrosine-based TCI: Temperament and Character Inventory
activation / inhibitory motifs TCM: tissue culture medium
KIR: killer immunoglobulin like receptor Th.: Helper T lymphocyte
KPSS: Kesseler Perceived Social Support TNF: Tumour necrosis factor
LoC: Locus of Control T-reg: regulatory helper T lymphocyte
LPS: lipopolysaccharide TUNEL: TdT-mediated dUTP nick end labelling
MCR: mineral corticoid receptor WHO: World Health Organisation
MHC: major histocompatiblity complex
NCR: natural cytotoxic receptors
NF B: nuclear factor kappa B

Ph.D. at University of London - 15 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Acknowledgements
I wish to thank all the participants who took parts in the project as study subjects.
The work in this thesis was funded by the Johrei Academy and Johrei Association. I am most grateful to
them for their generosity and for their kind support in developing materials of the Johrei intervention.

I would like to give special thanks to Dr. Don C. Henderson for his endurable close supervision and Prof.
John H. Gruzelier for allowing me to start and conduct the project. They have given me inspiration to see
the project through. Dr. Tannis M. Laidlaw, Dr. Prabudha Dwivedi and Mr. Bryan Bennett worked
together with me as a research team for the in vivo studies. I am very grateful to them. One of the
psychological interventions, the Self-hypnosis training and follow-up sessions, was planned and
conducted by Dr. Laidlaw. The mock neuro-feedback sessions (relaxation control) were conducted by Dr.
Dwivedi. Mr. Bennett was in charge of patient recruitment and organised meetings for the participants.
Without any of them, this project would undoubtedly have never been completed, or even started. I am
most indebted to their great contributions, personal help and warm support.

My special thanks also go to Prof. Adrian P. Burgess and Dr. Martin R. Goodier. They gave me very
useful and encouraging inputs at the transfer exam to Ph.D. from M.Phil. Dr. Alan W. Steel contributed to
the in vivo HIV-study as an independent researcher to select case matched controls. Dr. Simon E. Barton
kindly permitted and gave support to recruit HIV-patients in his clinic. Mr. Mohamed Shamji helped me
to start the in vitro experiments before the project commenced. I am indebted to their kind help and warm
support. I appreciate M.Sc. student researchers, Ijeoma Ugwu-Onuoha and Alex Gale, and B.Sc. student
researchers, Linda Farahani, Catrina Lynch, Nick Enzor, Christine Brincat, Tom French and Helena
Marconell, for their contribution to the studies. I appreciate the secretarial assistance given by Mrs. Ann
Ebberson throughout my Ph.D. course. Thanks also go to all the research staff / clinical laboratory staff /
students at Charing Cross Neuroscience Division, at Chelsea and Westminster Immunology Department
and St. Stephen’s AIDs Trust Kobler Centre who kept me going when I was struggling.

In addition, I am very grateful to Prof. Mark Jensen, Prof. Yukihisa Kurasawa, Dr. Graham Jamieson, Dr.
Gerald Stein, Prof. Frances Gotch, Dr. Nathalie Fouquet, Dr. Philippe Donatien, Dr. David Vernon, Dr.
Malcolm Hawken, Prof. Tom Sensky and Prof. Julia Buckingham for their comments on the manuscript. I
would like to thank my examiners, Prof. Phil Evans and Rev’d Prof. Nick Goulding, for their thoughtful
and useful suggestions. To the aforementioned and many other unnamed colleagues and friends, who
have also helped towards the completion of this thesis, I extend my sincere thanks.

Akira NAITO
London, August, 2006

Ph.D. at University of London - 16 - Imperial College London


Chapter I

Introduction

- 17 -
Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

CONTENTS OF CHAPTER 1
Introduction

1.1 Project overview 19

1.2 Theoretical framework


1.2.1 Immune system 22
1.2.1.1 Innate and adaptive immunity 22

1.2.1.2 Communication between immune cells 27

1.2.2 Neuro-endocrino-immune interaction 30


1.2.2.1 Brain-to-immune pathway 30

1.2.2.2 Immune-to-brain pathway 32

1.2.2.3 Circadian rhythm in the neuro-endocrino-immune network 33

1.2.2.4 Circadian rhythm and subjective well-being 35

1.2.3 Psychological input in the psycho-neuro-endocrino-immune network 36


1.2.3.1 Stressor 36

1.2.3.2 Stress perception 37

1.2.3.3 Psychological training intervention 39

1.2.4 Stress-associated changes 43


1.2.4.1 Chronological definition of stress response 43

1.2.4.2 Acute stress responses in the neuro-endocrino-immune network 44

1.2.4.3 Sustained stress and changes in the psycho-neuro-endocrino-


immune network 46

1.3 Approach taken 49

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

1.1 The project overview

Stress is inevitable in everyday life and it affects mental and physical well-being both
beneficially and detrimentally [Cohen & Herbert, 1996; Evans et al., 2000; Marmot, 2004]. This
project is based on the hypothesis that the detrimental effects of stress upon psychological well-
being and general health can be alleviated by psychological intervention [Text box 1].

Psychological intervention in the context of psycho-somatic medicine [Waldstein et al.,


2001; Walker et al., 1999] has both training and practice aspects, both of which aim to provide
other perspectives of life events (stress perception) and coping skills (stress management) in
order to reduce the levels of stress. Two psychological interventions used in this project were
Self-hypnosis and Johrei, a Japanese system encompassing stress management. These
interventions aimed to encourage the participants to secure their own time for themselves with
relaxation techniques as well as to reduce stress by providing alternative perspectives of, and
coping strategies for, stressful life events.

The fields of ‘psycho-neuro-endocrinology’ [de Kloet, 2000; Sapolsky et al., 2000] and
‘psycho-neuro-immunology’ [Ader & Cohen, 1993] are based on the concept of Descartes'
‘mind and body’ or ‘the way with the body-mind’ [Yuasa & Kasulis, 1987]. A growing body of
evidence suggests that there are cross regulatory influences between psychological, neurological,
endocrinological and immunological systems. This has led investigators to propose the
existence of an integrated psycho-neuro-endocrino-immune network, in which a change in one
facet has consequential influences upon other parts of the network. The primary mediators in the
network include catecholamines from the sympathetic-adreno-medulla (SAM) system, adrenal
hormones from the hypothalamus-pituitary-adrenal (HPA) axis and a myriad of components of
the immune system, including individual cells, cell surface receptors, and intracellular and
intercellular messengers (cytokines).

The field of stress research has not yet agreed upon a definition of stress that is used in all
studies. For example, the distinction between acute and sustained stress is ambiguous and varies
from study to study. In the current thesis, acute or sustained stress was distinguished on the
basis of time in a biological cycle. Acute stress was defined as when effect of stress or stress
response is contained within one day, i.e. a single circadian rhythm. In contrast, sustained stress
was defined as stress that the effect lasts for more than one day. This leads to a working
hypothesis that sustained stress is associated with altered circadian patterns of the psycho-

Ph.D. at University of London - 19 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

neuro-endocrino-immune network. Since the psychological interventions used in the project


require weeks of training and practice, this investigation focused on the sustained stress.

Previous research has shown that the stress-associated inappropriate response of the
immune system has been shown to comprise:
1. Impaired cellular immune responses, which lead to an increase in susceptibility to
infection and malignancy [Garssen, 2004]; and
2. Excessive inflammatory reactions, which may be due to impaired suppressive self-
regulation, or over-production of pro-inflammatory cytokines leading to an
augmented severity of symptoms of allergy or autoimmune diseases [Cleare, 2003].

This project focused on the first part - the stress-induced impairment of cellular immunity.

The effects of stress and psychological intervention upon the psycho-neuro-endocrino-


immune network were investigated in vivo in university students facing exams as an example of
individuals with time-limited sustained stress, and in HIV-infected adults as an example of
individuals with ongoing, life-long, disease-associated stress. General and mental well-being
were investigated through questionnaires designed to measure perceived stress, anxiety, quality-
of-life, and sleep quality. The effects of stress upon the psycho-neuro-endocrino-immune
network were examined through immune profiles including disease parameters. Associations
between the mediators of the psycho-neuro-endocrino-immune network were also investigated
in vitro in order to demonstrate any direct links between hormone levels and immune
parameters.

Ph.D. at University of London - 20 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Text box 1: PROJECT SYNOPSIS

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Ph.D. at University of London - 21 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

1.2 Theoretical framework

1.2.1 Immune system


Humans are constantly prone to infection by a vast range of organisms (viruses, bacteria,
fungi, and multi-cellular parasites) and to malignancy through mutations from ‘self’. It is the
functional responsibility of the immune system to recognise non-self and altered-self and to
produce an appropriate immune response to remove it in order to maintain the general health of
the body. Any immune response involves, first, recognition of the pathogen or mutation, and
then second, a reaction to eliminate it.

1.2.1.1 Innate and adaptive immunity


The immune system has been classified into two components: (1) innate (inborn: non-
specific) immunity; and (2) adaptive (acquired: pathogen-specific) immunity [Table 1].

Innate immunity
Innate immunity is non-specific and is defined as the in-born defence mechanism of the
body. The innate natural immune system is present at birth and is responsible for the early /
immediate defence against infection and malignancy. Although this form of immunity has no
memory of non-self, these cells are able to recognise non-self organisms (tumour cells or
infected cells) by ‘missing self’-signal [Moretta et al., 2002], and defend the body, even when
the trigger is not sufficient to activate the adaptive immune system. There are two elements to
innate immunity, i.e. humoral and cell-mediated immunity:

1. Humoral immunity: the secreted molecules, which include complement, lysozome,


fibronectin, acute phase proteins (e.g. C-reacted protein (CRP)), eicosanoids, natural
antibodies and pro-inflammatory cytokines (e.g. Interleukins (ILs), Interferons (INFs),
colony stimulating factors (CSFs) and tumour necrosis factors (TNFs)).

2. Cellular immunity: the principal cell types in innate immune system are phagocytes
(e.g. macrophages, monocytes and granulocytes including neutrophils) and natural
killer (NK) cells.

Ph.D. at University of London - 22 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Adaptive immunity
Adaptive immunity, on the other hand, is the defence mechanism that recognises and
fights against specific infectious organisms and develops memory of specific antigens of such
pathogens. This allows all subsequent responses to the same infection to proceed more rapidly
and effectively. This adaptive immunity also has humoral and cellular components.

1. Humoral immunity is mediated by a vast amount of pathogen-specific antibodies


produced by B-lymphocytes. Antibodies provide protection from pathogens by
neutralising toxic components of pathogens, by coating pathogens and thereby
targeting for disposal by phagocytes (this is known as opsonisation), and by activating
complement to trigger lyses and ingestion of pathogens by phagocytes. Specific
molecules produced by immune cells (namely, Th.1 and Th.2 cytokines) [see also
Table 1] also play a major role in regulation of the adaptive immune system as media
of cell-cell communication.

2. Cellular immunity is based on the activities of two types of lymphocytes, T- and B-


lymphocytes. Each lymphocyte can specifically recognise and react with one of the
thousands of foreign proteins, i.e. antigenic epitopes found on / in pathogens.

The B-lymphocytes are responsible for the production of specific antibodies, and
have B-cell antigen receptor (BCR). The BCR is a membrane-bound form of the
antibody that the B-lymphocyte will secrete after following activation by specific
antigen and differentiation into plasma cells.

The T-lymphocytes have been subdivided into two sub-populations according to


their functions, i.e. the cytotoxic T-lymphocyte (CTL) and the helper T-lymphocyte
(Th.). Both subsets express T-cell antigen receptors (TCR). The TCR is specially
adapted to detect antigens derived from foreign proteins and pathogens.

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Table 1: Classification of the main components of the immune system


HUMORAL CELLULAR
Cytotoxic NK-cells
Natural Killer (NK) cells
Cytokines (Pro-inflammatory) Regulatory NK-cells
Acute phase proteins Macrophages
INNATE Complement Monocytes
Lysozome Phagocytes Neutrophils
Natural antibodies Granulocytes Eosinophils
Basophils
Th. 1 Cytotoxic T-cells (CTL)
(cellular)
Cytokines T-lymphocytes Type I (Th. 1)
Th. 2 Helper T-cells Type II (Th. 2)
ADAPTIVE (humoral) Regulatory (T-reg)
IgM
Antibodies IgG
B-lymphocytes (and Plasma cells)
(Immunoglobulin) IgA
IgE

Natural Killer (NK) cells


The Natural Killer (NK) cells are one of the principal cell types in the innate cellular
immune system. They are large granular lymphocytes [Figure 1], which develop from common
lymphoid progenitor cells. They make up about 5-15% of the mononuclear cell fraction in
normal peripheral blood.

Figure 1: A typical Natural Killer cell


[Taken from Wasatch Health Group: http://www.wasatchhealth.com/Pages/NK-cell.html]

There are two subsets of NK-cells [Cooper, Fehniger & Caligiuri, 2001], i.e. 10-20% of
regulatory NK-cell subset (cytokine producing subset [Cooper, Fehniger, Turner et al., 2001])
and 80-90% of cytotoxic NK-cell subset [Jacobs et al., 2001], which has two possible killing
mechanisms:
1. Cytolitic granules make a hole in the membrane of target cells causing necrosis; and
2. Granules or a cell-cell contact (e.g. Fas - Fas-ligand contact) triggers a cascade for
‘programmed cell death’ (known as apoptosis) [Opferman & Korsmeyer, 2003] in the
target cell.

Ph.D. at University of London - 24 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

The cytolitic granule pathway employed by cytotoxic NK-cells is the same killing
mechanism as that applied by cytotoxic T-cells (CTL). Cytotoxic granules, which contain
Perforin and Granzyme A / B, are released onto the surface of the bound target cell, and the
Perforin makes a hole to destroy the cell membrane and Granzyme induces apoptosis. This
secretion of granules with contact to target cells takes a few seconds and is known as the ‘kiss of
death’ [Trambas & Griffiths, 2003]. Apoptosis is essential in the cell cycle to maintain cellular
homeostasis [Opferman & Korsmeyer, 2003]. The Fas-receptor [Hingorani et al., 2000] was
shown to trigger an intra-cellular cascade inducing apoptosis when the Fas-ligand from other
cells contacts [Alenzi & Warrens, 2003].

Target recognition by NK-cells


The main role of NK-cells is to recognise and kill virally infected-cells and tumour-cells
both of which have escaped recognition and death by other immune components. The exact
mechanism of the recognition by NK-cells is unclear, but there has been a hypothesis that NK-
cells can detect ‘missing self’ [Moretta et al., 2002]. In other words, NK-cells lack antigen
specificity but are able to identify ‘non-self’. All human cells express the major
histocompatiblility complex (MHC) class I molecules on the cell-surface. The levels of
expression of this MHC decrease when cells are infected or turn into tumour cells, and this
decrease of the MHC class I molecule is called the ‘missing self’ [Moretta et al., 2002].

Recognition of target cells by NK-cells is achieved by combination of two categories of


receptors, i.e. activating and inhibitory receptor groups [Middleton et al., 2002]. Recognition of
‘missing-self’ means that balance between activating and inhibitory signals weighs toward
activation due to a lack of the MHC class I. Recognition of ‘self’ through normal expression of
MHC Class I triggers NK-cell inhibitory signals, which switch off activating signals.

NK-cell receptors
The NK-cell surface receptors comprise two families:
1. Immunoglobulin super family, including killer immunoglobulin-like receptors (KIR);
and
2. C-type lectin like receptors including NKG2D and natural cytotoxic receptors (NCR)

A single signal from one of these receptors is not sufficient to trigger either the beginning
or the end of NK-cell activation. Instead, the trigger is the result of combined signals of these

Ph.D. at University of London - 25 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

NK-cell receptors [Biassoni et al., 2001; Mandelboim & Porgador, 2001]. The mechanisms of
the intra-cellular signals cascades are complex [Middleton et al., 2002] and not clearly defined.
Middleton et al. [2002] have recently described one model, which is based on the intra-cellular
structure of these two family receptors, i.e. the immuno-receptor tyrosine-based inhibitory
motifs (ITIM) and the immuno-receptor tyrosine-based activation motifs (ITAM) [Bakker et al.,
2000].

The ITIM are the receptor motifs with long cytoplasmic tails that are responsible for the
inhibitory signals associated with recognition of MHC class I molecules, whereas the ITAM are
the receptor motifs with short cytoplasmic tails and give rise to the activation signals. All
reactions seem to be the result of the balance between these inhibitory and activation signals.
Middleton explained the regulation by means of the length of their cytoplasmic tails, i.e. ITAM
has shorter and ITIM has longer/deeper cytoplasmic tails. Activation signals can be blocked by
inhibitory signals in deeper or more peripheral levels of intra-cellular cascade when there are
enough inhibitory signals from the ITIM to overcome activation signals from the ITAM. By
these complex mutual mechanisms, the NK-cell maintains its balance between insufficient
reaction against the target (non-self or altered-self) and over-reaction extending to the ‘self’.

T-lymphocytes
The T-lymphocytes are one of the principal cell types in the adaptive cellular immune
system. They make up about 60-85% of the mononuclear cell fraction in normal peripheral
blood (peripheral blood mononuclear cells: PBMCs). There are two subsets of T-lymphocytes:
35-80% of them are helper T-lymphocytes (Th.) which orchestrate both cellular and humoral
immunological responses by producing cytokines and by cell-cell contacts via T-cell receptor
(TCR); and 20-65% of them are cytotoxic T-lymphocytes (CTL).

The helper T-lymphocytes have been further subdivided into three phenotypes (Table 1):
1. Type I helper T-lymphocyte (Th.1);
2. Type II helper T-lymphocyte (Th.2); and
3. Regulatory T-lymphocyte (T-reg)
according to their roles in defence [Jonuleit & Schmitt, 2003; McHugh & Shevach, 2002].
The Th.1 cells principally orchestrate cellular immunity, the Th.2 cells enable humoral
immunity to react against pathogens, and the T-reg cells mainly regulate to suppress excessive
inflammatory reactions in cellular immunity.

Ph.D. at University of London - 26 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

In contrast to NK-cells which can attack ‘non-self’ in a non-specific manner as described


above, each T-lymphocyte has a unique recognition against a specific antigen. This specificity
of an antigen works by using the MHC class I (for cytotoxic T-lymphocyte) or class II (for
helper T-lymphocyte) molecules. Because any ‘non-self’ particles can be an antigen, the total
population of T-lymphocytes collectively bears a huge repertoire of receptors, and most of them
in circulation are inactive or resting. These repertoires are the results of the high level of
diversity in the prototype antigen receptor. This mechanism in production of huge variety of
antigen specific T-cell receptors is similar to the mechanism in production of a wide range of
antigen specific antibodies by B-cells and plasma cells.

Recognition and elimination of specific pathogens by T-lymphocytes


The role of T-lymphocytes is to recognise and arrange, either, directly or indirectly to
eliminate foreign pathogens. This recognition of pre-exposed antigen (recall antigen) is based
on a huge variety of antigen receptors, and the elimination of a pathogen depends on the ability
of rapid proliferation of one specifically recognised set of lymphocytes. The proliferative
response of T-lymphocytes consists of four processes:
1. Recognition of the foreign pathogen to match a pre-exposed / recall antigen;
2. Activating cell cycle (G1-S-G2-M) progression of the one type of cell matched;
3. Actual cell divisions and proliferation; and
4. Apoptosis of excessive cells generated through the above proliferation.
This process of T-lymphocyte proliferation is known to occur also in peripheral
lymphocytes both in vivo and ex vivo, particularly significant in response to in vitro stimulation
with a mitogen, a super-antigen, or specific recall antigens [Antia et al. 2003; Kaech and Ahmed
2003; Seder and Ahmed 2003].

1.2.1.2 Communication between immune cells


Immune cells (e.g. NK-cells, lymphocytes and phagocytes) communicate with each other
either directly by cell-cell contact, or indirectly via various secreted molecules, which interact
with cell surface molecules or intra-cellular (cytoplasmic or nuclear) receptors.

Many surface markers (receptors) have been identified and named according to the
clusters of differentiation (CD) system [Table 2]. This allows cell types (phenotypes) to be
identified according to their expression of cell surface markers, e.g. helper T-lymphocytes
(CD3+CD4+), cytotoxic T-lymphocyte (CD3+CD8+), B-cells (CD19+), NK-cells (CD3-CD56+)
and NKT-cells (CD3+CD56+). This identification can be performed by using a flow cytometer

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

which detects fluorescently conjugated specific antibodies bound to unique CD markers /


molecules expressed on the cell surface.

For example, NK-cells are characterised by the expression of CD56 molecules on the
surface and the absence of a common lymphocyte marker (CD3) which distinguishes it from
another cytotoxic lymphocyte sub-population (NKT-cells: CD3+CD56+ lymphocytes) [Seaman,
2000]. Further CD16 can be used to distinguish between NK-cell subsets, i.e. the cytotoxic NK-
cells (CD56Dim) express CD16 while the regulatory NK-cells (CD56Bright) are CD16 negative
[see also Table 2].

Table 2: CD expression by NK-cells and lymphocytes


Cytotoxic CD56Dim & CD16+
NK-cells CD3 (-) & CD56+
Regulatory CD56Bright & CD16 (-)

CD3+ & CD56+ NKT-cell

Cytotoxic CD8+
T-lymphocytes CD3+
Helper CD4+

B-lymphocytes CD19+

The secreted molecules which act as intra-cellular messengers between immune cells are
known as cytokines. Cytokines consist of three groups [see also Table 1]: (1) pro-inflammatory
(IL-1, IL-6, TNF- , INF- etc); (2) Th.1 (IL-2, IL-12 etc.); and (3) Th.2 (IL-4, IL-10 etc.)
cytokines. They have been classified into the cytokine families such as Interleukins (IL),
Interferons (INF), Colony stimulating factors (CSF), Tumour necrosis factors (TNF) and a
range of chemokine families [Janeway et al., 2001].

As new cytokines were discovered [Janeway et al., 2001], the distinctions between
various types of endogenous molecules, which are utilised for cell-cell communication, became
increasingly ambiguous due to overlapping functions [Table 3]. Initially these molecules were
considered to be produced by specific cells and to affect specific cells only in each organ system.
Recent evidence, however, has revealed that these molecules can be produced by a variety of
different cells and that their receptors are expressed by a vast range of types of cells in different
organ systems [Hansen et al., 1998; Marz et al., 1999; Miller et al., 1994; Turnbull & Rivier,
1999].

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Table 3: Definitions of various endogenous molecules


Hormones
Chemical substances produced in an organ or gland in the body that are
secreted into the blood and carried to other organs or parts of the body
Neurotransmitters Chemical substances released from neurons in synapses that bind to
(Catecholamines & Acetylcholine) corresponding receptors on nearby cell surfaces or activate secondary
messenger cascade, i.e. channels, pumps, kinases, or proteases
Cytokines Proteins that are the core of communication between immune cells
themselves, and with cells belonging to other tissue types
 .
Chemokines Proteins that signal leukocytes to move in a specific direction; in other
 . words, a class of chemotactic cytokines
Eicosanoids Biosynthesised molecules from arachidonic acid (poly-unsaturated
(Arachidonic acid derivatives) fatty acid), i.e. prostaglandins, prostacyclins, thromboxanes,
leukotrienes etc.; in other words, a class of oxygenated hydrophobic
cytokines

Communication and interaction between organ systems


The primary organ systems which are distributed throughout the body and in which
mediators are not-restricted in location are the nervous system, the endocrine system and the
immune system. They are considered to formulate independent cell-cell communication
networks by various signal transportation systems (e.g. electrical signals and molecules), and
these communication networks are shown to have their own independent regulatory
mechanisms including counter regulation, buffering mechanisms and/or feedback mechanisms.

For example, in the autonomic nervous system, the sympathetic and the parasympathetic
systems have opposite effects; the sympathetic nervous system suppresses the parasympathetic
nervous system and vice versa [Boeree, 2002].

In the endocrine system, the hypothalamus-pituitary-adrenal (HPA) axis has the


interactive modulation, namely ‘corticotrophin-releasing-factor (CRF) – adrenocorticotrophic-
hormone (ACTH) – glucocorticoid (cortisol in human)’ modulation. This ‘CRF-ACTH-cortisol’
modulation has feedback mechanisms, by which increased levels of cortisol secreted from
adrenal glands suppresses production of CRF and ACTH in the brain [Tsigos & Chrousos,
2002]. This modulation also contains buffering mechanisms where the levels of free cortisol can
be maintained in a narrow window range with a minimum time of over fluctuation as in the
following mechanism. The level of free cortisol in serum depends on the ratio of cortisol to
cortisol binding globulin (CBG) so that a sudden change of free cortisol level can be buffered
by changing this cortisol-CBG ratio, i.e. percentages of cortisol which bind to the CBG
[McEwen et al., 1997].

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In the immune system, counter-regulatory mechanisms of the Th.1 cells and the Th.2 cells
work to maintain long term balance between them [Kidd, 2003], i.e. Th.1 (cellular) cytokines
can suppress Th.2 (humoral) cytokine production and vice versa.

Furthermore, it has been revealed that cells also communicate over these different organ
systems [see below 1.2.2 for the neuro-endocrino-immune interaction], and it has been
suggested that these nervous, endocrine and immune systems have mutual regulatory
mechanisms between themselves as an interactive neuro-endocrino-immune axis [Elenkov et al.,
2000; Haas & Schauenstein, 2001; McEwen et al., 1997].

1.2.2 Neuro-endocrino-immune interaction


The interactive neuro-endocrino-immune axis has often been discussed in the context of
stress responses [Besedovsky & del Rey, 2001; Biondi, 2001], and it has been proposed that
psychological stress can modulate the immune system via various interactive mechanisms in
this neuro-endocrino-immune axis [Moynihan & Stevens, 2001] [Figure 2].

Figure 2: Schema of the neuro-endocrino-immune interaction.


(HPA: hypothalamus-pituitary-adrenal axis, SAM: sympathetic-adrenal-medulla modulation)

1.2.2.1 Brain-to-immune pathway


In recent decades, two major axes have been proposed as an important pathways in the
stress response: (1) the sympathetic nervous system-adreno-medulla (SAM) and (2) the
hypothalamus-pituitary-adrenal (HPA) axes [de Kloet, 2000; de Kloet et al., 1998; Downing &
Miyan, 2000; Heuser & Lammers, 2003; Yang & Glaser, 2002] [Figure 2]. This section
introduces the pathway from the brain to the immune system [Elenkov et al., 2000; Lawrence &
Kim, 2000].

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The ‘fight or flight’ response [Cannon, 1914; Cannon, 1932; Cannon, 1934] and emotions
like anxiety and/or fear [LeDoux, 1998] are known to be associated with increased activity in
the sympathetic nervous system. There are two primary pathways by which the sympathetic
signals are conveyed to target organs [Elenkov et al., 2000]. One pathway is direct neural
connections, where norepinephrine functions as a neurotransmitter in a neural terminal. The
other is known as the sympathetic-adreno-medulla (SAM) pathway, where epinephrine (as a
response to the sympathetic neural activity) released from the adrenal glands travels through the
blood stream and acts as a hormone in various organs. For example, the sympathetic neural
fibres descend from the brain into a neural terminal both in the primary lymphoid tissues (bone
marrow and thymus) and the secondary lymphoid tissues (spleen and lymph nodes) [Felten &
Felten, 1994]. Immune cells themselves have also been reported to express catecholamine
receptors, but their profiles are unique, i.e. NK-cells have high-density and high-affinity beta-2-
adrenergic receptors, B-cells have high-density but low-affinity, and T-cells have low-density
and low-affinity[Anstead et al., 1998]. This is in line with the findings that catecholamines,
norepinephrine and epinephrine activate the immune response, including an increase of NK-
cells and cytotoxic T-cells in circulation [Hennig et al., 2000] and an increase of pro-
inflammatory cytokine production [Elenkov et al., 2000]. This series of evidence strongly
suggests that the sympathetic nervous system (particularly with the SAM axis) directly
communicates with immune cells in the brain-to-immune pathway.

On the other hand, hormones in the hypothalamus-pituitary-adrenal (HPA) axis have also
been considered to play an important role as connectors or modulators between the brain and
immune system [Flier & Underhill, 1995; Reichlin, 1993]. Cortisol, the main secreted hormone
to the blood stream in the HPA axis, is believed to be a major stress-related hormone
[Hucklebridge et al., 1998; Rohleder et al., 2001]. It has been demonstrated that the major
immune organs, i.e. spleen and thymus, and immune cells themselves are rich in cortisol
receptors [Miller et al., 1994]. The effect of cortisol upon individual target cells, however, is
suggested to vary from suppressive to stimulatory, i.e. suppressive, preparative, permissive and
stimulatory actions [Sapolsky et al., 2000]. The various factors contributing to this
differentiation include:
Types of relevant target cells and their cortisol receptor profiles;
Timing of secretion, duration of exposure, and concentration of cortisol; and
Combination with cascading secretions of other molecules (hormones and
cytokines).

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In addition to this complexity, the exact mechanisms at the gene level are also yet to be
made clear. For example, cortisol is known to be antagonistic against the nuclear factor kappa B
(NF B) [Chrousos, 1995; Goulding, 2004], which is a known activator for the transcription of
pro-inflammatory cytokines (IL-1, IL-6 and TNF- ) [Barnes, 1997] as well as a genomic
inhibitor against the apoptotic cascades [Karin & Lin, 2002]. By contrast, the cortisol-induced
gene products are known to comprise a vast range of proteins including the Annexin I, which
has comprehensive anti-inflammatory intra-cellular actions [Goulding, 2004]. Nevertheless, the
evidence strongly supports the conclusion that the mediators of the HPA axis (particularly
cortisol) can exert direct influence upon immune cells in the brain-to-immune pathway.

Furthermore, in vivo laboratory stress studies have illustrated that there is a consistent
order of responses in the change levels of mediators between the SAM and the HPA axes, i.e. an
increase of cortisol levels was observed after an increase of catecholamine levels [Clow et al.,
1997; Clow et al., 2000; Hucklebridge et al., 1998; Madden, 2001; Rohleder et al., 2001;
Uchino et al., 2001]. This suggests that catecholamines may promote cortisol secretion, so that
these two main axes of the brain-to-immune pathway in the psycho-neuro-endocrino-immune
network may also communicate with each other.

1.2.2.2 Immune-to-Brain pathway


Reciprocally, it has been discovered that the pro-inflammatory cytokines (IL-1, IL-6 and
TNF- ) can activate the SAM and HPA axes [Besedovsky & del Rey, 2000; Buckingham et al.,
1996; Perlstein et al., 1993; Turnbull & Rivier, 1999; Wang & Dunn, 1999]. The blood-brain-
barrier generally prevents the transportation of large molecules from blood to the brain, however,
the pro-inflammatory cytokines (particularly IL-6) were shown to be capable of increasing
permeability of the blood-brain barrier [Castelnau et al., 1998; Mulla & Buckingham, 1999;
Turnbull & Rivier, 1999]. Furthermore, cytokines have been shown to be released from, and
affecting, peripheral nerves and the brain as well as peripheral immune cells [Hansen et al.,
1998; Marz et al., 1999]. The pro-inflammatory cytokines (IL-1 in particular) were also
demonstrated to initiate sickness behaviour (e.g. fever, fatigue, sleepiness and anorexia)
[Dantzer, 2004; Kelley et al., 2003; Vollmer-Conna, 2001; Watkins & Maier, 1999]. With the
finding that the IL-1 receptors have also been found on neurons and glial cells, particularly
dense in the hippocampus [Cunningham & De Souza, 1993], it was suggested that there is a
direct interaction between the immune cells and the brain in the immune-to-brain pathway
[Becher et al., 2000; Maier & Watkins, 1998] in the psycho-neuro-endocrino-immune network
[Figure 2].

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The hippocampus and the amygdala, which play a core role in learning, memory and
cognition [Blank et al., 2002], are also known to have an important role in the stress response
[de Kloet, 2000; Heuser & Lammers, 2003]. The hippocampus has been shown to be rich in
cortisol receptors [de Kloet et al., 1998; Downing & Miyan, 2000; Miller et al., 1994], and
cortisol was shown to enhance memory by means of the glucocorticoid receptors (GCRs) in the
hippocampus [de Kloet et al., 1999]. In addition, the GCRs inhibition (by antagonists) in the
amygdala was also reported to impair memory consolidation including stressful memories. This
impairment of memory consolidation was suggested to result in decreasing the levels of fear and
anxiety [McGaugh & Roozendaal, 2002; Roozendaal, 2000]. Furthermore, it has been
demonstrated that the sympathetic nervous system and the HPA axis managed to provoke
anxiety [Tanaka et al., 2000].

Hence, it was suggested that the immune-to-brain pathway [Maier, 2003] can extend to
psycho-behavioural responses or ‘mind-body’ interaction [Yuasa & Kasulis, 1987] as in the
psycho-neuro-endocrino-immune network.

Consequently, it is proposed that these inter-cellular interactions and intra-cellular


mechanisms in each level of the psycho-neuro-endocrino-immune network enable the immune
system to maintain homeostasis in health, i.e. the balance of reactions between incompetent
immune responses, which may lead to malignancy or infection, and excessive inflammation,
which may lead to auto-immune disease or allergy.

1.2.2.3 Circadian rhythm in the neuro-endocrino-immune network


Each organ system is known to follow its own homeostatic rest-alert cycles. These rest-
alert cycles synchronise with the sleep-wake cycle [Lavie, 2001]. They have been observed as
diurnal variations [Clerici et al., 1997; Glaser et al., 2001; Maschke & Hecht, 2004; Sakami et
al., 2002] or had their own circadian patterns [Folkard, 1990] in the levels of their mediators.

The biological sleep-wake cycle, a circadian rhythm, is originally driven from a


physiological cycle of changes in body temperature, i.e. one cycle of diurnal changes in the set-
point temperature [Foster & Kreitzman, 2004]. One example of the observed circadian patterns
in the neuro-endocrino-immune network are the shifts in dominance in the balance of
interactions within each nervous, endocrine and immune system [Laycock & Wise, 2003;
Marshall & Born, 2002]:

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1. The parasympathetic is dominant over the sympathetic at night, and vice versa
during the day in the nervous system;
2. A peak secretion of melatonin precedes sleep [Lavie, 2001] and a cortisol surge
precedes waking [Born et al., 1999; Spath-Schwalbe et al., 1992]; and
3. Th.1 cytokines (e.g. IL-2) are higher at night than during the day [Born et al.,
1997] and they can enhance sleep induction while Th.2 cytokines (e.g. IL-4 and L-
10) suppress sleep induction and disturb the sleep process [Marshall & Born, 2002].

It has also been shown that the circadian patterns of these secreted molecules in the neuro-
endocrino-immune network contribute to the sleep-wake cycle by promoting or inhibiting sleep
[Moldofsky, 1995]. For example, pro-inflammatory cytokines (e.g. TNF- IL-1 and IL-6) as
well as melatonin are known to induce sleep [Kronfol & Remick, 2000; Krueger & Majde, 1994,
2003]. In addition, cortisol and pro-inflammatory cytokine antagonists (e.g. IL-1ra) as well as
negative mood (e.g. anxiety and depression) can inhibit sleep [Sakami et al., 2002].

Reciprocally, disorganization of the sleep-wake cycle has been shown to induce


alterations:
1. In the levels of the neuro-endocrine hormones [van Reeth et al., 1994] including
impairment of melatonin secretion [Suzuki et al., 1993]; and
2. In immune parameters [Cruess et al., 2003; Moldofsky, 1995] including shifting the
balance of Th.1 / Th.2 cytokine production toward Th.2 dominance [Sakami et al.,
2002].
These alterations may be associated with each other since melatonin was reported to
stimulate Th.1 cytokine production [Garcia-Maurino et al., 1999; Inserra et al., 1998]. It has
also been shown that sleep deprivation, particularly the rapid eye movement (REM) sleep
deprivation, impairs immune responses [Casey et al., 1974]. The ‘sleep efficiency’, i.e. the
percentage of total sleep amount in available sleep-time (including awake time in bed), was
shown to correlate negatively with the clinical parameters representing disease severities both in
cystic-fibrosis patients [Milross et al., 2002] and in HIV-infected patients [Cruess et al., 2003].
The sleep efficiency was also reported to be significantly decreased in recurrent depressive
patients, and the percentage of the REM sleep was increased within the decreased time of sleep
[Thase et al., 1995]. This suggests the importance to health in maintaining an appropriate
amount of the REM sleep.

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In patients with depression, who often report sleep problems, melatonin secretion is
reported to be impaired, and exogenous melatonin has been shown to restore sleep quality and
mood [Leppamaki et al., 2003]. Exogenous melatonin was also shown to improve mood and to
increase the REM sleep in depressed patients during the next day of administration [Bauer et al.,
1995; Spath-Schwalbe et al., 1998] although the improvement in mood and increased amount of
the REM sleep disappeared in the following day. Further, a high cortisol to melatonin ratio
(caused by impairment of night-time melatonin secretion) has also been shown to correlate with
impaired cell-mediated immune responses and with an increase in depressive mood [Fiorina et
al., 1999]. Hence melatonin, as well as cortisol appeared to have an important role regarding
sleep regulation in the psycho-neuro-endocrino-immune network.

Consequently it has been suggested that the sleep process itself plays an important role in
interactions in the psycho-neuro-endocrino-immune network [Krueger et al., 2003; Marshall &
Born, 2002].

1.2.2.4 Circadian rhythm and subjective well-being


Psychologically, the sleep-wake cycle is a robust regulator of human alertness [Hull et al.,
2003]. At the beginning of sleep, the body reduces the input of external stimuli (neural sensory
inputs) and there is also less physical movement (neural motor outputs). This sleep induction
and its maintenance are known to be helpful for consolidating memory in the awake learning
process, so that effective cognitive function in daytime can be preserved [Stickgold et al., 2001;
Stickgold & Walker, 2005; Walker et al., 2005]. Alertness and the cognitive effectiveness in
function have been discussed in relation to subjective well-being ratings and quality of life
during the daytime and to subsequent sleep quality at night. The most commonly used self-
report measure of sleep quality is the Pittsburgh Sleep Quality Index (PSQI) [Buysse et al.,
1989].

Sleep quality, as measured by the PSQI, has been reported to correlate with amount of
sleep, namely the sleep efficiency, and with the percentages of REM sleep in the total sleep-
time [Milross et al., 2002]. Sleep quality has also been shown to reported to correlate with the
quality-of-life [Cohen et al., 1998], and it has been suggested that improved sleep quality may
contribute to better quality-of-life [Myers & Badia, 1995]. Reciprocally, daytime relaxation
exercise has shown to improve the sleep quality [Shapiro et al., 2003]. It has also been
demonstrated that subjects with a high amplitude of alpha waves in daytime brain waves, which
represents a calmer or relaxed state of mind, sleep significantly longer and deeper [Ehlers et al.,

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1998]. On the other hand, disorganization of the sleep-wake cycle was found to impair the
quality-of-life [Edell-Gustaffson, 2002; Edell-Gustaffson & Hetta, 2001].

Collectively, there may be an integrated relationship between night time well-being (sleep
quality) and day time well-being (quality-of-life), and the daytime well-being may contribute to
the night time well-being and vice versa in the psycho-neuro-endocrino-immune network.

1.2.3 Psychological input in the psycho-neuro-endocrino-immune network


The word ‘Stress’ was defined by Hans Selye, a physician and endocrinologist who
instigated stress research, as “the non-specific response of the body to any demand” [Selye,
1936]. Selye also emphasised the distinction between ‘Stressor’ (the cause) and ‘Stress’ (the
effect) [Selye, 1975a].

1.2.3.1 Stressor
Life events (such as trauma, disease, exercise and life-changing experiences) challenge
an individual’s capacity to adapt to inner and outer demands. These challenges may be
physiologically arousing and/or emotionally taxing, and these events may lead to cognitive and
behavioural responses.

Physiological stressors (including trauma, infection and starvation) affect human organ
systems directly through catabolic changes (energy consuming changes). Psychological
stressors(stimuli which arouse human emotions) can also affect human organ systems through
the effects of psychological stress, both directly via the psycho-neuro-endocrino-immune
network (i.e. appraisal of stressor acts as an input in the network) and by modulation of human
behaviour including eating habits, exercise, smoking and self-medication.

Stress related life-events have often been examined either in healthy populations using
academic examinations where NK-cells, in particular, were measured as a stress-related marker
[Deinzer & Schuller, 1998; Glaser et al., 1985; Gruzelier, Levy et al., 2001; Gruzelier, Smith et
al., 2001; Kiecolt-Glaser et al., 1986; Kiecolt-Glaser et al., 2001] [see also 1.2.4.2 and 1.2.4.3],
or in patient populations (i.e. clinically stressed individuals) who are persistently exposed to the
presence of life-long diseases. These life-long diseases often used in the literature include
malignancy (breast cancer in particular) [Antoni et al., 2001; Baider et al., 2003; Bakke et al.,
2002; Spiegel et al., 1989] and infections (particularly with the hepatitis virus or the human

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immunodeficiency virus: HIV) [Antoni et al., 2002; Antoni et al., 2000; Carbone et al., 2000;
Cruess et al., 2003; Cruess et al., 1999]. The HIV infection is a life-threatening disease directly
related to the immune cells, particularly CD4 T-cells, so it may be considered as a good
example of a life-long stressor to both one’s biology and psychology. In addition, stress has
been shown to detrimentally affect the HIV disease progression [Catalan et al., 1995; Cole et al.,
2003; Cole et al., 1997; Cole et al., 1996].

Hence, the two subject groups used for in vivo investigation in this project were:
1. University students, facing academic examinations, as an example of individuals
with time-limited sustained stress, and the primary outcome measure was NK-cells;
and
2. HIV-infected adults, not receiving anti-retrociral medication, as an example of
individuals with on-going disease-associated stress, and the primary outcome
measure was CD4 T-cells.

1.2.3.2 Stress perception


One individual’s perception of a particular stressor will differ from another’s. Factors
affecting stress perception can be classified into three categories [Folkman & Moskowitz, 2004]
Stressor-related: inherent quality (type), intensity and frequency;
Environment-related: physical and social resources; and
Individual-related: cognitive appraisal, coping skills, and personality.

Among these three factors, the individual-related factors are those most directly
susceptible to psychological influences [Han, 2002]. An individual’s appraisal of a stressor has
two elements [Lazarus & Folkman, 1984]:
1. Primary appraisal (perception of a demand, threat or stimulus); and
2. Secondary appraisal (judgement in one’s capability of coping with a threat).

Primary appraisal can be either neutral, benign or threatening [Text box 2]. When the
primary appraisal of a stressor was perceived as a threat, then consequently the secondary
appraisal would formulate and result in a coping action [Lazarus, 1999].

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Text box 2: APPRAISAL OF A STRESSFUL LIFE EVENT

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Two types of coping with a threat have been hypothesised: (1) emotion-focused and (2)
problem-focused [Folkman & Moskowitz, 2004]. These may be mainly associated with the
levels of primary and secondary appraisal, respectively. Recently, an additional independent
coping strategy, meaning-making focused, has been proposed [Text box 3]. The meaning-
making-focused element was proposed as an independent coping strategy in which the person
weighs values, beliefs and goals to modify the meaning of a stressful transaction [Frankl, 1970],
particularly under sustained stress that may not be amenable to the first two elements [Park &
Folkman, 1997]. For example, the meaning-making coping was illustrated as the strategy
caregivers most frequently reported to employ when they have to cope with behaviours of
demented care-recipients [Gignac & Gottlieb, 1996].

Text box 3: ELEMENTS OF COPING WITH A THREAT


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These three coping strategies have been integrated and paraphrased into the concept of the
sense of coherence (SoC) [Antonovsky, 1993], in which stressful perception can be altered and
reduced through three components [Geyer, 1997]:
1. Comprehensibility: To make a stressor rational, understandable and/or predictable.
2. Manageability: To become capable of influencing directly upon and taking in
control of his/her stress-related environment.
3. Meaningfulness: To obtain worthy awareness by commitment in and investment of
his/her own existing resources.

It has been suggested that comprehensibility can be constructed through the individual’s
own thoughts and theories, despite an insecure situation; that manageability can be achieved by
active information-seeking strategies, by social support and by positive reinterpretation of the
situation; and that meaningfulness can be created by a close-relationship and a faith (or a belief
and confidence in one self and/or something greater) [Frankl, 1970] as well as by a behavioural
commitment / effort [Strang & Strang, 2001].

1.2.3.3 Psychological training intervention


Since psychotherapy began to appear as a formally recognised intervention within
medicine (particularly among mental health professions), hundreds of forms of psychotherapies
have been introduced [Roth & Fonagy, 1996]. In recent decades, the style of psychotherapy has
shifted in its main focus from a paternalistic style (i.e. giving a therapist-orientated therapy to a
client / patient) to an empowering style (i.e. training or providing a client / patient with self-help
/ self-administrated techniques including skills to get support so that they can manage stress by
themselves).

The latter type of psychological intervention has focused upon a training aspect, i.e.
teaching specific skills / techniques and encouraging clients / patients to use these in daily life
[Baider et al., 2001; Batey et al., 2000]. This type of intervention aims to help patients reduce
stress levels and improve well-being through the modification of behaviour, cognition, or
emotion [Miller & Cohen, 2001]. This type of intervention includes stress management
(behaviour therapy and/or cognitive-behavioural therapy [Antoni et al., 2000; Cruess et al.,
1999; Cruess et al., 2000]), relaxation methods [Gillani & Smith, 2001; Gruzelier, Levy et al.,
2001; Keefer & Blanchard, 2002], self-hypnosis or visualisation [Gruzelier, Levy et al., 2001;
Gruzelier, Smith et al., 2001; Whitehouse et al., 1996], and biofeedback or conditioning
interventions [Miller & Cohen, 2001; Raymond et al., 2005].

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Based on the appraisal theories outlined above, it can be hypothesised that psychological
training and practice can alter stressful perception (primary and secondary appraisals) by
providing alternative or supplemental perspectives and coping skills for stressful life events.
Primary appraisal may be altered from a threat to a neutral or a benign stimulus by adjusting
individual reality, since one’s perception is based on one’s perspectives and experience. The
secondary appraisal may be altered by learning new coping skills in order to strengthen one’s
coping ability to the stressful perception.

Self-hypnosis
Among the many psychological interventions for self-administrated stress management,
one approach commonly used in clinical studies involves self-hypnosis training and practice
[Kiecolt-Glaser & Glaser, 1992; Kiecolt-Glaser et al., 2001]. Hypnosis (both with and without
self-hypnosis training) is arguably one of the more accepted therapies within complementary
medicine [Afari et al., 1999; Stewart, 2005], and self-hypnosis has been studied as a form of
psychological intervention for decades [Gruzelier, 2002a; Gruzelier, Levy et al., 2001; Schulz,
2001; Spiegel et al., 1989; Whitehouse et al., 1996].

One of the unique perspectives in hypnosis is the concept of the ‘hypnotic state’, i.e. a
state of mind in which a person's normal critical or skeptic nature is bypassed [Gruzelier, 1998].
The hypnotic state is usually taught as an altered level of awareness or a state of mind where
one can gain more control over him- or herself and one’s cognitions about or perceptions of
‘reality’. This may increase the comprehensibility in the coping strategies [see above 1.2.3.2].
Self-hypnosis typically includes a focussing of attention, followed by mental and physical
relaxation, and mental imagery of ‘deepening’ in and ‘absorption’ into a hypnotic state. As a
‘suggestion’ in a hypnotic state, attention focused on exercises of anxiety-control, guided
imagery of health and the feelings of confidence and happiness, i.e. an increase in inner sense of
empowerment. This process aims to provide coping skills to increase the manageability
[1.2.3.2]. For the purpose of enhancing the manageability, some skills from the cognitive
behavioural therapy (CBT) were also provided as a supplementary technique in this project
[Appendix A-2i].

Johrei
The other training intervention for a self-administrated strategy applied in this project was
Johrei, a Japanese non-contact healing method which looks similar to the laying-on-of-hands

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techniques. Johrei is classified under subtle energy medicine and as a category of


complementary medicine [Clarke, 2000] because it deals with imagery of ‘healing-light’ or
undiscovered ‘vital energy’. During a Johrei session, the practitioner visualizes a spiritual
healing-light coming from an imaginary source (as if light comes from the Sun) and transmits
this to the recipient through the palm of his/her outstretched hand. As with other subtle energy
medicine methods like Therapeutic touch [Meehan, 1998; O'Mathuna et al., 2002], Ki [Chang,
2003] or Spiritual healing [Patterson, 1998], Johrei has its own philosophical background, in
other words, Johrei provides an alternative perspective of stressful life events.

The philosophical background of Johrei includes two key principles aiming to supply
alternative perspectives:
1. ‘Process of Purification’ which encourages changing one’s perspectives during
stressful and/or difficult periods, shifting from focusing on negativities of the moment
to focusing upon future outcomes when the problem period is over; and
2. ‘IZUNOME’ which means a dynamic state of harmony in appropriate timing and
balance between two dichotomised concepts / attitudes, e.g. between endeavour to
persist making your own destiny with ‘Makoto’ (sincerity, integrity, truth and love
used for one’s self awareness) [Hardacre, 1988] and detachment to ‘let go’ (trust in
natural time course which is beyond one’s control).

‘Johrei’ literally means “purification of spirit”, so the approach may also provide an
alternative or additional perspective on life events with the concept of spirituality [Seaward,
2000]. Experience of Johrei is a self-contained time when one can quietly, mindfully and kindly
concentrate upon the recipient’s benefit, as well as his/her own, since the Johrei practice is
based upon the concept that “one can heal oneself by healing others” [Naito, 2003].

Hence, Johrei differs from self-hypnosis in that it is practised mainly in pairs, although
self-Johrei and distant Johrei techniques can be provided, and in fact, were provided at the end
of training sessions in the current study. In addition, teaching appreciation of surroundings and
human relationships (namely ‘Spiritual Cords’) is given as a one of major five principles to
practise Johrei [Appendix A-2ii]. These concepts may be collectively able to increase a sense of
receiving support (manageability) and to enhance the meaningfulness, both of which can
promote coping ability [1.2.3.2].

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Possible alterations in the neural part of the network


Hypnosis, by definition, comprises three inter-linked processes [Stewart, 2005]:
1. Focused attention,
2. Induction of a hypnotic state, and
3. Receptiveness to suggestion.

Positive emission topography (PET) studies have implied that these processes of hypnosis
are not processes of simply following instructions but they involve a change of perception in the
brain, such as in visual perception [Kosslyn et al., 2000] and in pain sensation [Faymonville et
al., 2000]. Particularly in pain perception, it was suggested that there is a strong relationship
between the psychological state of mind and neurological response to pain stimulus, i.e. a pain
stimulus was shown to cause different neurological responses whether an increase or a decrease
in blood flow, depending upon the state of mind in the ‘hypnotic state’ [Montgomery et al.,
2000; Patterson & Jensen, 2003]. Hence, it was suggested that self-hypnosis may directly be
able to alter the primary appraisal at the unconscious level as well as at the conscious level.

On the other hand, few studies have investigated the physiological processes associated
with a Johrei session. Two preliminary studies have demonstrated that experienced practitioners
were shown to increase the power of alpha waves, which indicates an experience of relaxation,
during the Johrei session [Dwivedi et al. in preparation]. Single-blinded studies comparing a
Johrei session (receiver was sitting with eyes closed, separated by a curtain from the practitioner,
and given Johrei from behind) and mock session (the same setting with the same arm
movements of the same practitioner but with no intention by the practitioner to practise Johrei)
has demonstrated that simultaneously recorded brain waves from both practitioner and receiver
exhibited significantly increased levels of ‘mutual information’ in the alpha wave band (an
experience of relaxation) in the Johrei session, but not in the mock condition [Dwivedi et al. in
preparation]. This indicates that the Johrei session may increase their levels of relaxation.
Consequently, it was suggested that the practice of Johrei may directly be able to reduce stress
levels.

In summary, the two psychological training interventions, self-hypnosis and Johrei, were
intended to provide subjects with techniques to alter the perception of stressors, to enhance
coping skills and to provide profound relaxation [Text box 4], and thereby to reduce the effects
of stress upon the psycho-neuro-endocrino-immune network.

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Text box 4: PSYCHOLOGICAL INTERVENTIONS IN THIS PROJECT


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1.2.4 Stress-associated changes


Published findings regarding stress-associated changes are complex and difficult to
summarize because of the huge variety of stressors, biological markers, and, time points at
which changes are observed or investigated that have been used in these studies. This latter
difference in published studies is particularly important with regard to the current project. It is
necessary, therefore, to be very specific regarding the timing of changes in stress responses that
will be measured.

1.2.4.1 Chronological definition of stress response


The chronological definition and distinction of stress responses are ambiguous and have
varied from study to study, even though Hans Selye introduced a concept that defined three
phases of stress responses as the name of the ‘general adaptation syndrome’ [Selye, 1975b]:
1. Alarm stage: triggering during which marshalled resources are organised;
2. Resistance stage: adaptation during which resistance to alarm rises above normal;
and
3. Exhaustion stage: when adaptation energy is used up.
Recently, it has been demonstrated by using a meta-analysis strategy that Selye’s three
categories of stress response may have counterparts within the immune system, and may
represent three different immunological modifications [Segerstrom & Miller, 2004]:
1. Up-regulation of innate immunity and suppression of adaptive immunity by short-
lasting (seconds to minutes) stress;
2. Cytokine shift from Th.1 to Th.2 [Elenkov & Chrousos, 1999] without consistent
changes in cellular immunity by temporal (hours to days) stress; and
3. Global immuno-suppression by long-lasting (weeks, months to years) stress.

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In contrast, there is a well-used dichotomised chronological distinction in stress, i.e. acute


and sustained or chronic stress. In terms of the general adaptation syndrome, acute, momentary
or short lasting stress (henceforth acute stress) is defined conceptually as the stress that falls in
the period from the alarm stage to the resistance stage. Sustained or chronic stress (sustained
stress) is from the resistance stage to the exhaustion stage.

Considering the sleep-wake cycle as a biologically fundamental period, acute or sustained


stress in this thesis is defined on the basis of this cycle. That is, acute stress is defined as stress
that has its effects within one day or, in other words, a single circadian rhythm. In contrast,
sustained stress is defined as stress that has its effects for more than one day, i.e. over more than
one circadian rhythm. This can be construed as “Sustained stress alters circadian patterns of the
psycho-neuro-endocrino-immune network.”

Due to the nature of psychological intervention in which training and practice occur over
a protracted period (days, weeks and months), the effects of sustained stress upon mediators in
the psycho-neuro-endocrino-immune network are the central focus of interest in this thesis. The
working hypothesis in the project is that sustained stress disturbs psychological balance with
consequential influences upon the immune system, and the detailed changes observed under
sustained stress supporting this working hypothesis are explained below in conjunction with the
stress responses in the psycho-neuro-endocrino-immune network.

1.2.4.2 Acute stress responses in the neuro-endocrino-immune network


In the psycho-neural part of the psycho-neuro-endocrino-immune network, the primary
appraisal of acute stress, particularly reward and fear reactions, are bi-directionally regulated via
neuro-hormonal secretions [Charney, 2004]. In the nervous system, acute stress is shown to
activate the secretory neurons through aminergic and GABA-ergic innervations in the
autonomic nervous system [Cole & Sawchenko, 2002]. This aminergic pathway has been
reported to project directly to the CRF neurons in the HPA, but many of the other neural
networks utilise neural synaptic signals via an inhibitory GABA-ergic network [de Kloet, 2003]
including neurons in the SAM. For example, the excitatory inputs from limbic-cortical regions
modulate the GABA-ergic inter-neuronal network [Cole & Sawchenko, 2002; Herman et al.,
2002]. This has been suggested to provide a stressor a specific neuro-chemical signature to the
secretory neurons [Pacak & Palkovits, 2001].

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In the neuro-endocrine part of the network, it was recently discovered that there are both
mineralocorticoid receptors (MCRs) and glucocorticoid receptors (GCRs) in the brain [de Kloet,
2000] as well as in the endocrine organs. Hence, de Kloet [2003] has hypothesised that the
MCRs and GCRs have inter-complementary effects against stress responses in the central
nervous systems. Specifically, it was hypothesized that (1) the sympathetic neurons and MCRs
respond to acute stress; and then that (2) soon after this response, the parasympathetic neurons
and GCRs facilitate:
1. Recovery of stress-induced acute immune responses;
2. Control of energy metabolism in the endocrine system; and
3. Promotion of information storage in the brain [de Kloet, 2003].

In the immune system, it has been elucidated via meta-analysis that acute stress increases
the number of NK-cells in the blood stream and NK cytotoxic activity, but that there is no
consistent effect upon the number of T-cells or B-cells [Segerstrom & Miller, 2004]. In addition,
it has been shown that the acute stress-induced increased NK cytotoxic activity, measured in
peripheral blood mononuclear cells (PBMCs), was not due to an increase in the per-NK-cell
cytotoxic activity but was due to an increase in percentages of NK-cells in PBMCs [Segerstrom
& Miller, 2004]. Furthermore, in the interaction between the endocrine and immune systems,
the acute stress-induced changes in immune cell distribution were shown to be associated with
the expression of the GCRs in mice [Dhabhar et al., 1996]. Diurnal changes in adrenal steroids
were known to be associated with changes of leukocyte distribution, i.e. the diurnal peak in
glucocorticoid (cortisol) levels coincided with a diurnal trough in the number of peripheral
blood lymphocyte and vice versa [McEwen et al., 1997].

Acute stress acting through the SAM and HPA axes of the neuro-endocrino-immune
network has been shown to have inter-complementary effects against stress responses, i.e. to
promote or to suppress inflammatory reactions [Chrousos, 1995; Huether, 1996]:
1. Promoting inflammation as the alarm or ‘fight or flight’ response [Cannon, 1914;
Goligorsky, 2001]: the SAM axis is stimulated to produce catecholamines which
act as immunological ‘alarm signals’ by promoting secretion of pro-inflammatory
cytokines [Elenkov et al., 2000] by mainly stimulated monocytes [Straub, Mayer et
al., 2000]; and
2. Suppressing inflammation as a response to the alarm reaction (the resistance stage
in the general adaptation syndrome theory): acute stress is known to increase the

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levels of cortisol [Rohleder et al., 2002] which can counteract the alarm signal and
suppress activation of the immune system [McEwen et al., 1997].

Historically, the concept of this counteracting mechanism in the endocrino-immune


pathway in the acute stress response was originally proposed by Besedovsky & Sorkin [1977]. It
was then paraphrased by Munck et al. [1984], who hypothesised that the stress-induced increase
in cortisol levels does not protect against the source of stress itself, but rather against the body’s
normal responses to stress. This process was suggested to prevent those stress responses from
excessive overshooting processes threatening homeostasis [Munck et al., 1984].

Although the whole picture of the mechanism of this cortisol-induced suppressive


immune response is yet to be investigated [Sapolsky et al., 2000], cortisol has been considered
as a main stress hormone which can suppress the immune response by affecting targets mainly
through its receptor (GCRs). This is considered to occur primarily by inhibiting the NF B’s
genomic functions to promote non-specific inflammation and to inhibit apoptosis [Karin & Lin,
2002]. In contrast to cortisol, another adrenal hormone in the HPA axis, dehydroepiandrosterone
(DHEA; and its sulphate DHEA-S), has complicated effects. DHEA has been reported to
stimulate Th.1 cytokine (IL-2 etc.) production [Clerici et al., 1997; Cutolo et al., 2000; Loria,
2002] and to behave as an antagonist to cortisol in this Th.1 / Th.2 regulation [Wolf &
Kirschbaum, 1999]. DHEA, however, has also been shown to have a similar and/or synergistic
effect with cortisol, i.e. inhibiting the NF B activation and suppressing pro-inflammatory
cytokines [Straub, Scholmerich et al., 2000].

Collectively, it has been suggested that adrenal hormones (cortisol in particular and
combined with DHEA/-S) play an important role in the endocrino-immune interaction with
regard to acute stress response in the psycho-neuro-endocrino-immune network.

1.2.4.3 Sustained stress and changes in the psycho-neuro-endocrino-immune network


Sustained stress has been demonstrated to induce various changes in the psycho-neuro-
endocrino-immune network. In the neuro-immune bi-directional interaction [Lawrence & Kim,
2000], the blood-brain-barrier permeability was found to be increased under Gulf War stress
[Freedman et al., 1996]. It has also been shown that inhibitory GABA-ergic nervous activity
was attenuated under sustained stress [Verkuyl et al., 2004]. These findings imply that the stress
responses in the neural level under sustained stress can be more hyperactive and then become
difficult to switch off, which may result in maintaining a vicious cycle of stress responses.

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In the immune system, sustained stress has been demonstrated to alter tissue specific
distribution of lymphocyte sub-populations in vivo [Sudo et al., 1997]. Specifically, peripheral
NK-cell levels have been shown to decrease under sustained stress [Borella et al., 1999;
Gruzelier, Smith et al., 2001; Inoue-Sakurai et al., 2000; Maes et al., 1992; Segerstrom & Miller,
2004]. In contrast, the effects of sustained stress upon in vivo distribution of lymphocytes are
not clear-cut and are inconsistent in the literature, i.e. some investigators report increases and
others report decreases in the sub-population counts (e.g. helper T-lymphocytes [de Gucht et al.,
1999; Maes, Van Bockstaele et al., 1999] and cytotoxic T-lymphocytes [Gruzelier, Smith et al.,
2001; Maes, Lin et al., 1999]). Nonetheless, in functional in vitro measures of the immune
system, sustained stress has been shown to suppress cellular immune responses [Bauer et al.,
2001; Bonneau et al., 1998; Dhabhar & McEwen, 1997; Koh, 1998; Segerstrom & Miller, 2004].
Hence, sustained stress has been believed to cause a detrimental effect upon immune cells in
vivo resulting in an increased susceptibility to malignancy [Garssen, 2004] and infection [Cohen
& Herbert, 1996; Kiecolt-Glaser & Glaser, 1999].

Notably, there was a report [Bonneau et al., 1998] demonstrating that adrenalectomy in
mice prevented the suppressive effect of sustained stress upon the acute stress-induced immune
activation (measured by in vitro cytotoxic activity and cytokine production). Hence, adrenal
hormones were suggested to play an essential role in the immuno-suppressive effect of
sustained stress. Further, there are contradictive clinical findings with regard to cortisol levels
under sustained stress, i.e. cortisol levels are high in patients with the post traumatic stress
disorder (PTSD) and depression, and low (known as a hypocortisolism) in patients with
somatoform disorders (chronic fatigue syndrome, fibromyalgia, burnout syndrome etc.) [Heim
et al., 2000]. There have been two interlinked hypotheses regarding the mechanisms which
contribute to suppress cellular immune responses under sustained stress [Text box 5]:
1. Sustained exposure to high levels of cortisol; and
2. Attenuated responses caused by habituation to repetitive stimulation.

The primary mechanism of the first hypothesis, sustained exposure to high levels of
cortisol, is that the stress reaction in the HPA axis stays active if coping with stress falls under
the sustained stress, therefore targets are exposed to elevated levels of cortisol for a prolonged
time [de Kloet, 2003]. This may be associated with multiple positive feedback loops in the HPA
axis [Gold et al., 2002], then this sustained high levels of cortisol may exacerbate the imbalance
of interactions within the psycho-neuro-endocrino-immune network [de Kloet, 2003], including

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loss of neurons in the hippocampus [Sapolsky, 2000]. Further, it is well-known that most
patients with depression have changed circadian patterns in the network, particularly in cortisol
levels, i.e. elevated trough and flattened peak levels with a narrowed range of fluctuation
[Goldsby et al., 2002; Weber et al., 2000]; as well as evidence of sympathetic hyperactivity
[Belanoff et al., 2002; Chrousos & Gold, 1992; Gold & Chrousos, 1999; Holsboer, 2001;
Schatzberg et al., 1985]. Furthermore, growing evidence suggests that the mediators of the SAM
and HPA axes are repeatedly elevated under sustained stress and often fail to shut off promptly
before experiencing another repetitive challenge by a stressor [McEwen et al., 1997].

The later mechanism, attenuated responses caused by habituation [Kirschbaum et al.,


1995; Schommer et al., 2003], is also known as a progressive diminution of the responses
against repetitive stimulation [Heim et al., 2000]. The attenuated response in hormonal secretion
has been observed in both the neural level (amygdala) [Carter et al., 2004] and the HPA axis
[Gaab et al., 2002; John & Buckingham, 2003; Pariante & Miller, 2001] as well as the cytokine
level [Gaab et al., 2005]. It has also been demonstrated that sustained stress attenuates the acute
stress responses in the endocrine and immune systems. As a commencing condition, acute stress
increases glucocorticoids and alter lymphocyte distribution in the blood stream of rats. The
greatest changes in these, glucocorticoid levels and alteration of lymphocyte distribution, were
seen on Day 1, but thereafter with repeated exposure to the same acute stress, the changes
decreased and reached their lowest at Day 35 [Dhabhar & McEwen, 1997]. Accordingly, it is
hypothesised that accumulated negative feedbacks from repetitive responses in both the SAM
and HPA axes result in impaired reactions in their hormone secretions.

Text box 5: SUMMARY OF HYPOTHESES Re: Stress-related changes of endocrine system which may
contribute to suppress cellular immune responses under sustained stress
/ (

( *

These may be able to represent the different stages of the sustained stress, i.e. the resistant
and the exhaustion stages in the Selye’s general adaptation syndrome. It should be noted,
however, that there remains confusion about the mechanisms that underlie sustained stress, and

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whether these may be best thought of, as two consecutive stages that occur after another, or as
two distinct responses that occur in parallel. Other factors like genetic differences (e.g. gender)
may also contribute to this confusion, but reports still remain inconsistent [Lundberg, 2005;
Sauro et al., 2003]. Nonetheless, the cellular immune responses are believed to be impaired
mainly by either or both above mechanisms in the psycho-neuro-endocrino-immune network.
Hence, this project focused on the hypothesis that psychological intervention can counteract, or
at least buffer, the negative effects of sustained stress upon the psycho-neuro-endocrino-immune
network, particularly the immune system.

1.3 Approach taken

This thesis is based upon the hypothesis that psychological intervention acting through the
psycho-neuro-endocrino-immune network will counteract the detrimental effect of stress on the
immune system.Investigation was performed by using a series of in vivo and in vitro studies.

The psychological interventions used to counteract stress were Self-hypnosis and Johrei,
both of which provide training and practice of self-help stress management techniques.
University students facing examination were used as an example of a time-limited stressful
situation, and the primary outcome measures were NK-cell percentages and NKCA levels.
HIV-infected patients were used as an example of ongoing disease-associated stressful situation,
and the primary outcome measures was CD4 T-cell counts. A number of validated
questionnaires was used to demonstrate that examinations and on-going disease both induce
stress, and to explore the effects of the psychological interventions upon stress perception.

In addition, in vitro experiments were performed to explore the direct influence of the
stress hormone, cortisol, on the peripheral NK-cells and T-lymphocytes.

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Chapter II

Materials and Methods

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CONTENTS OF CHAPTER 2
Materials and Methods

2.1 Participants with regards to outcomes and measurement time points


2.1.1 Student volunteers 53

2.1.2 Patient volunteers 56

2.1.3 Case matched patients from database 58

2.1.4 Laboratory volunteers 60

2.2 Psychological intervention


2.2.1 Self-hypnosis training 60

2.2.2 Johrei training 61

2.2.3 Mock intervention (Controls for the psychological trainings) 62

2.3 Self-report questionnaires


2.3.1 Stress perception 63
2.3.1.1 State anxiety score in the State and Trait Anxiety Inventory 63
2.3.1.2 Impact of Event Scale (IES) 63
2.3.1.3 Perceived Stress Scale (PSS) 63

2.3.2 Perceived quality of life 63


2.3.2.1 Locus of Control scale (LoC) 63
2.3.2.2 Mental Component Summary in the SF-36 (MCS) 64
2.3.2.3 Pittsburgh Sleep Quality Index (PSQI) 64

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2.4 Materials and methods in vitro


2.4.1 Materials and experiment kits 64
2.4.1.1 Chemicals and reagents 64

2.4.1.2 Biological reagents 65

2.4.1.3 Fluorescently conjugated antibodies for flow cytometry analyses 65

2.4.1.4 Commercially available laboratory kits for colorimetric analyses 66

2.4.2 Equipments used for in vitro investigations 66


2.4.2.1 Flow cytometry 66

2.4.2.2 Colorimeter 68

2.4.3 Methods in vitro 69


2.4.3.1 Preparation of tissue culture medium for cell culture in vitro 69

2.4.3.2 NK cytotoxic activity measured by flow cytometry 70

2.4.3.3 NK-cell characteristics during NKCA assay measured by flow


cytometry 73

2.4.3.4 Proliferative response measured by [3H]-thymidine incorporation 74


2.4.3.5 T-cell characteristics after in vitro incubation measured by flow
cytometry 75

2.5 Statistical analyses 77

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2.1 Participants with regards to outcomes and measurement time


points

2.1.1 Student volunteers

University students facing the academic examination period were recruited to investigate
the effects of a time-limited sustained stress (academic examinations) upon the perception of
stress and associated changes in immunological parameters, particularly NK-cell percentage and
NKCA levels. They were recruited through posters displayed in Imperial College London and
by word-of-mouth contacts using student research assistants (Psychology BSc students). Ethical
approval was granted from the Riverside Research Ethics Committee and all volunteers were
given detailed information sheets, interviewed and asked to sign an informed consent form [see
Appendix 1]. All subjects were asked to fast overnight before giving a morning blood sample
(between 8:00 and 9:00 a.m. in order to minimise diurnal effects [McGlone et al., 1991]), and
given a simple breakfast after their blood collection. At the end of the study, each participant
received £30 for travel expenses and inconvenience.

In total, 48 university students were recruited prior to their examinations. The median age
was 21 years and the range was 19-23 years with one participant of 37 years. There were 26
males and 22 females. Thirty nine of the participants were medical students at Imperial College
(from first to fifth year: specific years of the course were not recorded) and the remaining nine
were on other University of London courses (specific information was not recorded).

There were two examination periods and, for the Exam assessment point, students were
assessed within five days prior to their examinations [Figures 3 and 4]. Control (Non-exam)
periods for comparison with exam-periods were chosen as follows: for some students (Cohort
A), it was four weeks after examinations, and for the remainder (Cohort B), it was four weeks
after recruitment (which was four to eight weeks before Exams). Students were free to withdraw
from the study at any time without giving any specific reasons, and a total of 21 students
withdrew [Figures 3]. Blood samples were taken and questionnaires were completed at the
recruitment and at the Exam and Non-exam assessment points.

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4 weeks 4-8 weeks


Recruitment㩷 Exams Non-exam
(48) [Cohort A (21)] [Group A (11)]

Withdrew (10)
Non-exam Exams
[Cohort B (26)] [Group B (16)]

Withdrew (1) Withdrew (10)


Figure 3: Numbers of students recruited and remaining in the study at the Exam and Non-exam
assessment time points. The number of drop-out students is also indicated.

Cohort A Cohort B
12 12
Weeks from the Recruitment

8 8 Exam
Non-exam

4 4
Non-exam
Exam

Recruitment Recruitment
0 0

Figure 4: Timing of the Exam and Non-exam assessment time points with respect to recruitment for
individual students in the Cohorts A and B

Before combining Cohorts A and B to test the hypotheses that (1) academic examinations
cause stress in university students; and (2) psychological intervention can reduce stress effects,
comparability of the two groups at the Exam and Non-exam time points was confirmed by
statistical analyses comparing Cohorts A and B [Appendix 6: Tables A-1 to A-6]. The levels of
the PSS and the State anxiety score of the STAI, lymphocyte subsets distribution and NK
cytotoxic activity (NKCA) were compared. The levels of these measures were not different
between the two cohorts at the Non-exam time points, so the two cohorts were combined into a
single group in order to examine changes in the levels of these measurements.

A total of 27 students were studied, but not all of the participants provided complete data
sets. Some data were incomplete because of technical failure with the analyses and because
students did not turn up to provide samples or failed to complete questionnaires. Consequently,
the numbers in each analysis to test the first hypothesis that academic examinations cause stress
in university students were:

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Perceived stress scale (PSS) analysis: 22 students

State anxiety score analysis: 25 students

NK cytotoxic activity analysis: 21 students

CD4 and CD8 T-cells and NK-cell analyses: 26 students

In order to test the second hypothesis that psychological intervention can reduce stress
effects, the same 48 university students, as described above, were each randomly assigned to
one of three groups:
Relaxation control group: experiencing mock neuro-feedback sessions,
Self-hypnosis group: taught and practising Self-hypnosis with imagery, or
Johrei group: taught and practising a novel Japanese stress management system.

For both Cohorts A and B, training and practice of either intervention commenced at
recruitment and was encouraged to continue until their examinations, therefore, for Cohort A,
training and practice lasted four weeks and, for Cohort B, eight to twelve weeks. For analyses,
both examination data were combined [Figure 5].

Training-session Practice & follow-ups

4 weeks 4-8 weeks


Baseline㩷 Exams
(48) (21)
Exams
(16)

Withdrew (11)
Figure 5: Timing of training, follow-ups and data-collection sessions (Baseline and Exams assessment
points) and numbers of students at the sessions

Of 48 students recruited, 11 withdrew from the study. Complete datasets were not
obtained for the 37 students remaining. Hence, the number of subjects varied for each analysis
comparing between the psychological intervention groups and the Relaxation control group:

PSS 32 students

State anxiety score in the STAI 35 students

NK cytotoxic activity 31 students

Lymphocyte sub-populations and NK-cells 34 students

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2.1.2 Patient volunteers

After approval from the Riverside Research Ethics Committee, HIV-infected patients who
were not receiving anti-retroviral treatment (treatment naïve HIV-patients) were recruited to
investigate (1) the effects of on-going disease (HIV-infection) upon stress perception and
associated changes in immunological parameters, CD4 T-cell counts in particular; and (2)
whether psychological intervention can reduce stress effects. The clinical immunological data
were obtained from the patient record database at the Chelsea and Westminster Hospital.

A total of 63 HIV-infected individuals including three females, with a median age of 37


years (range 27-58 years) who were regular attendees to the St. Stephen’s clinic at the Chelsea
and Westminster Hospital, were recruited by a research assistant, Mr. Bryan M. Bennett, and
research nurses according to the inclusion criteria: (1) not receiving anti-retroviral treatment, (2)
no symptom regarding HIV-infection and (3) more than 200 cells per L of CD4 T-cells’ in
their peripheral blood. The HIV-infected participants were given detailed information sheets and
each gave signed informed consent. They participated for more than five months in the study,
during August 2003 to December 2004.

For the study investigating the effects of on-going disease (HIV-infection) upon stress
perception and associated changes in disease parameters, the rate of decline in CD4 T-cell count
was calculated between the recruitment time point and four months later as the primary outcome
measure of the study. The timing of clinical check-ups with regard to the two time points for the
psychological assessment (the Recruitment and After 4 months) was as follows [Figure 6]:

- Recruitment - After 4 months -


-
Term one Term two Term three
Baseline Term four

4 months
Figure 6: Timing of the measurement collection time points (Recruitment and After 4 months) and the
one-month blocks (Baseline, Term one to Term four) for clinical routine blood collections

Unfortunately, not all of the patients recruited had their CD4 T-cell counts measured at
each Term [Table 4].

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Table 4: Number and percentage of HIV-infected individuals who had CD4 T-cell counts check at the
monthly time periods from the Recruitment to four months after the recruitment (Term 4)
Numbers of patients (Total N = 63)
Valid number Missing
N Percent N Percent
Term 4 (four months after the Recruitment time point) 23 36.5% 40 63.5%
Term 3 (three months after the Recruitment time point) 18 28.6% 45 71.4%
Term 2 (two months after the Recruitment time point) 19 30.2% 44 69.8%
Term 1 (one month after the Recruitment time point) 14 22.2% 49 77.8%
Baseline (2 weeks before and after the recruitment) 22 34.9% 41 65.1%

The rate of decline in the CD4 T-cell counts (CD4 gradient) was therefore calculated from
those individuals who had at least two measurements made in the assessment period. Of the 63
participants recruited, only 38 patients (60.3%) had sufficient data from which to calculate the
rate of decline in the CD4 T-cells during the four month plus one month (five months
altogether) study period, and their data were used to investigate the association between stress
perception and disease progression.

The same 63 HIV-infected individuals were randomly assigned to one of three groups:
Control (wait-listed control: waiting for four months before being randomly
assigned to the Self-hypnosis or Johrei) group,
Self-hypnosis training and practice group, and
Johrei training and practice group.
Randomisation utilised random numbers generated by a researcher blind with a computer.
It was performed by using study numbers at recruitment, and then these anonymised
participants were assigned to one of the groups. Although the initial number of people in each
group at randomisation generated by a computer was almost equal, some subjects withdrew
before the training session was started (i.e. they did not turn up the first session) resulting in
different numbers of participants in each group. There were 23 participants in the Self-hypnosis
and 16 participants in the Johrei and 24 in the wait-listed control groups. The psychological
interventions were administered to seven Self-hypnosis and eight Johrei training cohorts.

At recruitment (Baseline) and after the intervention period (after 4 months) [Figure 7], the
participants were asked to complete psychological questionnaires. Of 63 patients, a total of
twelve patients either withdrew from the study or failed to complete some of questionnaires
before the end of the one-month training sessions. Hence, a total of 51 subjects (21 in Self-
hypnosis, 12 in Johrei and 18 in wait-listed controls) completed questionnaires. Lymphocyte
subpopulation and HIV viral load data were obtained from the pre-existing patient database, and
only 38 subjects (15 in Self-hypnosis, 10 in Johrei and 13 in wait-listed control groups) had

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sufficient data from which to calculate the rate of decline in the CD4 T-cells during the study
period.

- Recruitment - - Post-intervention -
Baseline Term one Term four

Training 㩷 㩷 㩷 Practice & Follow-ups

1 month 3 months
Figure 7: Timing of the two measurement points (Recruitment and Post-intervention) with regard to the
period of training and practice of the psychological interventions

2.1.3 Case matched patients from the database

The same HIV-infected individuals recruited to the above RCT study were analysed in
this case controlled study. The previous RCT study used a wait-listed control group, so after the
four months of the waiting period, they were randomly assigned to either the Self-hypnosis or
Johrei training group over the period from August 2003 to December 2004. This increased the
number in the two groups:
Self-hypnosis: 34 subjects consisting of seven training cohorts
Johrei: 29 subjects consisting of eight training cohorts

In addition to these participants, 58 matched database control patients were selected


blindly from the same HIV patient database with permission from the Ethics committee. This
selection was performed by an independent research doctor, Dr. Alan W. Steel, with the
following inclusion criteria:
1. CD4 T-cell counts > 200 cells / L
2. HIV viral load > 1000 viral copies /ml
3. Study period – same 12 months spread to minimise seasonal variation; and this
results in eight control cohorts with regard to commencing month for analysis
4. Equivalent mean CD4 T-lymphocytes counts with that of participants at the
commencing month of training intervention.
CD4 T-cell counts and viral load levels were obtained from the patient database. The
Training time point was set as the midpoint of the one-month training interventions and
subsequent terms were named after the number of months from the Training time point (e.g.
Term one is one month from the Training time point) [Figure 8].

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Training
Baseline Term one Term four

Training 㩷 㩷 㩷 Practice & Follow-ups

1 month 3 months
Figure 8: Time-points (Training time point and the Terms according to the Training).
Intervals between the doted-lines represent one term (one month)

The CD4 change gradients were compared between the intervention groups (Self-
hypnosis and Johrei) and the database control group. For each individual, a CD4 decline
gradient (cells per l per month) was calculated retrospectively from the preceding 12 months’
data held on the patient record database (Pre-intervention). These pre-intervention CD4
gradients were compared between the three groups. Similarly, a post-intervention CD4 gradient
was prospectively calculated. The CD4 gradients in the 12 month periods prior to and after the
Training (Pre-intervention vs. Post-intervention) [Figure 9] were compared within and between
the three groups.
Training
Pre-intervention Post-intervention

4 months
Figure 9: Comparison format with regard to the Training time point (Pre-intervention vs. Post-
intervention). Intervals between dot-lines represent one term (one month) as in Figure 8

Seven patients in the Self-hypnosis group, ten patients in the Johrei group and nine
patients in Database-controls were excluded because no routine blood samples were taken
during the 12 months either prior to or after intervention commenced. In these excluded patients,
several patients commenced anti-retroviral medication during the Post-intervention period, and
they were dropped from the study. Table 5 shows the numbers of patients who started drug
therapy in each group.

Table 5: Number and month of subjects who started anti-retroviral medication and dropped from the
study (NB: Term X represents that X months after the Recruitment time point)
Term 2 Term 5 Term 8 Term 11 Total
Database controls 0 1 3 3 7
Self-hypnosis 0 0 2 4 6
Johrei 1 0 1 1 3

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2.1.4 Laboratory volunteers

Thirty one healthy volunteers, of whom 21 were males and 10 females, with an age range
of 21-60 years, were recruited to donate blood in order to investigate the effects of in vitro
exposure to the stress hormone, cortisol, upon both NK-cells and T-lymphocytes.

This recruitment was through word-of-mouth contacts with the author and colleagues
from the Department of Immunology and the Division of Neuroscience and Mental Health. All
blood samples were collected between 9:30 and 11:30 a.m. to eliminate the effects of diurnal
variation [McGlone et al., 1991] and then either isolated peripheral blood mononuclear cells
(PBMCs) or whole blood were assayed straight after collection.

2.2 Psychological intervention

2.2.1 Self-hypnosis training


The self-hypnosis training, designed and conducted by an experienced clinical hypnotist
Dr. Tannis M. Laidlaw, consisted of four weekly sessions (two hours each).

The participants learnt first a Spiegel-type eye-roll induction (synchronising upward eye-
rolls with a deep inspiration from the nose, followed by holding the breath for a few seconds,
then eye-rolls down with expiration from the mouth) for ‘instant relaxation.’ They were also
taught a slower relaxation-type induction technique to achieve getting into a state of mind,
‘hypnotic state.’ All participants were provided with a standard audio-recording [Appendix 2-i]
which provided instructions for both inductions. Previous research has shown that university
students practising immune strengthen imagery in the self-hypnosis training had less cold
symptoms during their academic examination period than students practising relaxation imagery
[Gruzelier, Levy et al. 2001]. It was also shown in patients with genital herpes that self hypnosis
contributes to a reduction in disease-related symptoms [Fox et al. 1999; Gruzelier et al. 2002].
Hence, the induction technique was later combined with a specific imagery of immune
strengthening, which participants were told to practise under the ‘hypnotic state’. Further, all
participants were taught two anxiety management techniques:
1. How to use breathing techniques to control acute anxiety [Laidlaw, 1994]; and
2. The Interrupt Distraction Procedure (IDP) [Laidlaw, 1999] for worries and belief
change.

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Each participant was requested to practise self-hypnosis daily to learn and familiarise
themselves with the technique [Gruzelier et al., 1998]. During one-month training period, each
participant attended four group sessions with the hypnotist and each was expected to perform, at
least, more than ten self-hypnosis sessions. Diaries were kept by the participants to record their
practice frequency at home. After the four weekly training sessions, monthly follow-up group
sessions were continued for at least four months by HIV-infected individuals.

2.2.2 Johrei training


The Johrei training was planned and conducted by the author who is a trained practitioner
with more than 20 years of practice and who is also medically qualified. Original teaching
textbooks and support were provided by the Johrei Academy [http://www.johrei.org.uk/] and
Johrei Association [http://www.johreiassociation.co.uk/] so that the author could teach, edit and
adjust for each group of participants in the studies [Appendix 2-ii] [See also the Johrei
Foundation, http://www.johreifoundation.org/; Izunome, http://www.izunome.org/johrei.cfm;
and Johrei institute, http://www.johrei-institute.org/ourpeople.asp]

The Johrei intervention consists of four weekly training sessions (two hours each)
involving core principles needed to practise Johrei techniques such as “healing oneself by
healing others”; state of harmony, balance and timing (IZUNOME), and an introduction to the
three foundations of Johrei philosophy which emphasises importance of:
1. Awareness of spiritual well-being (Art of living / Art of Healing);
2. Appreciation of aesthetics (Art and Beauty) ; and
3. Appreciation of Nature including farming practice (Art of Nature).

In Johrei practice, the practitioner imagines an ethereal ‘healing-light’ entering his body
and being transmitted through his/her hands towards the recipient. The practitioner, without
touching a recipient, slowly moves his/her hands from the head down to the kidney area, front
and back. The procedure takes approximately 15 minutes in silence. The participants were
requested to practise Johrei daily with a partner, but at the end of training period, self-Johrei and
distant Johrei techniques were introduced as supplementary tools so that participants could
practice when a partner was not available.

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As in the hypnosis group, it was requested that each participant practise Johrei at least
four times a week to learn and familiarise themselves with the technique. Diaries were used to
record practice frequency at home. Group monthly follow-up sessions were continued for at
least four months.

2.2.3 Mock intervention (Controls for the psychological trainings)

This mock neuro-feedback condition was aimed to provide a control against possible
placebo effects from participants’ expectation and against factors of attention given and
experience of relaxation during their training sessions since this procedure was shown to
generate a relaxation response when used as a control condition [Egner et al., 2002].

This condition appeared to be high-tech, with two computers and electrodes fixed to
earlobes and the centre of the scalp, and with auditory relaxing feedback sounds (babbling
brook and ocean wave) heard over headphones. The sounds were supposedly representing alpha
and theta wave feedback from the participant’s own brain waves associated with relaxation.
Although the procedure was real, the feedbacks were false as the sounds were recorded
previously from another session.

The participants in this group had eight mock neuro-feedback sessions over one month,
and these sessions were performed by Dr. Dwivedi, a qualified clinical psychologist, with valid
neuro-feedback equipment.

2.3 Self-report questionnaires

Primary appraisal in stress perception (anxiety level and impact of a stressful event),
secondary appraisal, sense of ‘taking control of one’s own life’, psychological functioning
(mental quality of life) and sleep quality were measured by using the following published
questionnaires.

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2.3.1 Stress perception

2.3.1.1 State anxiety score in the State and Trait Anxiety Inventory [Spielberger et al., 1970]
The State and Trait Anxiety Inventory scale was designed to measure the levels of anxiety
both at the exact time the questionnaire was filled in (state anxiety) and as a general tendency
(trait anxiety). State anxiety level was measured and analysed in the current project. High scores
are associated with anxiety.

2.3.1.2 Impact of Event Scale (IES) [Horowitz et al., 1979; Joseph, 2000; Sundin & Horowitz,
2002]
The Impact of Event Scale (IES) measures the amount of stress which results from a
psychologically traumatising event, such as a diagnosis of HIV infection. High scores are
associated when event being perceived as a stressor. This IES was measured and analysed in the
study using HIV-infected individuals in this current project. High scores are associated with
high impact of the event.

2.3.1.3 Perceived Stress Scale (PSS) [Cohen et al., 1983]


The Perceived Stress Scale (PSS) is a scale designed to measure the secondary appraisal
of stress over a period (two weeks or a month) with the definition that “perceived stress is the
degree to which situations in one’s life are appraised as stressful (unpredictable, uncontrollable
and overloading.” High scores are associated with stress.

2.3.2 Perceived quality of life

2.3.2.1 Locus of Control scale (LoC) [Furnham & Steele, 1993; Wallston et al., 1978]
The Locus of Control scale (LoC) measures the perception of capability of controlling a
event [Schmitz et al., 2000]. Low scores are associated with high sense of taking control of
one’s own life, as saled from 0 (totally internal) to 24 (totally external) [Rotter, 1967]:
Individuals who have an internal locus of control believe that events result primarily
from their own behaviour and actions.
Those who have an external locus of control believe that powerful other partners, fate
or chance, primarily determine events. High scores are associated with low control.

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2.3.2.2 Mental Component Summary in the SF-36 (MCS) [Ware et al., 1994]*
The SF-36 is a scale developed to measure the levels of quality of life. This consists of
two subsets, i.e. Physical and Mental health clusters as scales of the Physical Components
Summary (PCS) and the Mental Components Summary (MCS). High scores in each subset are
associated with a high perceived quality of life physically (PCS) and mentally (MCS),
respectively. The MCS was measured and analysed in the study using HIV-infected individuals
in this current project. High scores are associated with high psychological functioning.

2.3.2.3 Pittsburgh Sleep Quality Index (PSQI) [Buysse et al., 1989]


The Pittsburgh Sleep Quality Index (PSQI) is a scale developed to measure the subjective
quality of sleep combined with behavioural items, e.g. sleep duration. High scores are
associated with low sleep quality.

All self-report data anonymised and depersonalised to protect the confidentiality of the
study participants. All data collection and entry was performed by research assistants blind to
the treatment conditions of the study participants.

* Note: The SF-36 scales, including the Mental Component Summary (MCS), are
scored such that higher scores represent better functioning (in this case, higher
psychological functioning), and lower scores represent poorer functioning. In all of
the other measurements used in this study, higher scores represent poorer functioning.

2.4 Materials and methods for in vitro investigation

2.4.1 Materials and experiment kits

2.4.1.1 Chemicals and reagents


The following Materials were purchased from Sigma (Dorset, U.K.,
http://www.sigmaaldrich.com/)
RPMI 1640
1% glutamine (200 mM)
1% penicillin (5000 IU/ml) / streptomycin (5000 mcg/ml)
1% Hepes buffer

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1% non-essential amino acids (100 x)


1% sodium pyruvate (100 mM).
Foetal calf serum (FCS)
Phosphate buffer saline (PBS)
Lymphoprep® (for peripheral blood mononuclear cells separation)
Paraformaldehyde (PFA)
0.05% Eosin dye
Propidium iodide (PI)
Phytohaemagglutinin (PHA: mitogen)
Staphylococcal enterotoxin B (SEB: super antigen)
Hydrocortisone

Radioactive [3H] thymidine was purchased from the Amersham [Amersham Pharmacia
Biotech, Buckinghamshire, U.K., http://www6.amershambiosciences.com/]

2.4.1.2 Biological reagents


The antigens of purified protein derivative (PPD: tuberculosis antigen) and Herpes
Simplex virus common antigen (Herpes) were provided by the Immunology clinical laboratory
at Chelsea and Westminster hospital.

The K562 cell line, a human chronic myelogenous leukaemia cell line for NK cytotoxic
activity measurement, was purchased from the European collection of cell cultures [ECACC,
Salisbury, U.K.].

2.4.1.3 Fluorescently conjugated antibodies for flow cytometry analyses


Antibody cocktails of CD45-FITC/CD3-PE/CD4-Cy-Chrome/CD8-APC and CD45-
FITC/CD3-PE/CD19-Cy-Chrome/CD56-APC, standard panels of fluorescently conjugated
antibody cocktails for lymphocyte subpopulation analyses, were purchased from Beckman
Coulter [Beckman Coulter, Bedfordshire, U.K., http://www.beckmancoulter.com/]

Individual antibodies of CD3, CD4, CD8, CD45, CD56, CD16, CD25, CD95, Nkp30,
Nkp46 and Annexin V (conjugated with FITC, PE, Cy-Chrome or APC) were purchased from
Becton Dickinson [Becton Dickinson, Oxford, U.K., http://www.bd.com/].

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2.4.1.4 Commercially available laboratory kits for colorimetric measurement


The CytoTox96®, a kit measuring NK cytotoxic activity, was purchased from Promega
[Promega U.K., http://www.promega.com/]. This CytoTox96® analysis includes measurements
of released Lactate dehydrogenase (LDH) in the supernant of the cultured medium (after 4-hour
incubation of mixed K562 cells and PBMCs) which contains certain levels of LDH from dead
cells. This kit was used in order to avoid using radio-active substance which is required in the
gold standard method, the 56Cr-release assay [Promega, 2004].

ELISA kits for measuring the levels of cortisol, DHEA-S and melatonin were purchased
from iDS [iDS U.K., http://www.idsltd.com/].

2.4.2 Equipments used for in vitro investigation

2.4.2.1 Flow cytometry


Flow cytometry is a method for quantitating components or structural features of cells
primarily by optical means. Flow cytometry measures one cell at a time, but it can process
thousands of cells in a few seconds. The cells are passed single-file through a laser beam by
continuous flow of a fine stream of the suspension. Each cell scatters some of the laser light,
and also emits fluorescent light excited by the laser come from the labelled fluorescent
conjugated antibodies designed to bond specific particle on the cell surface or in the cytoplasma.

The five main components of flow cytometry are:


1. Flow cell - liquid stream (sheath fluid) carries and aligns the cells so that they pass in
single file through the light beam
2. Light source - commonly used is high power water-cooled argon and/or krypton laser
3. Electronic detector which can quantitate the faint flashes of scattered and fluorescent light
4. Analogue to Digital Conversion (ADC) system which generates forward scatter (FSC)
and side scatter (SSC) signals as well as labelled fluorescence signals
5. Computer for analysis of the signals, and to record data for thousands of cells per sample,
and to display the data graphically and numerically
6. Statistical analysis can be done simultaneously or afterward using an analysis programme

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Data acquisition
The cytometer typically measures several parameters simultaneously for each cell:
Low angle forward scatter (FSC) intensity, proportional to cell diameter
Orthogonal (90 degree) scatter intensity, approximately proportional to the quantity of
granular structures within the cell (SSC)
SSC
Fluorescence intensities at several wavelengths
Granulocytes
(each FL-1, FL-2, FL-3 and so on associate with a
defined structure identified by a fluorescent probe)
Light scatter alone (FSC and SSC graph: e.g. Figure 10)
is commonly used to exclude dead cells, cell aggregates, and Debris Monocytes
cell debris from the fluorescence data. It is sufficient to Lymphocytes
distinguish lymphocytes from monocytes and granulocytes in FSC

blood leukocyte samples. Figure 10: FSC vs. SSC dot plot

Fluorescence intensities are typically measured at several different wavelengths


simultaneously for each cell. Fluorescent probes are used to report the quantities of specific
components of the cells. Fluorescent antibodies are often used to report the densities of specific
surface receptors, and thus to distinguish subpopulations of different cell types.

Gating and data analysis


In order to analyse characteristics of specific immune cell sub-population, selection of the
cells needs to be precise and efficient. This selection is performed by making a gate of region in
a dot-plot during flow cytometry analyses, for example:
1. After setting up flow cytometer for acquisition, create a dot plot with the X-axis as
forward scatter (FSC) and the Y-axis as side scatter (SSC).
2. Acquire data from cells, and draw a circle around a region of the lymphocytes based
on their light scatter characteristics on the plot (FSC vs. SSC). This becomes a region
1 (R1), the red circle of lymphocyte in the Figure 10 for example.
3. For further analyses, create another dot plot that encompasses the cells from the R1 in
the plot with fluorescents (FL1 vs. SSC or FL1 vs. FL2 etc.).

Precision of percentage in lymphocyte sub-population measured by flow cytometry


Methodological variation in lymphocyte percentages was assessed in three preliminary
blood samples from one volunteer. Nine sample tubes of blood were taken at each time point,

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and assessed for percentages of cells expressing CD45+CD3-CD56+ (henceforth NK-cells) and
CD45+CD3+CD4+ (CD4 T-cells) and CD45+CD3+CD8+ (CD8 T-cells).

Fluorescently conjugated single antibodies of CD3, CD4, CD8, CD45, CD56 and CD16
[Becton Dickinson, Oxford, U.K.] were used to determine NK cells (Cytotoxic and Regulatory
NK-cell subsets) and T-lymphocytes (CD4 T-cells and CD8 T-cells). Cell samples were
measured by a FACSCalibur flow cytometer [Becton Dickinson] and the data generated by flow
cytometer were analysed by a CellQuest software.

Means and ranges were within normal limits [Hannet et al., 1992] and standard deviations
were calculated in individual occasion and in total [Table 6].

Table 6: Mean percentages and counts (standard deviation: SD) of NK-cells, CD4 and CD8 T-cells and
total lymphocyte counts using a single volunteer (9 tubes collected and averaged on 3 separate occasions)
NK % CD4% CD8% NK count CD4 count CD8 count Total lymph count
(SD) (SD) (SD) (SD) (SD) (SD) (SD)
Time1. 9.7 48.1 29.6 94.8 438.4 270.0 894.3
(0.54) (0.97) (0.73) (9.3) (12.7) (11.3) (32.0)
Time2. 8.3 46.8 28.8 65.6 346.1 213.4 712.6
(1.31) (0.89) (0.33) (12.6) (10.7) (9.8) (36.2)
Time3. 14.3 44.4 28.2 130.3 381.0 249.3 859.2
(0.72) (0.52) (0.60) (8.7) (36.4) (11.6) (33.6)
Total mean 10.77 46.4 28.9 96.9 388.5 244.2 822.0
(SD) (0.86) (0.79) (0.55) (10.2) (19.9) (10.9) (33.9)

Hence, all changes in percentages of NK cells, CD4 T-cells and CD8 T-cells more than
twice of standard deviations in total means, i.e. 1.90, 1.58 and 1.10 % respectively, were
considered as in out of the 95 % confident interval. Changes in each cell sub-population more
than these percentages are considered as a difference between samples, and the statistical
analyses are performed with this consideration in the current project.

2.4.2.2 Colorimeter
The colorimeter is an apparatus that allows the absorbance of light at a particular wave-
length or frequency (colour) of visual light to be determined. Different chemical substances
absorb varying frequencies of the visible spectrum. Colorimeters rely on the principle that the
absorbance of a substance is proportional to its concentration, i.e. a more concentrated solution
gives a higher absorbance reading.

A quantitative reading for the concentration of a substance can be found by making up a


series of solutions of known concentration of the chemical under study, and plotting a graph of

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absorbance against concentration. By reading the absorbance of the specimen substance on the
graph, a value for its concentration is found.

Method of the enzyme linked immuno-sorbant assay (ELISA) [Voller, 1978]


The ELISA is ideally suited to assaying protein factors e.g. hormones but can be adapted
to measure small molecules as well. In the current project, various commercially available
ELISA kits [iDS U.K., http://www.idsltd.com/] and CytoTox96® [Promega U.K.,
http://www.promega.com/] were purchased and used for measurements of the hormone levels
and LDH levels to determine these concentrations.

2.4.3 Methods in vitro [See also Appendix 4. for detailed procedures]

2.4.3.1 Preparation of tissue culture medium for cell culture in vitro


Tissue culture medium (TCM) was used for incubation of cells in the project. It consists
of RPMI 1640 supplemented with 1% glutamine (200 mM), 1% penicillin (5000 IU/ml)/
streptomycin (5000 mcg/ml), 1% Hepes buffer, 1% non-essential amino acids (100 x), 1%
sodium pyruvate (100 mM).

Cortisol level in the tissue culture medium


In order to supply essential nutrition for cell culture, 10% Fetal Calf Serum (FCS:
Sigma®) was added in tissue culture medium (TCM) when cells were incubated in vitro.

ELISA assays show that the tissue culture medium containing 10% FCS contains 5.9nM
cortisol; and this was small in comparison to the amount of cortisol found in plasma in the 34
volunteers (29.5 - 265.8nM; mean 131.8, SD = 50.3) [Figure 11].
300

250

200

150

100

50

Figure 11: Individual cortisol levels in the tissue culture medium (left: 5.9 nM)
and in plasma from 34 volunteers (right: 29.5 - 265.8 nM)

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Conclusion:
The level of cortisol in the TCM was low, and thereafter in this thesis, TCM only was
considered to be cortisol free.

2.4.3.2 NK cytotoxic activity measured by flow cytometry [Godoy-Ramirez et al., 2000]


Detailed procedures are given in the Appendix 4.

Effector cells (PBMCs):


Peripheral venous blood samples were collected using Vacutainers [Becton Dickinson,
Oxford, U.K.]. Heparinized blood was centrifuged (1500 rpm for 10 minutes at room
temperature). The plasma was removed and the blood pellet reconstituted by the addition of
RPMI 1640. This was then mixed and overlaid onto Lymphoprep. PBMC were recovered from
the interface after centrifuging (1500 rpm) for 30mins at room temperature [Boyum, 1974].
Viability was assessed and adjusted by dye (Eosin 0.05 %) exclusion.

The separated effector cells (PBMCs) by LymphoPrep® were washed twice and
suspended in the TCM; then 10 l of anti-human CD45 monoclonal antibody directly
conjugated with fluorescein isothiocyanate (FITC) was added and incubated for 20 minutes.
These cells were washed again, and after the last wash, concentration of cells was assessed by
counting cells with dye exclusion, and the cell concentration adjusted to 5 x 106 cells / ml by
adding TCM prior to mixing with target cells, i.e. K562 cells, for the NKCA assay.

Target cells (K562 cell line):


K562 haematopoietic tumour cells served as the target cell for determining NK cytotoxic
activity. K562 cells are a human erythromyelocytic leukaemia cell line, which may transform if
allowed to overgrow resulting in losing their sensitivity to be killed by NK-cells. Hence, the
K562 cell line should be renewed every one to two months, by starting with a new batch of
frozen cells from the liquid nitrogen freezer.

In order to maintain a ready supply of cells in the optimal growth phase for the NKCA
assay, optimal duration and concentration of K562 cells during cell culture were examined by
determining the growth characteristics of this cell line.

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Growth characteristics of K562 cell line used as targets for assessing NKCA
K562 cells should be in the logarithmic growth phase for the use of NKCA assay, as cells
in the early (developing) and late (saturated) phase of their development are resistant to killing.
K562 cells (recovered from frozen sample) were cultured at various seeding concentrations of
1.5 x 104, 1.5 x 105 and 1.0 x 106 cells per ml in 10 ml flasks with tissue culture medium, and
each flask was placed at 37oC in a 5% CO2 humidified incubator for 3 days. The K562 viable
cell concentration in each flask was assessed on Day 0, 1, 2, and 3 and simultaneously the
percentage of dead cells was measured [Figure 12].
Cell numbers per mL (in Log-scale)

K562 growth curve in culture Dead K562 %in culture condition


100000000
100

Dead cell percentages


80 1.5x10^4 cells/ml
10000000
1.5x10^5 cells/ml
1.0x10^6 cells/ml
60
1000000

40
1.5x10^4 cells/ml
100000
1.5x10^5 cells/ml 20
1.0x10^6 cells/ml

10000
0
day0 day1 day2 day3 Day 1 Day 2 Day 3

Figure 12a: The growth rate curve of K562 cells Figure 12b: The dead cell % of K562 cells
(The red line in Figure 12b indicates the maximum acceptable level of dead K562 cells)
Figure 12: K562 cell growth at starting cell concentrations of 1.5x104, 1.5x105 and 1.0x106 cells per mL

Figure 12a shows that from Day 0 to 3, the K562 cells were in the log-growth phase
regardless of seeding concentration. The highest cell concentrations were obtained with a
seeding concentration of 1.5 x 105 cells per mL. Figure 12b illustrates that the lowest
percentage dead cells was obtained with a seeding concentration of 1.5 x 105 cells per mL.

Hence, it can be concluded that the cells should be passed every two to three days at
approximately 1.5 x 105 cells per ml of culture in TCM supplemented with 10% foetal calf
serum.

NKCA assay procedure:


Effector and target cells, i.e. fresh PBMCs and K562 cells, were added in duplicate in 12
x 75 mm round-bottom tubes [Beck Dickinson, Oxford, U.K.] to yield the Effector to Target
(E:T) ratio of 50:1, 25:1, 12.5:1 in 200 l. Control tubes were set up with only target or effector
cells for determining spontaneous cell death rates. Tubes were mixed by gently tapping, and

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incubated at 37°C for three hours (in the university student study) or four hours. After
incubation, the tubes were placed on ice until analysed. Twenty microliters of propidium iodide
(PI), which stains the nucleus of dead cells, were added to each tube 10-15 minutes before
acquisition.

Flow cytometric acquisition:


Flow cytometry was performed with a FACS Calibur cytometer [Beck Dickinson]. The
instrument was set for two-colour analysis using FACScomp software in conjunction with
Calibrite beads [Beck Dickinson]. Data were collected in list mode and analysis was performed
using CellQuest software [Beck Dickinson]. A region was set on a dot plot of SSC vs.
fluorescence 1 (FL-1: CD45) around the cluster of target cells identified in the control sample
tubes and percentages of PI-stained cell number in the region were counted. NKCA was
calculated by subtraction of target total from individual totals [Figure 13].

Figure 13a: K562 cells only tube acquisition Figure 13b: PBMCs only tube acquisition

M1

Figure 13d: K562 histogram by PI staining


(M1: PI staining positive cells)
Figure 13c: testing tube (mixed with K562 and PBMCs) acquisition

Figure 13: Acquision settings of the flow cytometer for measuring the levels of NKCA
A region setting (R1) for K562 [Figure 13a,b] and a process of dead K562 detection [Figure 13c]
followed by histogram percentage analysis (counting PI stained cell numbers) [Figure 13d]

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Precision of levels in the NK cytotoxic activity measured by flow cytometry


Methodological variation in NK cytotoxic activity was assessed in preliminary blood
samples from three volunteers. Three sample tubes of blood were taken from them, and all nine
samples were duplicated to measure NK cytotoxic activity. Changes in the levels of NKCA
more than twice of standard deviations (1.44 % killing) were considered as out of the 95 %
confident interval. Changes in NKCA level more than 1.44 were considered as a difference
between samples, and results from the statistical analyses were examined with this definition.

2.4.3.3 NK-cell characteristics during NKCA assay measured by flow cytometry


Expression of Natural cytotoxic receptor (NCR) measured by flow cytometry
Cell surface staining was performed by incubating with 5 l of particular monoclonal
antibodies for 15 min at room temperature in the dark. The analysis of natural cytotoxic receptor
(NCRs: Nkp30 or Nkp46) was performed using four-color flow cytometric acquision and
FlowJo® flow cytometry analyses [Figure 14].
FL-3: CD56-CyChrome staining

CD3-CD56+
(NK-cells)
CD56brightCD16-
FL-3: CD56-CyChrome staining

Regulatory NK-cells

FL-4: CD3-APC staining CD56dimCD16+


Cytotoxic NK-cells
Figure 14a: CD3-CD56+NK-cells in dot plot

Whole NCRs(+) %
lymphocytes
FL-1: CD16-FITC staining

FL2: Nkp30 or Nkp46-PE staining Figure 14b: NK-subsets of


Cytotoxic and Regulatory NK-cells in dot plot
Gated NKC
sample

Figure 14c: Ppercentage of NCR expressions

Figure 14: Acquision settings of the flow cytometer in the NCR analyses

A region was set on a dot plot of FL-3 (CD56) vs. FL-4 (CD3) to target CD3-CD56+ NK-
cells [Figure 14a], and further regions for NK sub-populations were determined by a dot plot of

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FL-3 (CD56) vs. FL-1 (CD16) [Figure 14b: CD56dimCD16+ Cytotoxic and CD56brightCD16-
Regulatory NK-cell subsets]. The percentage expressions of NCRs were then measured as a
percentage of FL-2 (NCRs) positive cells within the population of a selected region (Cytotoxic
or Regulatory NK-cells) by histogram [Figure 14c: percentage of cells expressing NCRs].

2.4.3.4 Proliferative response measured by [3H]-thymidine incorporation


PBMC cultures with stimulus:
Peripheral blood mononuclear cells (PBMCs) were cultured with microbacterial protein
(purified protein derivative: PPD) and Herpes antigen (memory recall antigens) for 6 days in
tissue culture medium (TCM: containing 10% fetal calf serum) with/out cortisol [hydrocortisone,
Sigma®] to measure lymphocyte proliferative responses. In addition, Three day cultures with 1)
Staphylococcal enterotoxin B (SEB) used as a stimulant, a super antigen, which can stimulate
both naïve and memory cells; and 2) Phytohaemagglutinin A (PHA) chosen as a mitogen which
stimulates all T-cells and thereby acts as a positive control for proliferative responses.

Whole Blood Cell cultures with stimulus [Hutchinson et al., 1999] [See also Appendix 4]:
The whole blood assay for measuring proliferative responses was applied in order to
reduce cell handling and manipulation. In brief, whole blood (diluted 1/4) was incubated for 3
days with 12.5 g/ml of SEB, super-antigen, or 12.5 g/ml of PHA, mitogen, to measure
lymphocyte proliferative responses. PHA or SEB were incubated with 1:10 diluted blood with
or without 250 nM cortisol in 96 U-shaped well plates in total amount of 200 L, and incubated
0, 24 and 48 hours followed by the [3H]-thymidine incorporation assay.

[3H]-thymidine incorporation
The plates were incubated at 37°C in a 5% CO2 humidified atmosphere, and the cells were
pulsed with 1 Ci tritium [mehyl-3H] Thymidine ([3H]-TdR) per well for the last four hours of
incubation. The supernatants were harvested using a Skatron AS Harvesting system [Shatron,
Suffolk] and the amount of [3H] incorporated with the cells determined on a Wallac 1205 Liquid
scintillation Counter [Pharmacia, Becks]. Results were presented as couts per minutes (cpm).

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2.4.3.5 T-cell characteristics after in vitro incubation measured by flow cytometry


Cell preparation for proliferation analyses
Previously titrated concentration of PPD or Herpes antigen was incubated with 2.0 x 105
cells per mL PBMCs in total amount of 1.2 mL per well in 24 well culture plates with/out 250
nM cortisol. 12.5 g/mL of PHA or SEB were incubated with 1:10 diluted blood with/out 250
nM cortisol in 24 U-shaped well plates in total amount 1.2 mL, and incubated for 0, 24 and 48
hours. The plates were incubated at 37°C in a 5% CO2 humidified atmosphere and, then cells
were allocated into FACS 12 x 75 round bottom tubes [Beck Dickinson, Oxford, U.K.] before
following procedures.

Staining cell surface markers upon T-lymphocyte sub-populations


Cell surface staining was performed by incubating with 5 l of particular monoclonal
antibodies for 15 min at room temperature in the dark. In a whole blood assay, erythrocytes
were lysed for 20 min with 1 ml of 10 x diluted lysing solution [Becton Dickinson] in the dark.
In both methods, cells were washed three times with the TCM and fixed in 100 l of 4% of
para-formaldehyde in PBS (fixation buffer) for 20 min at 4°C.

Expression of CD25 or CD95 upon T-lymphocytes measured by flow cytometry


Cell samples were run on a FACSCalibur flow cytometer (Becton Dickinson) and
analysed by CellQuest software. CD3-Cy-Chrome (FL-3) and CD8-APC (FL-4) fluorescent
conjugated antibodies were used to label lymphocyte sub-populations (CD3+CD8+ T-cells and
CD3+CD8- T-cells as CD4+ T-cells). At least 5000 events in the light-scatter (FSC/SSC)
lymphocyte region were acquired. Each lymphocyte sub-population was identified by a gate on
FL-3 versus FL-4 dot plots, i.e. CD3-CD8 scatter dot plots. The FITC-fluorescence intensities
(FL-1) of CD25 or CD95-labelled lymphocyte populations and isotype controls were displayed
and determined as mean channel values on a four-decade log scale in histogram plots.

The relative quantity of CD25 or CD95 expression was calculated by the percentage of
cell numbers in the positive intensity area, and mean fluorescence intensity (m.f.i.) was
calculated as the difference between mean values of CD25 or CD95 and isotype control labelled
samples. The instrument calibration was performed by CellQuest software using CaliBRITETM
beads.

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Expression of Annexin V- PI on T-lymphocytes measured by flow cytometry


The Annexin V-FITC (FL-1) antibody & propidium iodine (PI: FL-3) staining method for
detection of apoptosis was used to determine the effect of cortisol upon apoptotic processes.
Four-color flow cytometric acquision and FlowJo® flow cytometry analyses were performed. A
region was set on a dot plot of SSC vs. FL-2 (CD3-PE) to target T-lymphocytes, and further
percentages of necrotic cells, apoptotic cells and live cells were determined by a dot plot of FL-
3 (PI) vs. FL-1 (Annexin V) [Figure 15].

FL-1: Annexin V staining


Negative control
Whole lymphocytes (for gating purpose)
(gated by the FSC-SSC plot) Apoptotic
cells
Necrotic cells
Side Scatter (SSC)

Live cells

T-lymphocytes FL-3: PI staining


Figure 15b: setting-up a region for apoptotic cells
using negative isotype control in a dot plot
FL-1: Annexin V staining

Experiment samples

Apoptotic
cells
Necrotic cells
FL-2: CD3 staining
Figure 15a: a region for T-cells in a dot plot Live cells
FL-3: PI staining
Figure 15c: a region for apoptotic cells in a dot plot

Figure 15: Acquision settings of the flow cytometer in the Annexin V- PI analysis

A region was set on a dot plot of FL-2 (CD3) vs. SSC to target T-cells (Figure 15a) from
whole lymphocytes gated by the FSC-SCC plot previously, and further regions for apoptotic
cell subsets were determined by a dot plot of FL-3 (PI) vs. FL-1 (Annexin V) (Figure 15c:
apoptotic (Annexin V+ & PI-) and necrotic cell (PI+) subsets in the T-lymphocytes). Figure 15b
shows the method setting-up for the region of apoptotic cells by using negative isotype controls.

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2.5 Statistical analyses

All psychological and physiological laboratory samples were anonymised and processed
using an assigned study number to ensure that the analyses were performed blindly in terms of
the groups and subgroups to which the samples belong to.

To examine the a priori hypothesis that psychological intervention may counteract the
detrimental effect of stress on well-being, on-treatment analysis (analysis of results from only
those who completed the trial) was performed. When statistical differences were found in the
primary outcome measure in univariate analyses, Intention-to-treat analysis was performed in
order to confirm the results. The intention-to-treat is the analysis of results from every subject
enrolled in the trial regardless of whether they completed the trial, and all individuals who
withdrew were counted in the number of subjects who failed the hypothesis.

Univariate statistical analyses were performed to test specific hypotheses stated at each
point in the results section [3.1 and 3.2 in vivo studies; and 3.3 and 3.4 in virto experiments].
Sample sizes of each group were relatively small in the in vivo analyses [3.1 and 3.2], and
therefore essentially multivariate analyses are unlikely to achieve meaningful results. If
multivariate analyses were performed, standard errors would be relatively high giving very wide
95% confidential intervals (C.I.) indicating a lot of uncertainty in findings. For this reason, it
was felt inappropriate to perform multivariate analyses.

The Statistical Package of Social Science version 11.0 (SPSS®) [Field, 2003] was used
with a p value of less than 0.05 considered as a statistically significant value, and a p value of
less than 0.10 as a statistical trend. The Study Size Determination (SSD) programme [Lehmann,
2001] was used for sample size calculation at the 5% significance level and with the 90% power.
For independent normally distributed data, analyses of variance (ANOVA) were performed
followed by post-hoc Student t-tests. Repeated-measures ANOVA was used for longitudinal
data normally distributed [Tabachnick & Fidell, 1996]. For non-normally distributed data,
repeated-measure ANOVA followed by the 10% trimming method [Wilcox, 1997] was
performed. The 10% trimming method was applied in order to remove outliers so that the
ANOVA method can be applied. To confirm the finding from the ANOVA following the 10%
trimming method, the Non-parametric ANOVA (the Kruskal-Wallis rank tests) was used
[Tabachnick & Fidell, 1996]. The Wilcoxon rank tests were used for paired and non-normally
distributed data [Ajetunmobi, 2002].

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Chapter III

Results

- 78 -
Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

CONTENTS OF CHAPTER 3
Results

3.1 The influence of psychological intervention upon stress-related changes in


university students facing academic examinations.
3.1.1 Validation of academic examination as a stress inducer for university students 83
3.1.1.1 Impact of academic examination upon psychological measures of
stress and anxiety 83

3.1.1.2 Impact of academic examination upon immune parameters 84

3.1.2 Differences in the levels of lymphocyte subsets and NK cytotoxic activity


(NKCA) between university students with high and low perceived stress levels 85
3.1.2.1 Difference in the percentage of NK-cells and the levels of NK
cytotoxic activity (NKCA) between university students with high
and low perceived stress levels 86

3.1.2.2 Difference in the percentage of T-cells between university students


with high and low perceived stress levels 91

3.1.3 The influence of psychological intervention upon stress-related changes in


university students facing academic examinations 93
3.1.3.1 Effect of psychological intervention upon stress perception in
university students anticipating academic examinations 93

3.1.3.2 Effect of psychological intervention upon stress-related


lymphocyte sub-populations in university students anticipating
academic examinations 95

3.2 The influence of psychological intervention upon perceived stress and quality-of-
life and various immunological disease-associated parameters in HIV-infected
individuals
3.2.1 Disease-associated and stress-related changes in HIV-infected individuals in
psychology immunological parameters 103
3.2.1.1 Psychological profiles of the HIV-infected individuals recruited
into the study 103

3.2.1.2 Stress-related perception and disease progression markers of HIV-


infection 110

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3.2.2 The influence of psychological intervention upon disease progression


parameters in HIV-infected individuals (randomised controlled trial for four
months period) 117
3.2.2.1 Effect of psychological intervention upon perceived stress and
anxiety in HIV-infected individuals 117

3.2.2.2 Effect of psychological intervention upon CD4 T-cell counts in


HIV-infected individuals 119

3.2.2.3 Effect of psychological intervention upon other immunological


markers (log-transformed viral load levels and NK-cell counts) in
HIV-infected individuals 121

3.2.3 The influence of psychological intervention upon disease progression marker,


CD4 T-cells, in HIV-infected individuals (case controlled study over 24
months period) 122
3.2.3.1 Analysis of baseline differences between the three groups in the CD4 T-cell
count 122

3.2.3.2 Analysis of the rate of change in CD4 T-cell count (CD4 gradient) 123

3.3 In vitro investigation into the effect of exposure to stress hormones upon
Natural Killer cells
3.3.1 Development and optimisation of a flow cytometric method for measuring
NKCA 128
3.3.1.1 NKCA after incubation of PBMCs for 24 hours 128

3.3.1.2 NK-cell percentage in PBMCs after 24 hours incubation 129

3.3.1.3 NKCA after 24hrs incubation of PBMCs with cortisol 131

3.3.1.4 NK-cell percentage in PBMCs after 24 hours incubation with


cortisol 132

3.3.2 Comparison of two methods for measuring NK cytotoxic activity (NKCA): -


the flow cytometric method and the colorimetric method (CytoTox96®) 133

3.3.3 Investigation of NK-cell profiles and NKCA (colorimetric method) 134


3.3.3.1 NKCA, assessed by colorimetric method, after incubation of
PBMCs for 24 hours 134

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3.3.3.2 Repeat assessment of NK-cell percentage and examination of NK


subpopulations in PBMCs after 24 hours incubation 135

3.3.3.3 Expression of Natural Cytotoxic Receptors (Nkp46 and Nkp30) by


cytotoxic NK-cells (CD3-CD56dimCD16+) pre and post 24hours
incubation 136

3.3.3.4 NKCA, assessed by colorimetric method, after 24 hours incubation


of PBMCs with 250nM cortisol 138

3.3.3.5 NKCA levels between pre and post 24 hours incubation with or
without 250nM cortisol 139

3.3.3.6 CD56 NK-cell subset profiles in PBMCs after 24 hours incubation


with 250nM cortisol 141

3.3.3.7 Expression of Natural Cytotoxic Receptors (Nkp46 and Nkp30) by


cytotoxic NK-cells (CD3-CD56dimCD16+) after 24hours
incubation with or without 250nM cortisol 142

3.3.3.8 Nkp46 expression on cytotoxic NK-cells pre and post 24 hours


incubation with or without 250nM cortisol 143

3.3.3.9 NKCA and Nkp46 expression on cytotoxic NK-cells pre and post
24 hours incubation with or without 250nM cortisol 144

3.3.4 Endogenous stress hormone levels and NKCA (colorimetric method) 145
3.3.4.1 Endogenous hormone levels and NKCA at Time 0 point 146

3.3.4.2 Endogenous hormones levels and the increased levels of NKCA


after 24 hours in vitro incubation of PBMCs without exogenous
cortisol 146

3.4 In vitro investigation into the effect of exposure to stress hormones upon
T-lymphocytes
3.4.1 T-lymphocyte proliferative responses against common antigens 151
3.4.1.1 T-lymphocyte proliferation after incubation of PBMCs for six days
with and without exogenous cortisol 152

3.4.1.2 T-lymphocyte proliferative response against purified protein


derivative (PPD) after incubation of PBMCs for six days with or
without exogenous cortisol 153

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3.4.1.3 T-lymphocyte proliferative response against Herpes antigen after


incubation of PBMCs for six days with and without exogenous
cortisol 154

3.4.2 T-lymphocyte proliferative responses against super-antigen and mitogen 155


3.4.2.1 T-lymphocyte proliferative responses against staphylococcal
enterotoxin B (SEB) after incubation of diluted whole blood for
three days with or without exogenous cortisol 156

3.4.2.2 T-lymphocyte proliferative responses against phytohaemagglutinin


A (PHA) after incubation of diluted whole blood for three days
with or without exogenous cortisol 157

3.4.3 Cell surface markers on proliferating T-lymphocytes following stimulation


with PHA 158
3.4.3.1 Expression of an apoptosis (CD95: Fas) cell surface marker by T-
lymphocytes in vitro during PHA-induced proliferation with and
without exogenous cortisol 158

3.4.3.2 Expression of cell-surface Annexin-V and cytoplasmic Propidium


iodine (PI) in T-lymphocytes after incubation with PHA for three
days with or without cortisol 163

3.4.3.3 Expression of an activation (CD25: IL-2 receptor) cell surface marker


byT-lymphocytes during PHA-induced proliferation with and without
exogenous cortisol 164

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3.1 The influence of psychological intervention upon stress-related changes in


university students facing academic-exams.

3.1.1 Validation of academic examination as a stress inducer for university students

This study was designed to examine the hypothesis that anticipating an academic
examination induces stress and stress-related changes in university students. The levels of stress
perception (Perceived Stress Score and State anxiety score) and NK-cell percentages and NK
cytotoxic activity were examined in university students facing academic examination (Exams)
and compared with results obtained in the non-exam periods of university life (Non-exam) in
order to detect exam-stress associated changes. In addition, CD4 and CD8 T-cell percentages
were examined.

3.1.1.1 Impact of academic examination upon psychological measures of stress and anxiety

Perceived stress levels (PSS)


There was a significant increase (t = 2.1, df = 21, p = 0.045) in the levels of the PSS at the
Exams compared to the Non-exam time point [Table 7 and Figure 16].

Table 7: Mean (95% C.I.) PSS scores at the Non-exam and Exam time points
PSS
Non-exam Exams
Mean score 21.5 26.2
(95% C.I.) (18.6 - 24.5) (22.8 – 29.6)
n 22 22
40
Mean (95% C.I.) Perceived Stress Scores

p = 0.045

30
Mean(95%C.I.) of PSSscore

20

10

0
N= 22 22
Non-exams Exams

Figure 16: Mean (95% C.I.) PSS scores at the Non-exam and Exam time points

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Conclusion:
Perceived stress levels were increased in university students when faced with academic
examinations.

State anxiety levels


The students were shown to be more anxious (t = 2.1, df = 24, p = 0.048) at the Exam
time point than at the Non-exam time point [Table 8 and Figure 17].

Table 8: Mean (95% C.I.) State anxiety scores in the STAI at the Non-exam and Exam time points
State anxiety in the STAI
Non-exam Exams
Mean score 36.6 41.8
(95% C.I.) (32.7 - 40.5) (38.5 – 45.1)
n 25 25
Mean (95% C.I.) State Scores in the STAI

50
Mean (95%C.I.) of Statescorein STAI

40

30

20 p = 0.048

10

0
N= 25 25
Non-exams Exams

Figure 17: Mean (95% C.I.) State anxiety scores in the STAI at the Non-exam and Exam time points

Conclusion:
Academic examinations increased anxiety levels in university students.

3.1.1.2 Impact of academic examinations upon immune parameters


Previous research suggested that the levels of NK cytotoxic activity (NKCA) and NK-cells
may be associated with psychological stress, i.e. the hypotheses are that exam-induced stress
decreases the NK cytotoxic activity and the levels of NK-cells in the university students. Hence
this study was designed to examine these hypotheses with an additional exploratory
investigation of the effects upon the levels of CD4 and CD8 T-cells.

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NKCA levels
The NK cytotoxic activity levels were not statistically significantly different (t = 0.2, df =
20, ns) between the Non-exam and Exam time points [Appendix 6: Table A-7].

Conclusion:
Anticipation of academic examinations did not affect the levels of NKCA.

NK-cell and T-cell levels


The results from 26 students show that none of the NK-cell (t = 0.3, df = 25, ns), CD4 T-
cell (t =0.6, df = 25, ns) or CD8 T-cell (t =0.2, df = 25, ns) percentages were significantly
different between the Non-exam and Exam time points [Appendix 6: Table A-8].

Conclusion:
Anticipation of academic examinations did not affect NK-cell and CD4 and CD8 T-cell
percentage.

Summary:
The data indicated that an anticipation of academic examinations induces stress and
provokes anxiety in university students. However, the academic examinations did not appear to
affect the levels of NK-cells, CD4 and CD8 T-cells or NK cytotoxic activity in the university
students, in this study.

It is concluded that academic exams are a valid stressful life event (stressor) to increase
stress and anxiety levels for university students; but that academic exams did not appear to
provide concurrent changes in immunological parameters, i.e. levels of NK-cells, T-cells and
NK cytotoxic activity.

3.1.2 Differences in the levels of lymphocyte subsets and NK cytotoxic activity (NKCA)
between university students with high and low perceived stress levels

The previous section suggested that an anticipation of academic examinations did not
affect the levels of the immune parameters. However, as described in the introduction [1.2.3.2

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and 1.2.4.3], individual’s perception of stress has been shown to be more associated with
immune parameters, particularly NK-cell profiles, than a stressful life event itself. Hence, in this
section, immune parameters (NK-cells [3.1.2.1], T-lymphocytes [3.1.2.2]) were compared in
two subgroups of students assessed to be stressed and not-stressed individuals according to their
PSS scores. The university students, who filled the PSS questionnaires and had blood collection
four weeks after recruitment, were divided into two subgroups on the basis of the PSS (Median
= 22.5, Mean = 23.1, Standard deviation = 7.7) and labelled as Not-stressed and Stressed
individuals in this study:

Stressed individuals: PSS score was more than or equal to 22

Not-stressed individuals: PSS score was less than 22


There was no statistically significant difference in the numbers of Subjects between the
Not-stressed and Stressed subgroups with regard to gender distribution [Appendix 6: Table A-9].

3.1.2.1 Differences in the percentage of NK-cells and the level of NK cytotoxic activity
(NKCA) between university students with high and low perceived stress levels
Research has suggested that NK-cells are affected by stress perception [1.2.3.2 and
1.2.4.3]. Accordingly, this study was performed to determine if, in university students,
perceived stress level is associated with
1. Low NK-cell percentage
2. Low NK cytotoxic activity level, and
3. Low per-NK-cell cytotoxic activity.
In addition, the influence of gender upon these parameters was investigated.

NK-cell levels
There was no significant difference between the Stressed and Non-stressed subgroups in
the percentages of NK-cells (t = 0.7, df = 39, ns) [Appendix 6: Table A-10].

Gender did not appear to affect the results. There was no significant gender vs. stress-
perception interaction (F = 1.7, df = 39, ns; male: t = 1.1, df = 18, ns; and female: t = 1.5, df =
18, ns) [Appendix 6: Tables A-11 and 12]. Hence, it was concluded that there was no gender
bias with regard to the result from comparison analysis between the Stressed and Not-stressed
subgroups.

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With regard to gender difference, however, male students had a trend towards higher NK-
cell percentages than female students (t = 1.9, df = 38, p = 0.066) [Table 9 and Figure 18].

Table 9: Mean (95% C.I.) NK-cell percentages in male and female students
NK-cell (%)
Male Female
Mean level 11.0 8.0
(95% C.I.) (8.5 – 13.5) (6.1 – 10.0)
n 20 20
15
Mean (95% C.I.) NK-cell (%)

10

0
p = 0.066
male female

Figure 18: Mean (95% C.I.) NK-cell percentages in male and female students

Conclusion:
There was no difference in NK-cell levels between stressed and not-stressed students.
Male students appeared to have higher NK-cell percentages than females, but this difference
was not statistically significant.

Natural Killer cytotoxic activity (NKCA) levels


The Stressed students had significantly lower NKCA levels compared to the Non-stressed
students (t = 2.4, df = 39, p = 0.023) [Table 10 and Figure 19].

Table 10: Mean (95% C.I.) levels of NKCA (% killing) in the Not-stressed and Stressed subgroups
NKCA (% killing)
Not-stressed Stressed
Mean level 12.0 5.8
(95% C.I.) (7.7 – 16.3) (2.8 – 8.7)
n 20 21

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epoint 1
20

NK cytotoxic activity (%killing)


p = 0.023

NKCAlevels(95%C.I.)at Tim
15

10

0
N= 20 21
Non-stressed Stressed

Figure 19: Mean (95% C.I.) NKCA (% killing) in the Not-stressed and Stressed subgroups

Gender did not appear to affect the results. There was no significant gender vs. stress-
perception interaction (F = 0.6, df = 39, ns). This was confirmed when the Stressed and Not-
stressed subgroups were compared in each gender separately. The Stressed male students had
significantly lower NKCA levels compared to the Non-stressed male students (t = 2.2, df = 19, p
= 0.038) [Table 11 and Figure 20]. The same trend was shown in female students, but it did not
achieve statistical significance (t = 1.1, df = 18, ns) [Appendix 6: Table A-13]. With regard to
difference between male and female students, there was no significant difference in the levels of
the NKCA (t = 0.1, df = 39, ns) between male and female students [Appendix 6: Table A-14].

Table 11: Mean (95% C.I.) levels of NKCA (% killing) in the Not-stressed and Stressed male students
NKCA (% killing)
Not-stressed Stressed
Mean level 12.9 4.7
(95% C.I.) (6.6 – 19.2) (1.2 – 8.2)
n 11 10
NK cytotoxic activity (%killing)

20
p = 0.038
Mean (95% C.I.) NKCA in male (%killing)

15

10

Not-stressed Stressed

Figure 20: Mean (95% C.I.) NKCA levels (% killing) in the Not-stressed and Stressed male students

Hence, it was concluded that there was no gender bias with regard to the result from
comparison analysis between the Stressed and Not-stressed subgroups.

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Conclusion:
Stressed students had lower levels of NKCA compared to the not-stressed students.
NKCA levels were not significantly different between male and female students.

Per-NK-cell cytotoxic activity levels


It is known that NK cytotoxic activity (NKCA) is related to the number of NK-cells and
the promiscuity of the NK-cells. This promiscuity is dependent upon factors like:-
1. Relative concentrations of NK-cells, target cells and other isolated peripheral mono-
nuclear cells; and/or
2. Per-cell cytotoxic activity of individual NK-cells.

The first hypothesis was examined using a correlation analysis between NK-cell
percentage and NKCA level, and showed that there was a strong and statistically significant
positive association (r = 0.52, p = 0.001, n = 44). Hence, it was concluded that the number and
concentration of NK-cells were one of the most important factors for the NKCA levels.

Having demonstrated this positive correlation, the second hypothesis that low levels of
per-NK-cell cytotoxic activity associate with high stress perception was examined. This per-
NK-cell cytotoxic activity, which was calculated as a figure of a ratio of the NKCA to NK-cell
percentage as was suggested in the meta-analysis of Segerstrom and Miller [2004], were
compared between the Stressed and Not-stressed students. The results show that the Stressed
students had a trend towards lower levels of per-NK-cell cytotoxic activity (calculated as a ratio
of NKCA to NK-cell percentage) compared to the Non-stressed students (t = 2.0, df = 39, p =
0.056) [Table 12 and Figure 21].

Table 12: Mean (95% C.I.) ratios of NKCA to NK-cell in the Not-stressed and Stressed students
NKCA to NK-cell ratio
Not-stressed Stressed
Mean ratio 1.29 0.64
(95% C.I.) (0.74 – 1.84) (0.25 – 1.02)
n 20 21

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2.5
p = 0.056

Per-NK-cell cytotoxic activity

Mean (95% C.I.) ratios of NKCA to NKC %


2.0

1.5

1.0

0.5

0.0

Not-stressed Stressed

Figure 21: Mean (95% C.I.) per-NK-cell cytotoxic activity (calculated as a ratio: NK cytotoxic activity
(NKCA) / NK-cell percentage (NKC%)) in the Not-stressed and Stressed subgroups

Gender appeared not to influence the results significantly. There was no significant
gender vs. stress-perception interaction (F = 1.4, df = 39, ns). This was confirmed when the
Stressed and Not-stressed subgroups were compared in each gender separately. The Stressed
male students had lower per-NK-cell cytotoxic activity levels compared to the Non-stressed
male students (t = 2.3, df = 19, p = 0.035) [Table 13 and Figure 22]. There was, however, no
statistically significant difference between the Not-stressed and Stressed female students (t = 0.5,
df = 18, ns) [Appendix 6: Table A-15]. With regard to difference between male and female
students, there were no significant differences between male and female students in the ratios of
the NKCA to NK-cell percentage (t = 0.1, df = 40, ns) [Appendix 6: Table A-16].

Table 13: Mean (95% C.I.) ratios of NKCA to NK-cell in the Not-stressed and Stressed male students
NKCA to NK-cell ratio
Not-stressed Stressed
Mean ratio 1.38 0.33
(95% C.I.) (0.53 – 2.24) (0.14 – 0.52)
n 11 10
Mean (95% C.I.) ratios of NKCA to NKC % in male students

2.5
p = 0.035
Per-NK-cell cytotoxic activity

2.0

1.5

1.0

0.5

0.0

Not-stressed Stressed

Figure 22: Mean (95% C.I.) per-NK-cell cytotoxic activity (calculated as a ratio: NKCA / NK-cell
percentage) in the Not-stressed and Stressed male students

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Hence, it was concluded that there was no gender bias with regard to the result from
comparison analysis between the Stressed and Not-stressed subgroups.

Conclusion:
It appears that stressed students may have lower levels of per-NK-cell cytotoxic activity
compared to the not-stressed students, and possibly, because of the small sample size, this
achieved statistical significance only for male students.

3.1.2.2 Differences in the percentages of T-cells between university students with high and low
perceived stress levels
Associations between stress perception and T-cells have been inconsistently reported
[1.2.3.2 and 1.2.4.3]. Hence, this study was performed to determine if there are any associations
between perceived stress level and CD4 T-cell and/or CD8 T-cell percentages in university
students. In addition, the influence of gender upon these parameters was investigated.

There was no significant difference between the Stressed and Non-stressed individuals in
the CD4 or CD8 T-cell levels (CD4 T-cells: t = 1.4, df = 39, ns; CD8 T-cells: t = 0.8, df = 39,
ns) [Appendix 6: Table A-17]. There was no significant gender vs. stress-perception interaction
(CD4 T-cells: F = 0.8, df = 39, ns; and CD8 T-cells: F = 0.5, df = 39, ns). This was confirmed
when the Stressed and Not-stressed subgroups were compared in each gender separately. There
was no significant difference between the Not-stressed and Stressed students (male: CD4 T-
cells: t = 1.1, df = 18, ns; CD8 T-cells: t = 1.1, df = 18, ns [Appendix 6: Table A-18]; and
female: CD4 T-cell: t = 1.7, df = 18, ns; CD8 T-cell: t = 1.1, df = 18, ns [Appendix 6: Table A-
19]). Hence, it was concluded that there was no gender bias with regard to the result from
comparison analysis between the Stressed and Not-stressed subgroups.

Gender appeared to influence the results of CD4 T-cells (F = 6.0, df = 1, p = 0.019), but
not CD8 T-cells (F = 0.03, df = 1, ns). Female students have higher CD4 T-cell percentages than
male students (t = 2.4, df = 38, p = 0.020) [Table 14 and Figure 23]. There was no significant
difference between male and female students in the CD8 T-cell percentages (t = 0.4, df = 38, ns)
[Appendix 6: Table A-20].

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Table 14: Mean (95% C.I.) CD4 T-cell levels (%) of male and female
CD4 T-cell (%)
Male Female
Mean level 43.2 48.7
(95% C.I.) (39.9 – 46.4) (45.5 – 52.0)
n 20 20

50
Mean (95% C.I.) CD4 T-cell (%)

40
CD4 T-cell percentages

30
p = 0.020
20

10

male female

Figure 23: Mean (95% C.I.) CD4 T-cell levels (%) of male and female students

Conclusion:
There was no significant difference in CD4 or CD8 T-cell levels between stressed and
not-stressed students. Female students showed higher CD4 T-cell percentages than male
students in this study.

Summary:
The analyses of NKCA and per-NK-cell cytotoxic activity levels suggested that the
students with high stress perception (the stressed individuals) may have lower levels of NKCA
and per-NK-cell cytotoxic activity compared to the students who expressed a low level of stress
(the not-stressed individuals). There were no significant differences between the stressed and
not-stressed students in NK-cell or CD4 and CD8 T-cell percentages, and these results had no
gender bias; although male students had higher NK-cell and lower CD4 T-cell percentages than
females. Hence, it is suggested that gender may not need to be separately analysed when the
levels of the NKCA or percentages of NK-cells and CD4 and CD8 T-cells are compared in
relationship with the levels of stress perception.

This study has shown that there are associations between one’s perceived stress level and
one’s levels of NKCA; and these findings support previous reports [Introduction: 1.2.4.3] that

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there are interactions among the proposed integrated psycho-neuro-endocrino-immune network,


and that it is warranted to investigate this network further [3.3 and 3.4].

3.1.3 The influence of psychological intervention upon stress-related changes in


university students facing academic examinations

The objective of this series of investigations was to determine if the practice of


psychological coping skills would reduce:
1. Increased levels of perceived stress (PSS) and anxiety (State score of the STAI),
2. Stress-related low NKCA levels, and
3. Stress-related decrease in NK-cell percentages
in university students facing academic examinations. The levels were compared between the
recruitment baseline time point and the Exam time point. Psychological intervention was given
prior to the examinations. In addition, the effects of psychological intervention upon CD4, CD8
T-cell percentages were explored by using the same comparison method.

3.1.3.1 Effect of psychological intervention upon stress perception in university students


anticipating academic examinations
Perceived Stress levels (PSS)
The PSS questionnaire was not ready for use when the students were recruited for the
baseline assessments. However, 32 participants completed the questionnaire at the Exam time
point. ANOVA showed that there was no statistical difference between the three groups (F =0.5,
df = 29, ns), and exploratory post-hoc t-tests showed no significant differences (Relaxation
control vs. Self-hypnosis: t = 0.6, df = 19, ns; Relaxation control vs. Johrei: t = 0.3, df = 18, ns)
[Appendix 6: Table A-21]. Further, there was no significant differences in the PSS levels
between the three groups with regard to;-

Distribution of numbers of the participants in the Not-stressed and Stressed


subgroups (No differences: Pearson's Chi square: Relaxation cntrols vs. Self-
hypnosis = 0.2; Relaxation controls vs. Johrei = 2.6) [Table 15]; and

Gender bias [Appendix 6: Tables A-22 and A-23].

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Table 15: The numbers of participant in the three groups (Self-hypnosis, Johrei and Relaxation control) in
the Not-stressed and Stressed subgroups based on the PSS scores at the Exam time point
Not-stressed Stressed Total
Group Self-hypnosis 5 7 12
Johrei 7 4 11
Controls 3 6 9
Total 15 17 32

Conclusion:
Students practising psychological interventions prior to academic exams did not show any
significant differences in perceived stress levels at the Exam time point when compared with
students in the Relaxation control group.

State anxiety levels


Thirty five participants completed the State score of the STAI questionnaires at baseline
and the Exam time point, allowing for an analysis of anxiety levels in the three groups before
the psychological intervention period and later at the exam stress period.

Anxiety levels increased significantly at the Exams stress time point compared to baseline
in all three groups (F = 11.0, df = 32, p = 0.002). Although repeated measure ANOVA showed
that there was no significant difference in the State anxiety scores in the STAI between the three
groups at baseline and Exam time point, i.e. there was no time x group interaction (F = 0.02, df
= 32, ns), post hoc t-tests showed that the increases were statistically significant for the
Relaxation control (mean change = 6.8, t = 3.3, df = 9, p = 0.027) and Self-hypnosis (mean
change = 4.9, t = 3.3, df = 12, p = 0.006) groups but not for the Johrei groups (mean change =
4.2, t = 1.1, df =11, ns) [Table 16 and Figure 24]. Analyses of the data by gender showed the
same trends [Appendix 6: Tables A-24 and 25].

Table 16: Mean (95% C.I.) State anxiety scores in the Self-hypnosis, Johrei and Relaxation control
groups at baseline and the Exam time point
State score in the STAI
Baseline Exams
Self-hypnosis Johrei Controls Self-hypnosis Johrei Controls
Mean score 37.3 34.8 31.5 42.2 39.0 38.3
(95% C.I.) (34.0 - 40.6) (29.7 – 40.0) (28.5 - 34.5) (39.1 - 45.4) (33.9 – 44.1) (31.9 - 44.7)
N 13 12 10 13 12 10

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Not significant
Baseline
Exams

Mean (95% C.I.) State scores in the STAI 40

30

p = 0.006
20
Not significant p = 0.027

10

Self-hypnosis Johrei Controls

Figure 24: Mean (95% C.I.) State anxiety scores in the STAI of the three groups at baseline and the Exam
time point

Conclusion:
Anxiety levels increased in university students facing academic examination, and the
training and practice of Johrei appeared to help students to control these provoked levels of
anxiety although this anti-anxiety effect did not achieve a statistical significance.

3.1.3.2 Effects of psychological intervention upon stress-related lymphocyte sub-populations in


university students anticipating academic examinations
NK cytotoxic activity (NKCA) levels
NKCA levels were determined in peripheral blood from 31 participants at the Exam time
points. Due to technical difficulties, the NKCA levels could not be collected at the baseline. The
mean level of the NKCA in the Relaxation control group were not significantly different from
those levels in the Self-hypnosis and the Johrei groups (Relaxation control vs. Self-hypnosis: t =
1.3, df = 18, ns; Relaxation control vs. Johrei: t = 0.2, df = 18, ns) [Appendix 6: Table A-26].
Gender did not appear to influence [Appendix 6: Tables A-27 and A-28]. It was confirmed that
there was no gender bias.

Conclusion:
Psychological intervention did not appear to influence NKCA levels of university students
facing academic examinations.

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Peripheral NK-cell and CD4 and CD8 T-cell percentages


Peripheral NK-cell and CD4 and CD8 T-cell percentages were determined for 34
participants (19 male and 15 female) [Table 17] at baseline and the Exam time points allowing
analysis of the influence of psychological intervention upon exam-stress associated changes.

Table 17: Numbers of participants in the three groups at the Exam time point

Group
Total
Relaxation
Self-hypnosis Johrei
controls
male 7 7 5 19
female 5 4 6 15
Total 12 11 11 34

NK-cell percentages
There was no difference between the three groups in the levels of NK-cell percentages at
baseline (Self-hypnosis and Relaxation control: t = 0.5, df = 21, ns; Johrei and Relaxation
control: t = 0.7, df = 20, ns).

Repeated measures ANOVA showed that there is a significant difference in the changes
from baseline to the Exams assessment point among the three groups (F = 5.8, df = 31, p =
0.007). At the Exams period, there was a decrease in NK-cell percentage in the Relaxation
control group (mean change = 2.1, SD = 4.7, t = 1.5, df = 10, ns), but this did not achieve
statistical significance. However, the change in the NK-cell percentages in the psychological
intervention groups were significantly different, i.e. there was a significant time x group
interaction (F = 5.8, df = 31, p = 0.007). This was confirmed by following post-hoc t-tests.
There was no significant change in the mean NK-cell percentage in the Self-hypnosis group
(mean change = 1.0, t = 0.7, SD = 4.5, df =11, ns), but there was a significant increase in the
Johrei group (mean change = 3.2, t = 6.9, SD = 1.7, df =10, p < 0.001) at the Exam time point
[Table 18 and Figure 25]. Analyses of the data by gender showed the same trends [Appendix 6:
Tables A-29 and 30].

Table 18: Mean (95% C.I.) levels of NK-cell (%) in the Self-hypnosis, Johrei and Relaxation control
(Controls) groups at baseline and the Exam time point
NK-cells (%)
Baseline Exams
Self-hypnosis Johrei Controls Self-hypnosis Johrei Controls
Mean level 9.6 7.6 8.8 8.6 10.8 6.7
(95% C.I.) (7.7 - 11.5) (4.9 – 10.3) (6.3 - 11.3) (5.7 - 11.5) (8.1 – 13.6) (4.7 - 8.7)
n 12 11 11 12 11 11

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20
p = 0.025
Not significant

NK cell percentages (95% C.I.)


15 Not significant Not significant

10

5
Baseline

p < 0.001
0 Exams
N= 12 12 11 11 11 11
Hypnosis Johrei Control (relaxation)

Figure 25: Mean (95% C.I.) levels of NK-cells of the three groups at baseline and the Exam time point

Figure 26 shows the changes from baseline NK-cell percentages to that at the Exam time
point for individual students in each group. In the Johrei group, there was a clear increasing
trend with eight individuals increased while three stayed the same (less than 2.6% changes: 99%
Confidence Interval of the measure in NK-cell %). There was no clear trend in the Relaxation
control group with two individuals increased, four stayed the same and five decreased. In the
Self-hypnosis, three increased, five stayed the same and three decreased.
20 20 20

15 15 15
NK-cell percentage

10 10 10

5 5 5

0 0 0
Baseline Exam Baseline Exam Baseline Exam

Self-hypnosis Johrei Relaxation control


Figure 26: The individual levels of NK-cell percentages in the three groups at baseline and the Exam
time point

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This finding was investigated by the Intention-to-treat analysis comparing percentages of


individuals between the two groups (Maintaind (increase or stay the same) NK-cell levels and
Decreased NK-cell levels). All the data from the subjects who withdrew were added into the
number in the group in which the subjects decreased their NK-cells [Table 19].

Table 19: Percentages (numbers) of subjects whose NK-cell counts maintained or increased (Maintained)
and decreased (Decreased) in the Self-hypnosis and Johrei and Relaxation control groups for the
Intention-to-treat analysis (missing data were added into the number in the Decreased group)
Group Maintained NK-cells Decreased NK-cells Total
Self-hypnosis 44% ( 7/16) 56% ( 9/16) 16
Johrei 75% (12 /16) 25% ( 4 /16) 16
Controls 38% (6 /16) 62% (10/16) 16
Total 25 23 48

The percentage of the students who maintained the NK-cell counts at the Exam time point
in the Johrei group (75%) was higher than that of Relaxation control group (38%) (Pearson’s
chi-square = 4.6, p = 0.033).

Conclusion:
As has been shown in the literature [see also Introduction: 1.2.4.3] but not in this study
[3.1.1.4], exam stress appeared to result in a decrease in peripheral blood NK-cell percentage in
university students in the Relaxation control group although this decrease did not achieve
statistical significance. However, training in and practise of Johrei reversed this effect and led to
an overall increase in NK-cell percentages. In contrast, training and practice of Self-hypnosis
did not significantly change the levels of NK-cells.

CD4 T-cell percentages


The results show that there was no difference between the three groups in the levels of
CD4 T-cell percentages at baseline (Self-hypnosis and Relaxation control: t = 0.2, df = 21, ns;
Johrei and Relaxation control: t = 0.5, df = 20, ns).

Repeated measures ANOVA showed that there is a trend toward difference in the changes
from baseline to the Exams assessment point between the three groups (F = 3.2, df = 31, p =
0.054). At the Exams period, there was an increase in CD4 T-cell percentage in the Relaxation
control group (mean change = 1.6, t = 1.2, SD = 4.5, df = 10, ns), but this did not achieve
statistical significance. The change in the CD4 T-cell percentages in the psychological
intervention groups had trends towards different changes from that of the Relaxation control
group (F = 3.2, df = 31, p = 0.054). There was no change in the mean CD4 T-cell percentage in
the Self-hypnosis group (mean change = 0.1, t = 0.1, SD = 3.5, df =11, ns), and there was a

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trend toward decrease in the Johrei group (mean change = 3.4, t = 1.8, SD = 6.2, df =10, p =
0.094) [Table 20 and Figure 27]. Analyses of the data by gender showed the same trends
[Appendix 6: Tables A-31 and A-32].

Table 20: Mean (95% C.I.) levels of CD4 T-cell (%) in the Self-hypnosis, Johrei and Relaxation control
(Controls) groups at baseline and the Exam time point
CD4 T-cells (%)
Baseline Exams
Self-hypnosis Johrei Controls Self-hypnosis Johrei Controls
Mean level 45.6 46.5 45.3 45.8 43.1 46.9
(95% C.I.) (42.4 - 48.9) (42.8 – 50.3) (41.8 - 48.7) (41.1 - 50.4) (37.7 – 48.6) (38.5 - 51.1)
n 12 11 11 12 11 11
50 Baseline
Exams
Mean (95% C.I.) levels of CD4 T-cells (%)

40

Not significant Not significant


30 p = 0.094

20
Not significant

10
Not significant
0

Self-hypnosis Johrei Controls

Figure 27: Mean (95% C.I.) levels of CD4 T-cells of the three groups at baseline and the Exam time point

Figure 28 shows the changes from baseline CD4 T-cell percentages to that at the Exam
time point for individuals in each group. There appeared to have no clear trend.

In the Johrei group, one individual increased, five stayed the same (less than 2.4%
changes: 99% confident interval of the measure in CD4 T-cell %) and five decreased, while in
the Relaxation control group, five individuals increased, four stayed the same and two decreased.
In the Self-hypnosis group, three increased, seven stayed the same and two decreased.

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64 64 64

58 58 58

52 52 52
CD4 T cell percentage

46 46 46

40 40 40

34 34 34

28 28 28
Baseline Exam Baseline Exam Baseline Exam

Self-hypnosis Johrei Relaxation control


Figure 28: The individual levels of CD4 T-cell percentages in the three groups at baseline and the Exam
time point

Conclusion:
Exam stress did not affect peripheral blood CD4 T-cell percentages. In addition, neither of
the psychological interventions affected CD4 T-cell percentages significantly.

CD8 T-cell percentages


The results show that there was no significant difference between the three groups in the
levels of CD8 T-cell percentages at baseline (Self-hypnosis and Relaxation control: t = 0.8, df =
21, ns; Johrei and Relaxation control: t = 0.2, df = 20, ns).

There was no significant difference statistically in the changes between the three groups
(F = 2.3, df = 31, ns). At the Exam time points, there was no significant change in CD8 T-cell
percentage in the Relaxation control and Johrei groups. However, an exploratory analysis with
paired t-tests showed that the CD8 T-cell percentages increased in the Self-hypnosis group
(mean change = 2.5, t = 3.1, SD = 2.8, df = 11, p = 0.010) [Table 21 and Figure 29]. Analyses of
the data by gender showed the same trends [Appendix 6: Tables A-33 and A-34].

Table 21: Mean (95% C.I.) levels of CD8 T-cell (%) in the Self-hypnosis, Johrei and Relaxation control
(Controls) groups at baseline and the Exam time point
CD8 T-cells (%)
Baseline Exams
Self-hypnosis Johrei Controls Self-hypnosis Johrei Controls
Mean level 25.1 26.8 27.2 27.6 27.6 26.8
(95% C.I.) (20.9 - 29.4) (24.0 – 29.6) (24.4 - 30.1) (24.0 - 31.2) (24.3 – 30.9) (23.9 - 29.8)
N 12 11 11 12 11 11

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Baseline
Exams
30

Mean (95% C.I.) levels of CD8 T-cells (%)


20

Not significant
p = 0.010
Not significant
10

Not-significant

0 Not-significant
Self-hypnosis Johrei Controls

Figure 29: Mean (95% C.I.) levels of CD8 T-cells of the three groups at baseline and the Exam time point

Figure 30 shows the changes from baseline CD8 T-cell percentages to that at the Exam
time point for individual students in each group. In the Self-hypnosis group, there was an
increasing trend with seven individuals increased, four stayed the same (less than 1.7% changes:
99% confident interval of the measure in CD8 T-cell %) and one decreased. There was no clear
trend in the Johrei with four increased, five stayed the same and two decreased; and in the
Relaxation control groups, two individuals increased, six stayed the same and three decreased.
40 40 40

34 34 34
CD8 T cell percentage

28 28 28

22 22 22

16 16 16
Baseline Exam Baseline Exam Baseline Exam

Self-hypnosis Johrei Relaxation control


Figure 30: The individual levels of CD8 T-cell percentages in the three groups at baseline and the Exam
time point

Conclusion:
Exam stress did not appear to affect peripheral blood CD8 T-cell percentages. However,
the Self-hypnosis appeared to increase CD8 T-cell percentages.

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Summary:
Practising Self-hypnosis or Johrei prior to academic exams, when compared with the
passive relaxation experience, did not appear to affect perceived stress levels in university
students facing academic exams. Johrei, however, appeared to decrease the increased anxiety
levels in university students facing at academic exams.

NKCA levels in the university students practising either Self-hypnosis or Johrei at


academic exams were similar to the levels in the students following the control relaxation
experiences. On the other hand, academic exam stress appeared to be associated with a decrease
in NK-cell levels in university student with relaxation experience although this decrease did not
achieve statistical significance, and interestingly this decrease was reversed in the students
trained in and practising Johrei. Self-hypnosis did not appear to differ significantly from
Relaxation control in the effect on NK-cell levels. On this occasion, exam stress was not found
to affect the levels of CD4 and CD8 T-cells, and the levels of these cells were not affected by
Johrei intervention while Self-hypnosis increased CD8 T-cell percentages when students faced
academic exams.

These findings partially supported the hypothesis that psychological intervention may
counteract the effect of stress upon the proposed integrated psycho-neuro-endocrino-immune
network; and warrant the need to investigate this hypothesis further, in a patient population.
Hence, the next study employs HIV-infected individuals [3.2].

3.2 The influence of psychological intervention upon perceived stress and


quality-of-life and various immunological disease-associated parameters in
HIV-infected individuals

The objective of this study was to determine if the training and practice of psychological
stress coping skills would influence stress perception as well as immunological parameters
particularly the disease progression marker, CD4 T-cell counts, in HIV-infected individuals.

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3.2.1 Disease-associated and stress-related changes in HIV-infected individuals

This part investigates the proposed integrated psycho-neuro-endocrino-immune network


by examining psychological and immune (disease) parameters and associations between the
parameters, i.e. between the levels of stress perception and perceived quality-of-life and CD4 T-
cell counts as primary outcome measures.

3.2.1.1 Psychological profiles of the HIV-infected individuals recruited into the study
The HIV-infected individuals recruited into the study were asked at recruitment and four
months later to complete the psychological questionnaires listed below in order to assess their
levels of stress perception and perceived quality-of-life:
General and HIV-infection related stress perception
Perceived Stress Score (PSS): secondary appraisal scale
State anxiety in the State Trait Anxiety Inventory (STAI): anxiety scale
Impact Event Scale (IES): primary appraisal against a stressor scale
Quality-of-life (QoL) perception
Locus of Control (LoC): sense of taking control of one’s own life scale (recall:
inner or outer locus of control = low or high score)
Mental Component Summary in the SF-36 (MCS): mental quality-of-life scale
Pittsburgh Sleep Quality Inventory (PSQI): sleep quality scale

With all of these questionnaires, except the MCS, lower scores mean better functioning.
Analyses of these data involved, first, a comparison of the PSS and the State anxiety scores of
the participants in this study with those of the university students in the previous study. Second,
the other psychological profiles collected specifically to this study were analysed by comparing
the two stress-related subgroups (labelled as Not-stressed and Stressed subgroups) defined by
the midline split of the PSS at recruitment, i.e. individuals scoring more than 31 in the PSS were
placed in the Stressed subgroup and individuals scoring equal to or below 31 were placed into
the Not-stressed subgroup.

Perceived stress levels (PSS)


The HIV-infected individuals had higher PSS scores (mean difference = 7.2, t = 4.5, df =
81, p < 0.001) than the university students facing examinations, and the PSS scores did not
change significantly (F = 0.02, df = 46, ns) from recruitment to the four months later time point
in HIV-infected individuals [Table 21].

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Table 22: Medians, means and standard deviations of the PSS of the university students at non-exam and
exams and of the HIV-infected individuals at recruitment and four months later
University students HIV-infected individuals
Non-exam Exams Recruitment After 4 months
Median 22 25 32 31
Mean 23.3 24.4 31.6 31.7
SD 8.8 7.5 7.0 5.6
n 34 32 51 51

Conclusion:
Patients with HIV have higher stress perception than university students facing exams.

The subgroups of Not-stressed and Stressed were defined by the judgement of a midline
split of the PSS score at recruitment. The Stressed subgroup had 26 subjects (including two
females) and the Not-stressed subgroup had 25 subjects (one female). The results from 51
subjects (three females and 48 males) and the results from 48 male subjects only are similar and
they have the same trend, so only the results from 48 male subjects are presented.

The result from 48 HIV-infected male individuals shows that there were differences
between the Stressed and Non-stressed subgroups in the levels of PSS at recruitment and four
months later [Table 23].

Table 23: Mean (95% C.I.) levels of the PSS in the Not-stressed and Stressed subgroups at the
recruitment and four months later
PSS levels
Recruitment After 4 months
Not-stressed Stressed Not-stressed Stressed
Mean score 26.0 36.8 27.4 35.5
(95% C.I.) (24.6 – 29.5) (34.9 – 38.6) (25.8 – 28.9) (33.2 – 35.5)
n 24 24 24 24

There were significant differences between the two subgroups both at recruitment (t = 9.2,
df = 46, p < 0.001) and four months later (t = 5.9, df = 46, p < 0.001). There was a significant
time x subgroup interaction (F = 5.8, df = 49, p = 0.020), but the PSS scores did not change
significantly over the four months study period in the Not-stressed (t = 1.2, df = 23, ns) or in the
Stressed subgroup (t = 1.6, df = 23, ns) [Figure 31].

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40 Recruitment
After 4
months
p < 0.001
30

Mean (95% C.I.) PSS levels Not-significant


20

Not-significant
10

p < 0.001
0

Not-stressed Stressed

Figure 31: Mean (95% C.I.) PSS levels of the Not-stressed and Stressed subgroups at recruitment and
four months later

Conclusion:
PSS levels in HIV-infected individuals did not change significantly over the four months
of the study period, and this consistency in the levels of PSS scores was also shown when the
Stressed or Not-stressed subgroups were separately compared.

State anxiety levels


In contrast to the PSS, anxiety levels in the HIV-infected individuals were similar to the
levels of the university students (mean difference = 1.1, t = 0.4, df = 85, ns), and state anxiety
level in the HIV-infected individuals did not change significantly (F = 0.7, df = 46, ns) from
recruitment to four months later [Table 24].

Table 24: Median, mean and standard deviation of the State anxiety score of the university students at
non-exam and exams and of the HIV-infected individuals at recruitment and four months later
University students HIV-infected individuals
Non-exam Exams Recruitment After 4 months
Median 37 40 36 38
Mean 37.9 39.7 38.9 40.8
SD 9.4 8.3 12.5 14.4
n 37 36 51 51

Conclusion:
This result suggested that there was little difference in the State anxiety scores in HIV-
infected individuals compared with the university students facing academic exams.

When comparisons were made between the 48 HIV-infected male individuals in the Not-
stressed and Stressed groups based on a median split of the PSS score, the individuals in the

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Stressed subgroup had significantly higher anxiety levels than the individuals in the Not-
stressed subgroup both at recruitment (t = 7.0, df = 49, p < 0.001) and four months later (t = 6.1,
df = 49, p < 0.001). There was no significant time x subgroup interaction (F = 1.7, df = 49, ns),
and the State anxiety scores did not change significantly over the study period in the Not-
stressed subgroup (mean change = 2.2, t = 1.1, df = 23, ns) or in the Stressed subgroup (mean
change = - 0.3, t = 0.3, df = 23, ns) [Table 25 and Figure 32].

Table 25: Mean (95% C.I.) levels of the State anxiety score of the STAI in the Not-stressed and Stressed
subgroups at the recruitment and four months later
State anxiety score of the STAI
Recruitment After 4 months
Not-stressed Stressed Not-stressed Stressed
Mean level 30.2 49.6 30.5 47.4
(95% C.I.) (26.9 – 33.4) (45.1 – 54.1) (27.6 – 33.3) (42.6 – 52.2)
n 24 24 24 24

60 p < 0.001 Recruitment


after 4
months
50
Mean (95% C.I.) State anxiety scores

40

Not-significant
30

20
Not-significant

10
p < 0.001
0

Not-stressed Stressed

Figure 32: Mean (95% C.I.) levels of the State anxiety scores in the STAI of the Not-stressed and Stress
subgroups at recruitment and four months later

Conclusion:
HIV-infected individuals reporting low levels of stress also report lower scores of the
State anxiety than individuals reporting high levels of stress.

Primary appraisal against a specific life event levels (IES)


The primary appraisal of the awareness of carrying HIV-infection was measured by the
IES. The mean IES scores of the Stressed individuals were significantly higher than the levels
for the Not-stressed individuals at recruitment (t = 3.2, df = 23, p = 0.003) and after four months
(t = 3.2, df = 23, p = 0.003). The IES scores decreased significantly over the study period (F =

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18.8, df = 46, p < 0.001), and this was observed in both the Stressed subgroup (mean change =
7.3, t = 3.5, df = 23, p = 0.002) and the Not-stressed subgroup (mean change =6.2, t = 2.7, df =
23, p = 0.013), but there was no significant time x subgroup interaction (F = 0.5, df = 49, ns)
[Table 26 and Figure 33].

Table 26: Mean (95% C.I.) levels of the IES in the subgroups of the Not-stressed and Stressed at the
recruitment and four months later
IES
Recruitment After 4 months
Not-stressed Stressed Not-stressed Stressed
Mean 16.5 31.4 10.4 24.2
(95% C.I.) (10.0 – 23.1) (24.7 – 38.1) (5.9 – 14.8) (16.6 – 31.7)
n 24 24 24 24

40 Recruitment
After 4
p = 0.003 months

p < 0.001
30
Mean (95% C.I.) IES levels

20

10 p = 0.002

0 p = 0.003
Not-stressed Stressed

Figure 33: Mean (95% C.I.) levels of the IES in the Not-stressed and Stress subgroups at recruitment and
four months later

Conclusion:
The impact in the primary appraisal from diagnosed as carrying HIV-infection decreased
over the study period. Although the decreased levels of impact in the HIV-infected individuals
were not different between the stressed and not-stressed subgroups, individuals with a high
perceived stress level consistently showed higher levels of impact than the individuals in the
not-stressed subgroup both at recruitment and four months later.

Locus of control (LoC: Sense of control of one’s own life levels)


The sense of taking control of one’s own life was measured by using the LoC. The LoC
scores were significantly higher in the Stressed individuals than the Not-stressed individuals at

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recruitment (t = 2.8, df = 23, p = 0.007) and four months later (t = 1.0, df = 23, p = 0.002), but
there was no significant time x subgroup interaction (F = 0.1, df = 49, ns), and the scores did not
change significantly over the study period (F = 0.4, df = 46, ns) [Table 27 and Figure 34].

Table 27: Mean (95% C.I.) levels of the LoC in the Not-stressed and Stressed subgroups at the
recruitment and four months later
LoC score
Recruitment After 4 months
Not-stressed Stressed Not-stressed Stressed
Mean level 9.3 12.6 8.7 12.6
(95% C.I.) (5.9 – 14.8) (11.0 – 14.2) (7.5 – 11.0) (11.0 – 14.2)
n 24 24 24 24

15 Recruitment
Not-significant After 4
months
Mean (95% C.I.) LoC levels

10

Not-significant
5
p = 0.002

p = 0.007
0

Not-stressed Stressed

Figure 34: Mean (95% C.I.) levels of the LoC in the Not-stressed and Stressed subgroups at recruitment
and four months later

Conclusion:
The locus of control did not change significantly over the study period, and less stressed
HIV-infected individuals reported the lower scores (a higher sense of taking control of one’s
own life) than did higher stressed individuals.

Mental aspect of quality of life levels (MCS: psychological functioning)


The psychological functioning in the HIV-infected individuals not receiving anti-
retroviral medication and not experiencing disease-related symptoms in this study was
examined by measuring mental aspect of quality of life, i.e. the MCS scores. The MCS scores
were significantly higher in the Not-stressed individuals than in the Stressed individuals at
recruitment (t = - 5.8, df = 23, p < 0.001) and four months later (t = - 4.6, df = 23, p < 0.001),

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but there was no significant time x subgroup interaction (F = 1.2, df = 49, ns), and the scores did
not change significantly over the study period (F = 0.8, df = 46, ns) [Table 28 and Figure 35].

Table 28: Mean (95% C.I.) levels of the MCS in the Not-stressed and Stressed subgroups at recruitment
and four months later
MCS score
Recruitment After 4 months
Not-stressed Stressed Not-stressed Stressed
Mean 51.8 35.3 52.1 37.6
(95% C.I.) (48.7 – 54.8) (30.5 – 40.1) (48.8 – 55.4) (32.2 – 42.9)
n 24 24 24 24

60 p < 0.001 Recruitment


After 4
months
50
Mean (95% C.I.) MCS levels

40

30
Not-significant
20

Not-significant
10

p < 0.001
0

Not-stressed Stressed

Figure 35: Mean (95% C.I.) levels of the MSC in the Not-stressed and Stressed subgroups at recruitment
and four months later

Conclusion:
Psychological functioning, as measured by the mental health component of the SF-36
(MCS), did not change significantly over the study period, and HIV-infected individuals in the
stressed subgroup reported lower levels of psychological functioning than individuals in the
non-stressed subgroup both at recruitment and four months later.

Sleep quality levels (PSQI)


The PSQI scores of the Stressed individuals were significantly higher than that of the Not-
stressed at recruitment (t = 2.1, df = 46, p = 0.039) and after four months (t = 2.9, df = 46, p =
0.005), but there was no significant time x subgroup interaction (F = 0.6, df = 49, ns), and the
scores did not change significantly over the study period (F = 0.6, df = 46, ns) [Table 29 and
Figure 36].

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Table 29: Mean (95% C.I.) levels of the PSQI in the Not-stressed and Stressed subgroups at recruitment
and four months later
PSQI
Recruitment After 4 months
Not-stressed Stressed Not-stressed Stressed
Mean 8.1 10.3 7.5 10.2
(95% C.I.) (6.9 – 9.4) (8.7 – 11.8) (6.6 – 8.4) (8.6 – 11.8)
n 24 24 24 24
Recruitment
12 p = 0.039 After 4
months
10
Mean (95% C.I.) PSQI scores

Not-significant
6

4
p = 0.005

2
Not-significant

Not-stressed Stressed

Figure 36: Mean (95% C.I.) levels of the PSQI in the Stress and Not-stressed subgroups at recruitment
and four months later

Conclusion:
The sleep quality scores did not change significantly over the study period, and HIV-
infected individuals in the stressed subgroup reported worse sleep quality than HIV-infected
individuals in the non-stressed subgroup at recruitment and four months later.

3.2.1.2 Stress-related perception and disease progression markers of HIV infection


CD4 T-cell counts and HIV viral load levels are routinely measured for the purpose of
clinically monitoring disease progression. Therefore, relationships between the CD4 T-cell
counts and HIV viral load levels and levels of stress perception were examined.

Rate of decline in CD4 T-cell count (CD4 gradient)


CD4 T-cell counts form a major part of the routine monitoring of HIV-infected patients.
As described in the methods section [2.1.2], of the 63 participants recruited, only 38 patients
(60.3%) had sufficient data from which to calculate the CD4 gradients during the (four month
plus one month, five months altogether, i.e. Baseline to Term four) study period. Within the 38
patients, 32 participants (30 males and two females) completed their questionnaires. The results
obtained from 32 subjects and the results from 30 male subjects only are similar and they have

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the same trend, so only the results from 30 male subjects were presented. Mean CD4 gradient of
30 male subjects was a decline of 3 cells per l per month.

The CD4 gradient over the five months was investigated by correlation analyses for
associations with the levels of the stress-related perceptions (State anxiety score of the STAI,
the Perceived Stress Score: PSS and Impact Event Scale: IES) and the perceived quality-of-life
(Locus of Control: LoC, Mental Component Summary in the SF-36: MCS and Pittsburgh Sleep
Quality Index: PSQI). The CD4 gradients over the five months was not significantly associated
with the scores of State anxiety, IES or PSS at either recruitment or after the four months time
points, and that there was no significant associations with scores of the MCS or PSQI at either
recruitment or after four months [Appendix 6: Table A-35]. There was a trend towards a
positive correlation (r = 0.32, n = 30, p = 0.085) between the CD4 gradients and LoC scores at
recruitment, but this relationship was not statistically significant at four months after the
recruitment [Appendix 6: Table A-35].

Conclusion:
The scores of stress perception, quality of life and sleep quality either at recruitment or at
four months had no significant association with the CD4 gradients over the five months period,
i.e. none of the scores at a one assessment point in this study could be used to predict or to
indicate the rate of change in the CD4 counts in HIV-infected patients not receiving anti-
retroviral treatment.

The CD4 gradient represents the rate of change in CD4 counts over a five month period,
therefore change scores of the various perceived stress scales (State anxiety scores, PSS and
IES) over the same period were analysed with the CD4 gradient. The CD4 gradients had no
significant association with the State anxiety, IES or PSS [Appendix 6: Table A-36].

Further, change scores of the various perceived quality-of-life were analysed, and it was
found that the CD4 gradients had a significant negative correlation with the change scores of the
LoC; and a significant positive correlation with the change scores of the MCS, while there was a
trend towards a negative correlation with the change scores of the PSQI [Table 30].

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Table 30: Correlations between the CD4 gradient (cells per l per month) and the change scores of
perceived quality-of-life scales in:
LoC* MCS* PSQI
R - .40 .44 - .32
2-tailed p 0.029 0.015 0.086
N 30 30 30
*: p < 0.05

The correlations between CD4 gradient and the change scores in the (1) LoC, (2) MCS
and (3) PSQI can imply that:
1. Individuals with an increased sense of taking control of one’s own life (decreased
their LoC scores) showed an increase in their CD4 T-cell counts (recall that low LoC
= inner locus of control), and individuals who decreased the sense of control
(increased their LoC scores) showed a decrease in their CD4 T-cell counts;
2. Individuals who improved their psychological functioning (increased MCS levels)
showed an increase in their CD4 T-cell counts, and individuals who reported a
decrease in psychological functioning (decreased MCS scores) showed a decrease in
their CD4 T-cell counts; and
3. Individuals with improvement in their sleep quality (decreased PSQI scores) showed
an increase in their CD4 T-cell counts (recall that low PSQI = better sleep quality),
and individuals who reported a decrease in sleep quality (increased PSQI scores)
showed a decrease in their CD4 T-cell counts.

Therefore, further analyses were performed after grouping the patients according to
change scores of these perceived quality-of-life scales (increased or decreased), and similar
trends of associations between changes in these scales and the CD4 gradients were
demonstrated. There were significant differences in the CD4 gradient when CD4 gradients were
compared between the two subgroups according to change scores of:
1. LoC (t = 2.1, df = 30, p = 0.049): Increased sense of control of one’s own life
(Increased control: 16 individuals with a decreased LoC score) and Decreased sense
of control (Decreased control: 14 individuals with an increased LoC score) subgroups
[Table 31 and Figure 37];
2. MCS (t = 2.4, df = 30, p = 0.027): Improved psychological functioning (Improved
QoL: 16 individuals with an increased MCS score) and Decreased psychological
functioning (Decreased QoL: 14 individuals with a decreased score) subgroups [Table
32 and Figure 38]; and

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3. PSQI (t = 2.1, df = 30, p = 0.047): Improved sleep quality (23 individuals had a
decreased PSQI score) and Decreased sleep quality (7 individuals had an increased
score) subgroups [Table 33 and Figure 39].

Table 31: Mean (95% C.I.) CD4 T-cell change over the five month (cells per l per month) in HIV-
infected individuals of the Increased control and Decreased control subgroups
CD4 T-cell change (cells per l per month)
Increased control Decreased control
Mean CD4 gradient +9 - 15
(95% C.I.) (- 2.8 to + 21.7) (- 34.7 to - 5.2)
n 16 14
40
p = 0.049
Mean (95% C.I.) CD4 gradient (cells/ul/month)

20

-20

-40

Increased control Decreased control

Figure 37: Mean (95% C.I.) CD4 gradients (cells per l per month) in the Increased control (decreased
scores of the LoC) and Decreased control (increased scores of the LoC) subgroups

Table 32: Mean (95% C.I.) CD4 T-cell change over the five month (cells per l per month) in HIV-
infected individuals of the Improved QoL and Decreased QoL subgroups
CD4 T-cell change (cells per l per month)
Improved QoL Decreased QoL
Mean CD4 gradient + 11 - 19
(95% C.I.) (- 1.4 to 21.4) (- 40.5 to + 2.5)
n 16 14

40 p = 0.027
Mean (95% C.I.) CD4 gradient (cells/ul/month)

20

-20

-40

Improved QoL Decreased QoL

Figure 38: Mean (95% C.I.) CD4 gradients (cells per l per month) in the Improved mental psychological
functioning (Improved QoL: increased scores of the MCS) and Decreased psychological functioning
(Decreased QoL: decreased scores of the MCS) subgroups

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Table 33: Mean (95% C.I.) CD4 T-cell change over the five month (cells per l per month) in HIV-
infected individuals of the Improved sleep quality and Decreased sleep quality subgroups
CD4 T-cell change (cells per l per month)
Improved sleep quality Decreased sleep quality
Mean CD4 gradient +4 - 26
(95% C.I.) (- 8.4 to + 18.2) (- 48.9 to - 1.9)
n 23 7
60
Mean (95% C.I.) CD4 gradient (cells/ul/month)

40 p = 0.047

20

-20

-40

-60

Improved sleep quality Decreased sleep quality

Figure 39: Mean (95% C.I.) CD4 gradients (cells per l per month) in the Improved sleep quality
(decreased scores of the PSQI) and Decreased sleep quality (increased scores of the PSQI) subgroups

Conclusion:
These findings suggest that the rate of decline in the CD4 T-cell count of HIV-infected
individuals is associated with change scores in the perceived control, psychological functioning
and sleep quality scales rather than actual scores on one occasion, i.e. those who increased CD4
T-cell counts over the five month period improved in the scores of LoC, MCS and PSQI.

Rate of change in HIV viral load (HIV viral load gradient)


Viral load levels are another major part of the routine monitoring of the HIV-infected
patients, particularly with regard to drug treatment. Although none of the HIV-infected
individuals in this study had taken any anti-retroviral treatment, the rate of change in viral load
levels (log-transformed figures) was calculated between the recruitment time point and four
months later. Similar to the results of the CD4 T-cell blood collection, not all of the HIV-
individuals recruited had viral load levels measured during the study period [Appendix 6: Table
A-37]. Of the 63 participants recruited, only 38 patients (60.3%) had sufficient data from which
to calculate the viral load level (log-transformed) gradients during the study period. Within the
above 38 patients, 32 participants (29 males and three females) completed their questionnaires.
The results obtained from 32 subjects and the results from 29 male subjects are similar and they
have the same trend, so only the results from 29 male subjects are presented.

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This viral load level gradient over the five months was investigated by correlation
analyses for association with the scores of the State anxiety score, PSS, IES, LoC, MCS and
PSQI. There was no significant correlation at either recruitment or after four months time points
[Appendix 6: Table A-38]. Similar to the analyses of the CD4 gradient, change scores of the
stress perceptions (State anxiety scores, PSS and IES) and perceived quality-of-life scales (LoC,
MCS and PSQI) over the same period were correlated with the viral load level gradient.
However, the viral load level gradients were not significantly associated with the change scores
of any self-report variable [Appendix 6: Table A-39].

Conclusion:
Viral load level was not significantly associated with any of the perceived stress or
quality-of-life measures used in this study.

Rate of change in NK-cell count (NK gradient)


Because NK-cell levels were shown to be associated with the stress perception in the
university students [3.1], for exploratory purposes, the NK gradient was also calculated from the
same blood sample data as the CD4 T-cell gradients. One sample datum was missing for NK-
cell calculation. Hence 37 subjects had sufficient data from which to calculate the NK gradients
during the study period. Within the above 37 patients, 31 participants (29 males and two
females) completed their questionnaires. The results obtained from 31 subjects and the results
from 29 male subjects are similar and they have the same trend, so only the results from 29 male
subjects were presented.

The NK gradient over the five months and the scores of the psychological measures were
further investigated by correlation analyses for associations with the levels of the stress-related
perceptions (State anxiety score, PSS and IES) and the perceived quality-of-life and sleep (LoC,
MCS and PSQI). Results show that the NK gradients over five months were not significantly
associated with the scores of State anxiety, IES, PSS LoC, MCS or PSQI at either recruitment or
after four months, except with the PSQI at recruitment [Appendix 6: Table A-40]. There was a
significant correlation (r = 0.44, p = 0.017) between the NK gradient and the PSQI score at
recruitment but this correlation was not replicated at the four month assessment point [Appendix
6: Table A-40]. Similar to the analyses of the CD4 gradient, change scores of the stress
perceptions (State anxiety scores, PSS and IES) and quality-of-life scales (LoC, MCS and
PSQI) over the same period were correlated with NK gradients. However, none of these
correlations were statistically significant [Appendix 6: Table A-41].

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Conclusion:
The levels of or changed levels of NK-cell count were not significantly associated with
any of the stress perception or quality of life measures used in this study.

Summary:
Patients with HIV diagnosis have higher stress perception than university students facing
exams, and the higher levels did not change significantly over the four months of the study
period. On the other hand, there was little difference in the State anxiety levels between the
HIV-infected individuals and the university students facing academic exams, and HIV-infected
individuals who reported low levels of stress also reported lower State anxiety scores than
individuals who reported high levels of stress. The impact of being diagnosed as carrying HIV-
infection decreased over the study period. Although the impact levels decreased, individuals
with high perceived stress consistently showed higher scores of the impact than the individuals
who reported low stress.

The perceived quality-of-life (locus of control, psychological functioning and sleep


quality) scales did not change significantly over the study period, and lower stressed HIV-
infected individuals constantly reported having a higher sense of taking control of one’s own
life, better psychological functioning and better sleep quality than the higher stressed
individuals.

Notably, it was concluded that the scores of stress perception and perceived quality-of-life
scales at a single assessment point (recruitment or four months later) were not associated with
the rate of change in the CD4 counts in HIV-infected patients (not receiving anti-retroviral
treatment); but that the rate of decline in the CD4 T-cell count of HIV-infected individuals was
associated with changes in the perceived quality-of-life (LoC, MCS and PSQI) scores.

These results support the central hypothesis that stress causes detrimental effects upon
well-being; and further, that an improvement in perceived quality-of-life may have beneficial
effect upon disease progression, i.e. psychological improvement may be beneficial for health.
This leads onto the next investigation of psychological intervention upon the psycho-neuro-
endocrino-immune network, particularly upon the perceived quality-of-life scales and the
disease progression marker, CD4 T-cell count [3.2.2].

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3.2.2 The influence of psychological intervention upon disease progresion parameters in


HIV-infected individuals (randomised controlled trial over a four month period)

This study investigates the hypothesis that psychological intervention can counteract
detrimental effect of stress upon disease progression, i.e. decline of the CD4 T-cell counts,
acting through the proposed integrated psycho-neuro-endocrino-immune network.

3.2.2.1 Effect of psychological intervention upon perceived stress and anxiety in HIV-infected
individuals
The influence of the psychological intervention upon mental well-being (perceived stress
and quality-of-life) was examined by comparing the scores on the self-report questionnaires
between the Self-hypnosis, Johrei groups and the wait-listed control group.

The State anxiety scores or the PSS scores did not change significantly from the Baseline
to the Post-intervention time point (four months after the Baseline) in any group, and the scores
did not differ between groups at either assessment point [Appendix 6: Table A-42]. The Impact
Event Scale (IES) scores improved significantly between the pre- and post- intervention time
points (F = 14.1, df = 48, p < 0.001). There were, however, no significant group differences in
this change, i.e. no significant time x group interaction (F = 0.8, df = 48, ns) [Table 34].

Table 34: Mean (95% C.I.) levels of the IES in the HIV-individuals in the Self-hypnosis, Johrei and wait-
listed control groups at the Baseline and Post-intervention time points
IES
Baseline Post-intervention
Self-hypnosis Johrei Controls Self-hypnosis Johrei Controls
Mean score 23.1 23.5 28.4 16.0 20.1 21.2
(95% C.I.) (19.2 - 27.0) (18.3 - 28.7) (24.2 - 32.6) (12.3 - 19.7) (15.2 - 25.0) (17.2 - 25.2)
N 21 12 18 21 12 18

The Locus of Control (LoC) (F = 1.1, df = 48, ns), Mental Components Summary of SF-
36 (MCS) (F = 0.8, df = 48, ns) or Pittsburgh Sleep Quality Inventory (PSQI) (F = 1.9, df = 48,
ns) scores did not change significantly from the Baseline to the Post-intervention time point in
any group, nor did it differ between groups at either assessment point, i.e. no significan time x
group interactions [Appendix 6: Table A-43]. Hence, it was concluded that psychological
intervention did not affect stress perception or perceived quality-of-life significantly.

However, having shown in the previous section that the worsining in the levels of the LoC,
MCS and PSQI over the study period were positively associated with the decline in the CD4 T-

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cell count [3.2.1.3], the distributions of the number of people who were improving or worsening
in the levels of (1) LoC [Table 35], (2) MCS [Table 36] and (3) PSQI [Table 37] were further
examined using the Intention-to-treat analysis (The participants who failed to provide self-report
questionnaire data were added in the numbers of the Decreased control, Decreased QoL or
Decreased sleep quality groups) for the exploratory purpose.

Table 35: Summary of the numbers (%) of the HIV-individuals whose changes in the LoC between the
Baseline and post-intervention either decreased (Increased control: increased sense of control in one’s
own life) or increased (Decreased control) in the Self-hypnosis, Johrei and wait-listed control groups
Increased control Decreased control Total
Self-hypnosis 10 / 21 (48%) 11 / 21 (52%) 21
Johrei 9 / 12 (75%) 3 / 12 (25%) 12
Control 10 /18 (55%) 8 /18 (45%) 18
Total 29 22 51

Table 36: Summary of the numbers (%) of the HIV-individuals whose changes in the MCS between the
Baseline and post-intervention either increased (Improved QoL: improved psychological functioning) or
decreased (Decreased QoL) in the Self-hypnosis, Johrei and wait-listed control groups
Improved QoL Decreased QoL Total
Self-hypnosis 11 / 21 (52%) 10 / 21 (48%) 21
Johrei 7 / 12 (58%) 5 / 12 (42%) 12
Control 7 /18 (38%) 11 /18 (62%) 18
Total 25 26 51

Table 37: Summary of the numbers (%) of the HIV-individuals whose changes in the PSQI between the
Baseline and post-intervention either decreased (Improved sleep quality) or increased (Decreased sleep
quality) in the Self-hypnosis, Johrei and wait-listed control groups㩷
Improved sleep quality Decreased sleep quality Total
Self-hypnosis 15 / 21 (71%) 6 / 21 (29%) 21
Johrei 9 / 12 (75%) 3 / 12 (25%) 12
Control 10 /18 (55%) 8 /18 (45%) 18
Total 34 17 51

The percentages of the HIV-infected individuals who improved the LoC. MCS or PSQI
scores in the Johrei group were all higher than those in the wait-listed control group, although
these different percentages between the three groups did not achieve statistical significance
(Self-hypnosis vs. wait-listed control: chi-square = 0.2, and Johrei vs. wait-listed control: chi-
square = 1.2 for the LoC; Self-hypnosis vs. wait-listed control: chi square = 0.7, and Johrei vs.
wait-listed control: chi-square = 1.1 for the MCS; and Self-hypnosis vs. wait-listed control: chi-
square = 1.1, and Johrei vs. wait-listed control: chi-square = 1.2 for the PSQI).

Sample size calculation shows that it would need 121, or 133, subjects per group in order
to show that 75%, or 58% in the Johrei group, is significantly different to the 55%, or 38% in
the wait-listed control group, respectively.

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Conclusion:
Neither Self-hypnosis nor Johrei appeared to alter anxiety or perceived stress levels in the
HIV-infected individuals (not receiving anti-retroviral treatments). The impact of being aware
of carrying HIV-infection decreased over the study period, but neither the intervention of the
Self-hypnosis nor Johrei affected the decrease over that found in the wait-listed control group.
None of the LoC, MCS and PSQI scores changed, and the Johrei and Self-hypnosis group did
not alter these scores significantly over the study period, although the Johrei group showed the
highest percentages who improved in their scores in all the perceived quality-of-life scales.
Sample size calculation showed that the numbers in each group were too few to demonstrate
statistically significant changes.

3.2.2.2 Effect of psychological intervention upon CD4 T-cell counts in HIV-infected


individuals
The influence of psychological intervention upon the change in the CD4 T-cell counts
(CD4 gradient: the disease parameter) was examined in order to test the hypothesis that
psychological intervention may counteract the detrimental effect of stress upon disease
progression in HIV-infected individuals.

CD4 gradients were constructed for 15 participants in the Self-hypnosis, ten in the Johrei
and 13 in the wait-listed control groups. A mean decline of 12 cells per l per month in CD4 T-
cell counts was shown in the wait-listed control group. The Kruskal-Wallis rank test showed a
trend towards difference among the three groups (chi-square = 5.5, df = 2, Monte Carlo
significance = 0.064). These different trends among the three groups were confirmed by
following chi-square analyses. There was a significant decrease in the rate of decline of CD4 T-
cells in participants who were trained in and practising Johrei compared with individuals in the
wait-listed control (chi-square = 3.9, df = 1, two-tailed p = 0.047) and Self-hypnosis (chi-square
= 4.5, df = 1, two-tailed p = 0.033) groups. There was, however, no difference in CD4 gradient
between the Self-hypnosis and wait-listed control groups (chi-square = 0.1, df = 1, ns) [Table 38
and Figure 40].

Table 38: Mean (95% C.I.) levels of CD4 gradients (counts per l per month) in HIV-infected individuals
in the Self-hypnosis, Johrei and wait-listed control groups
CD4 gradient (counts per l per month)
Self-hypnosis Johrei Controls
Mean change -9 + 17 - 12
(95% C.I.) (-17 to -1) (+ 6 to + 28) (- 20 to - 3)
n 15 10 13

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50

CD4 gradient (95%C.I.) cells/month


p = 0.033 p = 0.047

Not significant
-50
N= 15 10 13
Hypnosis Johrei Control

Figure 40: Means (95% C.I.) CD4 gradients over the five months study period from the Baseline to the
Post-intervention time point

This finding was further investigated by the Intention-to-treat analysis comparing


percentages of individuals between the two groups (decrease or maintained the CD4 T-cell
counts). All the data from the subjects who withdrew were added into the number in the group
in which the subjects decreased their CD4 T-cells [Table 39].

Table 39: Percentages (numbers) of participants whose CD4 T-cell counts maintained or decreased
over the five months in the Self-hypnosis and Johrei and wait-listed control groups for the
Intention-to-treat analysis (missing data were added into the number in the Decreased group)
Group Maintained CD4 T-cells Decreased CD4 T-cells Total
Self-hypnosis 29% (6 /21) 71% (15/21) 21
Johrei 58% (7 /12) 42% (5 /12) 12
Controls 33% (6 /18) 67% (12/18) 18
Total 19 32 51

The percentage of the HIV-infected individuals who maintained the CD4 T-cell counts in
the Johrei group (58%) was higher than that of wait-listed control group (33%), but the different
percentages between the three groups did not achieve statistical significance. The sample size
calculation shows that it would need 84 subjects per group to show that 58% in the Johrei group
is significantly different to the 33% in the wait-listed control group.

Conclusion:
As shown in the previous analysis [3.2.1], CD4 T-cell counts in HIV-infected individuals
who were not receiving anti-retroviral treatment decreased in the wait-listed control group.
However, this decreasing trend of CD4 T-cell count appeared to be reversed by the training and
practice of Johrei, although this was not supported by the intention-to-treat analysis, while the

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training and practice of Self-hypnosis did not appear to affect this trend. Sample size calculation
showed that the numbers of each group were too few to demonstrate statistically significant
changes.

3.2.2.3 Effect of psychological intervention upon other immunological markers (log-


transformed viral load levels and NK-cell counts) in HIV-infected individuals
Viral load level (log-transformed) gradients were constructed for 14 participants in Self-
hypnosis, 12 in Johrei and 12 in the wait-listed control groups; and NK gradients were
constructed for 15 participants in Self-hypnosis, 10 in Johrei groups and 12 in the wait-listed
controls. The viral load level (log-transformed) did not change significantly over the five
months nor did NK gradients, and there were no significant difference between the three groups
[Appendix 6: Tables A-44 and A-45, respectively].

Conclusion:
As shown in the previous analysis [3.2.1], neither viral load level nor NK-cell count
changed significantly in HIV-infected individuals who were not receiving anti-retroviral
treatment, and the training and practice of Johrei or Self-hypnosis did not affect the viral load
level or the NK-gradient.

Summary:
Although the results showed a possibility that Johrei may have helped to improve
perceived quality-of-life scores, neither Self-hypnosis nor Johrei appeared to alter the levels of
stress perception or perceived quality-of-life significantly in the HIV-infected individuals.

The major disease progression marker of HIV-infection, CD4 T-cell counts, was shown to
decrease over the study period in the wait-listed control group as expected. The training and
practice of Self-hypnosis did not appear to affect this decrease. In contrast, this decreasing trend
appeared to be reversed significantly by the training and practice of Johrei in those individuals
who completed the study. Significance was lost in the intention-to-treat analysis, but this was
suggested to be related to the small sample size. Hence, this counteracting effect of Johrei on
decreasing trend was further investigated in the next study [3.2.3] examining the rate of change
in CD4 T-cell counts in the 12 months periods both prior to (in order to examine baseline
difference) and after participants starting either of their trainings (in order to examine the effect
of the intervention trainings).

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These results provide support for the hypothesis that learning and practising psychological
intervention, which provides alternative perspectives of, and coping skills for, stressful life
events, improves psychological and physical well-being.

3.2.3 The influence of psychological intervention upon CD4 T-cells (disease progression
marker) in HIV-infected individuals (case controlled study over 24 months period)

This study aimed to replicate and explore the above findings [3.2.2.4] by increasing the
sample size and extending the investigation period from five months to a total of 24 months
(CD4 gradients were calculated over 12 months prior to and after the psychological
interventions commenced).

Matched anonymised patients from the database [see Methods section 2.1.3] were used as
controls for this study. Comparability was confirmed by comparing CD4 T-cell counts and CD4
gradiens of the controls with the study groups as follows [3.2.3.1].

3.2.3.1 Analysis of baseline differences between the three groups in the CD4 T-cell count
There were no differences in absolute CD4 T-cell counts between the three groups at the
time point the interventions commenced (F = 0. 7, df = 94, ns; Self-hypnosis vs. Database-
control: t = 0.6, df = 74, ns; Johrei vs. Database-control: t = 0.9, df = 66, ns) [Table 40].

Table 40: Mean (95% C.I.) levels of CD4 T-cell (counts per l) in HIV-infected individuals in the groups
of the Self-hypnosis, Johrei and Database-controls at the Training period
CD4 T-cells (counts per l)
Self-hypnosis Johrei Controls
Mean 397 346 375
(95% C.I.) (330 - 464) (278 - 414) (346- 404)
n 27 19 49

Further, there were no statistical differences in CD4 gradients between the three groups at
the Pre-intervention period (F = 0.2, df = 93, ns; Self-hypnosis vs. database control: t = 0.5, df =
75, ns; Johrei vs. database control: t = 0.2, df = 67, ns); and the average CD4 gradient for all 96
HIV-infected individuals was a decline of seven cells per l per month [Table 41].

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Table 41: Mean (95% C.I.) CD4 gradients in HIV-individuals in the Self-hypnosis, Johrei and Database
control groups, and in all the 96 individuals (ALL) at the Pre-intervention period
CD4 gradient (cells per l per month)
ALL Self-hypnosis Johrei Controls
Mean -7 -7 -9 -5
(95% C.I.) (- 11 to - 3) (-10 to -3) (- 14 to - 4) (- 8 to - 3)
n 95 27 19 49

Conclusion:
There were no statistical differences in absolute CD4 T-cell counts at Month 0 and CD4
gradients during the Pre-intervention period between the three groups.

3.2.3.2 Analysis of the rate of change in CD4 T-cell count (CD4 gradient)
The objective of this study was:
To explore the influence of the psychological intervention, particularly of Johrei,
upon the rate of decline by comparing the CD4 gradients in each group between the
periods over the 12 months prior to (Pre-intervention) and after the interventions
commenced (Post-intervention).

The CD4 gradients from all subjects were calculated as described in the method section
[2.1.3]. Two individual examples of CD4 gradient regression lines are presented in Figures 41
and 42.

Figure 41 shows the CD4 gradient of one database control patient as an example of the
natural course of the CD4 T-cell counts in HIV-infected individuals. For this patient, there is an
average decline of six (cells per l per month) in the Pre-intervention period and four (cells per
l per month) in the Post-intervention period. The Self-hypnosis participants gave similar
profiles to those shown for the database control patients, so that an individual example of their
CD4 gradient regression lines is not presented here.

In contrast, Figure 42 shows the CD4 gradient of one Johrei participant. There is a decline
of 10 (cells per l per month) in the Pre-intervention period, and an increase of seven (cells per
l per month) in the Post-intervention period.

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Pre-intervention Post-intervention

Mean decline of 6 cells per l per month Mean decline of 4 cells per l per month

Training
Months (Pre (-) and Post (+) from the Training)

Figure 41: CD4 gradients of one control subject at the Pre-intervention and Post-intervention periods

Pre-intervention Post-intervention

Mean decline of 10 cells per l per month Mean increase of 7 cells per l per month

Training
Months (Pre (-) and Post (+) from the Training)
Figure 42: CD4 gradients of one Johrei subject at the Pre-intervention and Post-intervention periods

The different trends in CD4 gradients between Pre- and Post- intervention periods was
examined by ANOVA repeated-measure analyses followed by using the 10% trimming method
[Wilcox, 1997] as described in the Methods section [2.5]. This subsequent ANOVA included 78
subjects (20 in the Self-hypnosis, 14 in the Johrei and 44 individuals in the Database-controls).

Figure 43 shows the individual changes in the CD4 gradients between the Pre- and Post-
intervention periods in the Self-hypnosis, Johrei groups and Database-control groups.

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Gradient changes in Hypnosis Gradient changes in Johrei Gradient changes in Control

30 30 30

20 20 20

10 10 10

0 0 0
Pre Post Pre Post Pre Post

-10 -10 -10

-20 -20 -20

-30 -30 -30

Self-hypnosis group (n =20) Johrei group (n =14) Database-controls (n =44)


Figure 43: Individual changes in CD4 gradients (cell counts per l per month) in the Self-hypnosis, Johrei
and Database-control groups between the Pre-intervention and Post-intervention periods

There was a significant decrease in the CD4 gradients from the Pre-intervention period to
the Post-intervention period (F = 19.4, df = 75, p < 0.001). The greatest improvement in the
CD4 gradients was seen in the Johrei group among the three groups (F = 2.4, df = 75, p = 0.095).

Having this result, further explorative analyses were performed as follows. Table 42
shows the summary of the increasing and decreasing trends in CD4 gradients with the
assumption that a change of four or less in CD4 gradient was defined as staying the same since
four CD4 T-cells change per l per month was within the 95% C.I. of the mean in 95 patients in
the Pre-intervention period. Accordingly, individuals in the Improving subgroup improved the
rate of decline more than four cells per l per month in CD4 gradient between Pre- and Post-
intervention periods, individuals in the Worsening subgroup increased the rate of decrease by
more than four CD4 T-cells per l per month, and the rest were in the Staying-the-same
subgroup.

Table 42: Percentages of HIV-individuals in the Self-hypnosis, Johrei and Database-control groups whose
changes in CD4 gradients between the Pre-intervention and Post-intervention periods were either
increased, stayed the same or decreased with a judgement that a change of four of less cells per l per
month was defined as the same change (Staying-the-same subgroup), more than four increase (Improving
subgroup), and more than four decrease (Worsening subgroup)
Group Improving (%) Staying-the-same (%) Worsening (%) Total
Self-hypnosis 9 / 20 (45%) 7 / 20 (35%) 4 / 20 (20%) 20
Johrei 11 / 14 (79 %) 2 / 14 (14%) 1 / 14 (7%) 14
Database-control 11 / 44 (25%) 21 / 44 (48%) 12 / 44 (27%) 44
Total 31 30 17 78

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This 11/14 (79%) of the Improving subgroup in the Johrei group is significantly different
to the 11/44 (25%) in the Database-control group (Pearson's chi-square = 12.9, df = 1, p <
0.001) while the 9/20 (45%) in the Self-hypnosis group was not significantly different to that in
the Database-control group (Pearson's chi-square = 2.6, df = 1, ns).

Having shown these findings using trimmed data set, further analyses using all of the 96
subjects were performed in order to confirm the results that Johrei may have maintained CD4 T-
cell counts. The analyses include comparisons between:
1. Three groups at the Post-intervention period (by using the Kruskal-Wallis rank tests);
2. Pre- and Post-intervention periods in the Database-control group (by using the
Wilcoxon Signed Rank tests); and
3. Pre- and Post- intervention periods in the Self-hypnosis and Johrei groups (by using
the Wilcoxon Signed Rank tests) [Table 43 and Figure 44].

Table 43: Mean (95% C.I.) CD4 gradients in HIV-individuals in the Self-hypnosis, Johrei and Database-
control groups at the Pre-intervention and Post-intervention periods
CD4 gradient (cells per l per month)
Pre-intervention Post-intervention
Self-hypnosis Johrei Controls Self-hypnosis Johrei Controls
Mean -7 -9 -5 -1 +3 -2
(95% C.I.) (-10 to -3) (- 14 to - 4) (- 8 to - 3) (- 4 to + 3) (- 2 to + 7) (- 5 to + 1)
N 27 19 49 27 19 49

30 Pre
Post
Mean (95% C.I.) CD4 gradients (cells/mcl/month)

p = 0.040
20

Not significant Not significant


10

-10

-20
Not significant
-30 Not significant

Self-hypnosis Johrei Database


control

Figure 44: Means (95% C.I.) CD4 gradients (count per l per month)
in the Self-hypnosis and Johrei and Database-control groups in the Pre- and Post- intervention periods

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The CD4 gradients of the individuals in the Database-control group showed a declining
trend in both the Pre-intervention and the Post-intervention periods (z = -1.5, df = 48, ns). In
contrast, the declining trend at the Pre-intervention period was halted and reversed in the Johrei
group at the Post-intervention period (z = - 2.1, df = 18, two-tailed p = 0.040), while patients in
the Self-hypnosis group continued to show a declining trend (z = - 1.1, df = 26, ns). Surprisingly,
however, there was no statistically significant difference between the psychological
interventions and Database-control in CD4 gradients at the 12 month of the Post-intervention
period (Self-hypnosis vs. Database-control: t = 0.2, df = 75, ns; Johrei vs. Database-control: t =
1.0, df = 67, ns).

Summary:
The results support the previous findings that there is a steady decline in peripheral blood
CD4 T-cell counts in HIV-infected individuals (not receiving anti-retroviral treatment); and that
the decline of CD4 T-cell counts appeared to be decreased or alleviated by the training and
practice of Johrei, but not by Self-hypnosis. Hence, it was suggested that learning and practising
Johrei may have improved disease progression in HIV-infected individuals.

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3.3 In vitro investigation into the effect of exposure to stress hormones upon
Natural Killer cells

Background
NK-cells are important cellular components of the innate immune system in defence
against infection and malignancy, and they are known to be affected by stress as shown in
previous study [3.1.2] and in the literature [Segerstrom & Miller, 2004]. Pharmacological levels
of exogenous glucocorticoids are known to suppress inflammation in vivo and to suppress NK
cytotoxic activity in vitro [Zhou et al., 1997].

In this series of in vitro experiments, the effects of exposure to the major stress hormone,
cortisol, and other stress hormones (DHEA-S and melatonin), particularly within physiological
levels, on NK-cells were examined.

Aims
A. To confirm the suppressive effect of a major stress hormone, cortisol, upon NK
cytotoxic activity (NKCA), and to explore the underlying mechanisms at the cellular
level.
B. To examine the relationships between other endogenous stress associated hormones,
DHEA-S and melatonin, upon NKCA.

Working hypothesis
“An in vitro model of sustained stress impairs NK cytotoxic activity.”
In vitro model of sustained stress is defined as “more than 24 hours of exposure to upper
physiological levels of cortisol”.

3.3.1 Development and optimisation of a flow cytometric method for measuring NKCA

3.3.1.1 NKCA after incubation of PBMCs for 24 hours


To investigate the effect of cortisol upon NK-cells, PBMCs were cultured for 24 hours
with cortisol. Therefore this experiment was designed to determine if incubation itself of
peripheral blood mononuclear cells (PBMCs) had an effect upon NK cytotoxic activity (NKCA).

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The flow cytometry method was used. PBMCs from 12 healthy subjects were incubated for 24
hours prior to assessing the NKCA.

Figure 45a and Figure 45b show mean (95% C.I.) and individual levels of NKCA pre and
post incubation for 24 hours. NKCA levels were increased in six individuals, decreased in three
and stayed the same in three. The repeated measures ANOVA showed a trend towards increased
levels of the NKCA (F = 4.3, df = 11, p = 0.063) when PBMCs were incubated in vitro for 24
hours.
Individual NKCA levels (% killing at E:T ratio = 50:1)

NKCA levels (95% C.I.) by flowcytometry


60
NKCA (50:1 ratio)
60 50
p = 0.063
50
40

40
30
30
20
20
10
10
0
0 N= 12 12

Time 0 24hrs Time 0 24hrs

Figure 45a: Individual NKCA levels at Figure 45b: Mean (95% C.I.) NKCA levels
Time 0 and after 24hour incubation at Time 0 and after 24hour incubation

Conclusion:
The levels of the NKCA of PBMCs after 24 hours in vitro incubation showed a trend
towards increased levels compared with Time 0, but that the increase was short of statistical
significant.

3.3.1.2 NK-cell percentage in PBMCs after 24 hours incubation


Having shown that incubation of PBMCs for 24 hours resulted in a trend towards an
increase in NK cytotoxic activity, this experiment was performed to determine if this was
associated with an increase in total NK-cell percentage, perhaps due to adherence or loss by
selective cell death of other sub-populations of PBMCs.

Lymphocyte sub-populations within PBMCs were analysed by flow-cytometry performed


prior to and after 24 hours culture.

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Figure 46a shows that there was no consistent change in individual levels of total NK-cell
percentages in PBMCs after 24hrs in vitro incubation, i.e. five increased, four decreased and
three stayed the same regardless of their NKCA changes. Figure 46b shows that there was no
significant difference in the mean percentage of NK-cells after 24hrs incubation.
30
CD56 NKcell%
30
Individual NK-cell percentages in PBMCs

25

CD56 NK cell% (95% C.I.)


20
20

15
10
10

5
0
0 N= 12 12

Time 0 24hrs Time0 24hrs

Figure 46a: Individual NK-cell percentage Figure 46b: Mean (95% C.I.) NK-cell percentage
in PBMCs at Time 0 and after 24hours in PBMCs at Time 0 and after 24hours

Conclusion:
The trend towards an increase in NK cytotoxic activity in PBMCs after 24 hours
incubation was not associated with an increase in the percentage of NK-cells.

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3.3.1.3 NKCA after 24hrs incubation of PBMCs with cortisol


The aim of this experiment was to examine the effect of 24 hours of incubation of PBMCs
with cortisol at physiological and higher levels of NKCA. PBMCs were incubated with two
concentrations (250nM and 2500nM) of cortisol for 24 hours and NKCA was measured by
flow-cytometry. Due to technical problems, one sample from one volunteer was lost to the study,
so that results from 11 samples were analysed.

Figure 47a shows in each individual the effect of exposure of PBMCs to cortisol (250 and
2500nM) on the levels of the NKCA. Figure 47b shows a decrease in the mean level of the
NKCA when cortisol was present. The repeated measure ANOVA showed a significant
decrease in the levels of NKCA with cortisol concentration (F = 10.50 df = 10, p = 0.009). This
decrease was confirmed by post-hoc paired t-test comparisons (mean difference = 6.06, standard
error = 1.74, p = 0.018: between the NKCA with 0nM and with 250nM; and mean difference =
7.02, standard error, 2.17, p = 0.027: between the NKCA with 0nM and with 2500nM). There
was no significant difference between NKCA with 250nM and 2500nM.
Individual NKCA (% killing at E:T ratio = 50:1)

NKCA levels (95% C.I.) by flowcytometry

NKCA (50:1 ratio) 60


60
p = 0.018
50 50
p = 0.027
40 40

30
30
20
20
10
10
0 㪥㩷㪔 㪈㪈 㪈㪈 㪈㪈
0 nM 250nM 2500nM 0 nM 250 nM 2500 nM

Figure 47a: Individual NKCA levels with cortisol Figure 47b: Mean (95% C.I.) NKCA levels
at 0nM, 250nM, and 2500nM with cortisol at 0nM, 250nM, and 2500nM

Conclusion:
The incubation of PBMCs with 250nM or more cortisol for 24 hours had a suppressing
effect upon NKCA.

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3.3.1.4 NK-cell percentage in PBMCs after 24 hours incubation with cortisol


Having shown that cortisol at 250nM or more had a suppressive effect upon NKCA, this
experiment was performed to determine if this suppressive effect was associated with a change
in the percentage of NK-cells in the PBMCs. NK-cells within PBMCs were analysed by flow-
cytometry, performed after 24 hours incubation with two cortisol concentrations (0nM, 250nM
and 2500nM).

There were no significant differences in the NK-cell percentage in the PBMCs after 24hrs
in vitro incubation with cortisol (0nM, 250nM and 2500nM). Figure 48a and Figure 48b show
individual and means levels of total NK-cell percentages in the PBMCs, respectively.
30
CD56 NKcell%
30
Individual NK-cell percentages

25
CD56 NK cell% (95% C.I.)

20

20

15
10
10

0
0 N= 12 12 12
0 nM 250nM 2500nM 0 nM 250 nM 2500 nMcortisol

Figure 48a: Individual NK-cell percentage Figure 48b: Mean (95% C.I.) NK-cell percentages
with cortisol at 0nM, 250nM, and 2500nM with cortisol at 0nM, 250nM, and 2500nM

Conclusion:
The cortisol associated suppression of NKCA in PBMCs was not associated with a
change in NK-cell percentage.

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3.3.2 Comparison of two methods for measuring NK cytotoxic activity (NKCA):


- the flow cytometric method and the colorimetric method (CytoTox96®) -

The flow cytometric method of assessing NK cytotoxic activity [Godoy-Ramirez et al.,


2000] was initially adopted to avoid the conventional radio-active 51Cr-release assay. However,
the flow cytometric method is laborious and requires a high degree of skill. A simpler
colorimetric method, namely the CytoTox96®, has been developed [Promega, 2004]. This
method measures levels of released lactic dehydrogenase (LDH) from killed K562 cells. The
amount of LDH is proportional to the numbers of killed K562 cells. The LDH level can be
accurately and simply measured colorimetrically.

The flow cytometric and the colorimetric assays were compared in this experiment.
PBMCs were separated from 45 healthy individuals and NKCA analysed, in parallel, by both
the flow-cytometric and the colorimetric methods.

Figure 49 shows the scatter graph of the correlation between the two methods. The
Pearson correlation analysis shows that there is a significant correlation between the two
methods (r = 0.367, 2-tailed p = 0.013).
60
NKCAby CytoTox (LDH) assay

50

40

30

20
p = 0.013
10 n = 45
r = 0.367
0 Rsq = 0.1349
0 10 20 30 40 50 60

NKCA by flowcytometry assay

Figure 49: Correlation between flow-cytometry method and LDH-releasing method of the NKCA

Conclusion:
It was determined that the levels of the NKCA should be measured by the less laborious
but still valid colorimetric method in subsequent experiments and to repeat the previous
experiments with a larger sample size to confirm the previous findings.

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3.3.3 Investigation of NK-cell profiles and NKCA (colorimetric method)

The flow cytometric assessment of NKCA showed that incubation of PBMCs for 24 hours
resulted in increased NK cytotoxicity. This repeat experiment was performed with PBMCs from
a larger number of volunteers using the colorimetric method with the objective of confirming
these results.

3.3.3.1 NKCA, assessed by colorimetric method, after incubation of PBMCs for 24 hours
PBMCs were obtained from 31 healthy subjects and incubated for 24 hours prior to
assessing the NKCA at effector: target ratios of 50:1 and 25:1 by the colorimetric assay.

Individual and mean (95% C.I.) NKCA levels with effector: target ratios of 50:1 and 25:1
pre and post incubation are shown in Figure 50 (a), (b) and Figure 51 (a), (b), respectively. In
agreement with previous findings, there was a significant increase in the mean NKCA level
after incubating PBMCs for 24 hours. The repeated measure ANOVA shows that the levels of
NKCA were increased after 24hrs incubation in a 50:1 ratio (mean difference = 6.38 (15.67 to
22.05), sd = 14.9, p = 0.024, t = 2.38, df = 30) and 25:1 ratio (mean difference = 10.47 (8.78 to
19.25), sd = 11.1, p < 0.001, t = 5.32, df = 30).

Time course of NKCA50:1


Individual NKCA (% killing at E:T ratio = 50:1)

50 40

p = 0.024
Mean (95% C.I.) NKCA levels at E:T = 50:1

40
30

30

20

20

10
10

0 0
Time 0 24 hours incubation
Time 0 After 24hours

Figure 50a: Individual NKCA (50:1) levels Figure 50b: Mean (95% C.I.) NKCA (50:1) levels
(% killing) at Time 0 and after 24hrs incubation (% killing) at Time 0 and after 24hrs incubation

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Time course of NKCA25:1 40

Individual NKCA (% killing at E:T ratio = 25:1)


50

Mean (95% C.I.) NKCA levels at E:T = 25:1


40 30
p < 0.001

30
20

20

10

10

0
0
Time 0 24 hours incubation Time 0 After 24hours

Figure 51a: Individual NKCA (25:1) levels Figure 51b: Mean (95% C.I.) NKCA (25:1) levels
(% killing) at Time 0 and after 24hrs incubation (% killing) at Time 0 and after 24hrs incubation

Conclusion:
The colorimetric assay results confirmed previous flow cytometric findings that
incubation of PBMCs for 24 hours leads to a significant increase in the levels of NKCA.

3.3.3.2 Repeat assessment of NK-cell percentage and examination of NK subpopulations in


PBMCs after 24 hours incubation
Although previous results showed no association between the increase in NKCA after 24
hours incubation of PBMCs and NK-cell (CD3-CD56+) percentages, the association was re-
examined pre and post 24 hours incubation with this increased number of subjects. In addition,
the distribution of NK cells according to the level of expression of CD56 and CD16 was
examined. The level of expression of these markers can be used to define cytotoxic and
regulatory NK-cells. However, due to technical problems with the flow cytometer, only the last
16 subjects from the above 31 were analysed.

Figure 52 (a), (b) and (c) show mean (95% C.I.) percentages of NK-cells (CD3-CD56+)
with subpopulations of cytotoxic NK-cells (CD3-CD56dimCD16+) and regulatory NK-cell (CD3-
CD56brightCD16-) in the PBMCs at Time 0 and after 24hrs incubation, respectively. There were
no significant differences in NK-cell profiles between Time 0 and 24hrs after incubation.

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Regulatory NK (CD56bright) cells% in PB


40 40 40

Cytotoxic NK (CD56dim)% in PBMCs


Total CD56+ NK cell% in PBMCs

30 30 30

20 20 20

10 10 10

0 0 0
N= 16 16 N= 16 16 N= 16 16
Time 0 Incubated 24hrs Time0 Incubated 24hrs Time 0 Incubated 24hrs

Figure 52a: NK (%) in PBMCs Figure 52b: Cytotoxic NK (%) Figure 52c: Regulatory NK (%)
at Time 0 and after 24hrs at Time 0 and after 24hrs at Time 0 and after 24hrs
Figure 52: Percentages of NK-cell in total and each subset at Time 0 and after 24 hours incubation

Conclusion:
The increase in NK cytotoxic activity in PBMCs after 24 hours incubation was not
associated with increases in the percentage of NK-cells or alteration in the cytotoxic / regulatory
NK-cell subpopulations.

3.3.3.3 Expression of Natural Cytotoxic Receptors (Nkp46 and Nkp30) by cytotoxic NK-cells
(CD3-CD56dimCD16+) pre and post 24hours incubation
Having shown that the increase in NKCA in PBMCs after 24 hours incubation was not
associated with increases in the percentage of total NK-cells or cytotoxic and regulatory NK-
cells, this experiment was performed to determine if the increase in NKCA was associated with
an increase in the expression of the Natural Cytotoxic Receptors (Nkp46 and Nkp30) on the
cytotoxic NK-cells. The expression (mean florescent intensity: m.f.i.) of the Nkp46 (major
cytotoxic receptor) on cytotoxic NK (CD3-CD56dimCD16+) cells within PBMCs from the same
16 healthy subjects were analysed by flow-cytometry performed prior to culture and after 24
hours.

Figure 53 (a) and (b) show that there was a significant increase (+44%) in the expression
of the Nkp46 on cytotoxic NK-cells after 24hrs in vitro incubation (Repeated measures
ANOVA: F = 38.1, p < 0.001, n = 16; mean difference = 27.0, se = 4.4). Figure 54 (a) and (b)
show that there was a significant decrease (-22%) in the expression of the Nkp30 on cytotoxic
NK-cells after 24hrs in vitro incubation (F = 18.0, p = 0.001, n = 16; mean difference = 5.4, se =
1.3).

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125
Nkp46 m.f.i. on Cytotoxic NK cells
140 p < 0.001

N k p 4 6 m .f.i. o n C y to to x ic N K cells
120
100
Nkp46 m.f.i. on Cytotoxic NK-cells

100
75

80

50
60

40
25

20
0
N= 16 16
0
Time0 24hrs
Time 0 24hrs

Figure 53a: Individual Nkp46 (m.f.i.) Figure 53b: Mean (95% C.I.) Nkp46 (m.f.i.)
at Time 0 and after 24hrs incubation at Time 0 and after 24hrs incubation
125
Nkp30 m.f.i. on Cytotoxic NK cells
140
N kp 30 m .f.i. on C y to tox ic N K cells

100
120
Nkp30 m.f.i. on Cytotoxic NK-cells

100
75

80

50
60 p = 0.001

40 25

20
0
N= 16 16
0
Time 0 24hrs
Time 0 24hrs

Figure 54a: Individual Nkp30 (m.f.i.) Figure 54b: Mean (95% C.I.) Nkp30 (m.f.i.)
at Time 0 and after 24hrs incubation at Time 0 and after 24hrs incubation

Conclusion:
These results show that the increase in NKCA in PBMCs after 24 hours incubation was
associated with an increase in the expression of Nkp46 on cytotoxic NK-cells, and it was also
associated with a decrease in the expression of Nkp30 although the amplitude of this decrease
was small.

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3.3.3.4 NKCA, assessed by colorimetric method, after 24 hours incubation of PBMCs with
250nM cortisol
This experiment aimed to confirm the results from the previous experiment 3.3.2.3 by
using a colorimetric method with a larger number of subjects, i.e. to determine if 24hrs
incubation of PBMCs with 250nM cortisol inhibits an increase in the levels of the NKCA.

PBMCs from 31 healthy subjects were incubated for 24 hours with or without 250nM
cortisol prior to assessing the NKCA by the colorimetric method at effector: target ratios of both
50:1 and 25:1.

Individual (a) and mean (b) NKCA levels after exposure of PBMCs to cortisol are shown
in Figure 55 (a), (b) and 56 (a), (b). As previously found, there was a significant reduction
following exposure of PBMCs to 250nM of cortisol in the mean NKCA levels at effector: target
ratio = 50:1 (mean difference = 6.69, SD = 6.9, p < 0.001, t = 5.41, df = 30) and 25:1 (mean
difference = 9.73, SD = 8.1, p < 0.001, t =6.66, df = 30), respectively [Figure 55b and Figure
56b].

NKCC % (LDH assay) 50:1 40


50
Individual NKCA (% killing at E:T ratio = 50:1)

p < 0.001
NKCA levels (95% C.I.) 50:1 ratio

40 30

30
20

20
10

10

0
N= 31 31
0
0nM 250nMcortisol
Without drug Cortisol 250 nM

Figure 55a: Individual NKCA (50:1) levels Figure 55b: Mean (95% C.I.) NKCA (50:1) levels
(% killing) of PBMCs incubated (% killing) of PBMCs incubated
with or without cortisol with or without cortisol

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Individual NKCA (% killing at E:T ratio = 25:1)

NKCC % (LDH assay) 25:1 40


50

NKCA levels (95% C.I.) 25:1 ratio


p < 0.001
40 30

30
20

20
10

10

0
0 N= 31 31
0nM 250nMcortisol
Without drug Cortisol 250 nM

Figure 56a: Individual NKCA (25:1) levels Figure 56b: Mean (95% C.I.) NKCA (25:1) levels
(% killing) of PBMCs incubated (% killing) of PBMCs incubated
with or without cortisol with or without cortisol

Conclusion:
Incubation of PBMCs for 24 hours with cortisol (250nM) causes an inhibition of NKCA.

3.3.3.5 NKCA levels between pre and post 24 hours incubation with or without 250nM cortisol
Having shown that there was an increase in the levels of NKCA (p < 0.001, n = 31)
following in vitro culture of PBMCs for 24 hours; and that the addition of 250nM cortisol to the
culture medium inhibited this rise (p < 0.001, n = 31), the association between these degree of
the increases and the inhibition were analysed.

The independent t-tests showed that the NKCA levels of the PBMCs before 24 hours
incubation were not different from the levels of PBMCs after 24 hours of incubation with
250nM cortisol [Figure 57].

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30
Not significant

NKCA 25:1 ratio (95%C. I.)


20

10

p < 0.001
0 p < 0.001
N= 31 31 31
Time 0 24hrs No drug 24hrs Cortisol

Figure 57: Mean (95% C.I.) of the NKCA at Time 0 and after 24hrs incubation of PBMCs
with/out 250nM cortisol in the target: effector ratio of 25:1

Conclusion
The degrees of an increase in NKCA level after 24 hours of incubation and an inhibition
of this increase in NKCA of the PBMCs incubated with 250nM cortisol are statistically the
same.

This finding was also confirmed by correlation analyses, in which the association between
time changes (after 24 hours incubation without cortisol - Time 0) and differences (after 24
hours incubation without cortisol – with cortisol) both at the ratio of 50:1 (p = 0.042, r = 0.37, n
= 31) and 25:1 (p < 0.001, r = 0.72, n = 31) [Figure 58].
Increasein NKCA25:1 (24h - Time0)

40

30

20

10
p < 0.001
n = 31
0 r = 0.72

-10 Rsq = 0.5220


-30 -20 -10 0 10 20 30

Reduction of NKCA25:1 (No-drug - Cortisol)

Figure 58: Correlations between the changes and differences of the NKCA (% killing: between at
Time 0 and after 24hrs incubation of PBMCs with/out 250nM cortisol in the ratio of 25:1)

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Conclusion:
The degree of inhibition in the NKCA by the in vitro exposure to the 250nM cortisol was
almost the same degree of increase in the levels of NKCA after 24 hours in vitro incubation of
PBMCs.

3.3.3.6 CD56 NK-cell subset profiles in PBMCs after 24 hours incubation with 250nM cortisol
Having confirmed in the larger cohort of subjects that 24 hours incubation with cortisol
inhibited NKCA, the experiment to determine if this was due to a change in NK-cell percentage
was repeated. In addition, NK-cell subset profiles (percentages of cytotoxic and regulatory NK-
cells within the PBMC) were examined. Due to technical problems in flow cytometer, only the
last 16 subjects from the above 31 subjects were analysed.

Figure 59 (a), (b) and (c) show (a) mean (95% C.I.) percentages of NK-cell (CD3-CD56+),
(b) cytotoxic NK-cell (CD3-CD56dimCD16+) and (c) regulatory NK-cell (CD3-CD56brightCD16-)
in the PBMCs after 24hours incubation with and without 250nM cortisol, respectively. As found
previously, there were no significant differences in NK-cell percentages and subpopulations in
PBMCs after 24hrs incubation with and without 250nM cortisol.
Reg u lato ry N K (CD 5 6b righ t) cell% in PBM
Cyto tox ic NK (CD5 6dim ) cell% in PBM Cs

40 40 40
Total NK (C D56) cell% in PB M Cs
NK-cells (%) in PBMCs

30 30 30

20 20 20

10 10 10

0 0 0
N= 16 16 N= 16 16 N= 16 16
No drug Cortisol No drug Cortisol No drug Cortisol

Figure 59a: NK (%) in the PBMCs Figure 59b: Cytotoxic NK (%) Figure 59c: Regulatory NK (%)
after 24hrs with/out cortisol after 24hrs with/out cortisol after 24hrs with/out cortisol
Figure 59: Percentages of NK-cell in total and each subset in the PBMCs after 24hours incubation with
and without 250nM cortisol

Conclusion:
The cortisol associated suppression of NKCA in PBMCs after 24 hours incubation was
not associated with a change in NK-cell profiles.

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3.3.3.7 Expression of Natural Cytotoxic Receptors (Nkp46 and Nkp30) by cytotoxic NK-cells
(CD3-CD56dimCD16+) after 24hours incubation with or without 250nM cortisol
Having shown that the inhibition of an increase in NKCA in PBMCs after 24 hours
incubation with 250nM cortisol was not associated with changes in the percentage distributions
of total or cytotoxic NK-cells, this experiment was performed to determine if the cortisol
induced inhibition was associated with the expression of the major cytotoxic receptor on
cytotoxic NK-cells. In experiment 3.3.4.3, this was shown to be increased after 24 hours
incubation.

The expression (mean florescent intensity: m.f.i.) of the Nkp46 and the Nkp30 on
cytotoxic NK (CD3-CD56dimCD16+) cells within PBMCs from 16 healthy subjects were
analysed by flow-cytometry performed after 24 hours incubation with or without 250nM
cortisol.

Figure 60 (a) and (b) show that there was a significant inhibition of an increase in the
expression of the Nkp46 on cytotoxic NK-cells after 24hrs in vitro incubation (Repeated
measures ANOVA: F = 58.4, p < 0.001, n = 16; mean difference = 13.3, se = 1.7). Figure 61 (a)
and (b) shows that there was a significant, but slight decrease in the expression of the Nkp30 on
cytotoxic NK-cells after 24hrs in vitro incubation (F = 13.2, p = 0.002, n = 16; mean difference
= 2.3, se = 0.6).
125
Nkp46 m.f.i. on CytotoxicNK cells
140
N k p 4 6 m .f.i. o n C y to to x ic N K cells

120 100

100
75

80

50
60

40
25 p < 0.001
20
0
N= 16 16
0
No drug cortisol
No drug Cortisol

Figure 60a: Mean (95% C.I.) Nkp46 (m.f.i.) Figure 60b: Individual Nkp46 (m.f.i.) after
after 24hrs incubation of PBMCs with/out cortisol 24hrs incubation of PBMCs with/out cortisol

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125
Nkp30 m.f.i. on Cytotoxic NK cells
140

N k p 3 0 m .f.i. o n C y to to x ic N K c ells
100
120

100
75

80

50
60 p = 0.002

40 25

20
0
N= 16 16
0
No drug cortisol
No drug Cortisol

Figure 61a: Mean (95% C.I.) Nkp30 (m.f.i.) Figure 61b: The individual Nkp30 (m.f.i.) after
after 24hrs incubation of PBMCs with/out cortisol 24hrs incubation of PBMCs with/out cortisol

Conclusion:
These results suggest that the inhibition of an increase in NKCA in PBMCs after 24 hours
incubation with cortisol was associated with an inhibition of an increase in the expression of
Nkp46 on cytotoxic NK-cells.

3.3.3.8 Nkp46 expression on cytotoxic NK-cells pre and post 24 hours incubation with or
without 250nM cortisol
Having shown that there was an increase in the expressions of Nkp46 (m.f.i.) on the
cytotoxic NK-cells (p < 0.001, n = 16) following in vitro culture of PBMCs for 24 hours; and
that the additive of 250nM cortisol to the culture medium inhibited this increase, the degree of
these increases and the inhibition were analysed. The independent t-tests showed that the
expression of Nkp46 (m.f.i.) on cytotoxic NK-cells before 24 hours incubation were lower than
that on cytotoxic NK-cells after 24 hours of incubation with 250nM cortisol [Figure 62].

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125
p < 0.001

Nkp46 m.f.i. on Cytotoxic NK cells


100

75

50

25 p < 0.001 p < 0.001

0
N= 16 16 16
Time0 24hrs No drug 24hrs cortisol

Figure 62: Mean (95% C.I.) and individual expression of Nkp46 (m.f.i.) on the cytotoxic NK-cells
(CD56dimCD16+) at Time 0 and after 24hrs incubation of PBMCs with/out 250nM cortisol

Conclusion:
Although there was an increase in the expressions of Nkp46 (m.f.i.) on the cytotoxic NK-
cells following 24 hours incubation, the addition of 250nM cortisol inhibited this rise. This
inhibition did not reach complete suppression.

3.3.3.9 NKCA and Nkp46 expression on cytotoxic NK-cells pre and post 24 hours incubation
with or without 250nM cortisol
Having the discrepancy between the changes in NKCA levels and changes in the Nkp46
expression pre- and post- 24 hours incubation with or without 250nM cortisol, the same 16
volunteers whose Nkp46 were examined out of the 31 subjects were re-analysed according to
their levels of NKCA. A trend of changes that were similar to the results in Nkp46 analyses was
obtained in this analysis of NKCA levels. Although there was an increase in the levels of
NKCA (p < 0.001) following in vitro culture of PBMCs for 24 hours, and the addition of
250nM cortisol to the culture medium inhibited this rise, this inhibition did not reach complete
suppression [Figure 63].

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Means (95%C.I.) of NKCAin 25:1 ratio


40

p < 0.001 p = 0.001


30

20

10

0 p = 0.012
N= 16 16 16
Time 0 24hrs No drug 24hrs cortisol

Figure 63: Mean (95% C.I.) and individual level of NKCA at Time 0 and after 24hrs incubation of
PBMCs with/out 250nM cortisol at target: effector ratio of 25:1

Conclusion:
The inhibition of an increase in NKCA levels after 24hrs in vitro incubation with 250nM
cortisol has association with the inhibition of an increase in Nkp46 on cytotoxic NK-cells after
the 24hrs incubation.

3.3.4 Endogenous stress hormone levels and NKCA (colorimetric method)

Having shown that exposure of PBMCs to exogenous cortisol for 24 hours appeared to
block the culture associated rise in the levels of NKCA, the levels of endogenous cortisol in the
plasma of subjects was measured to determine if prior in vivo exposure affected NKCA levels.
In addition, the plasma levels of DHEA-S and melatonin were also measured in order to
examine the hypothesis that prior in vivo exposure to either of them is associated with NKCA
levels. The plasmas from the separated PBMCs were analysed to measure endogenous levels of
cortisol, DHEA-S and melatonin by ELISA assays. Correlation analyses between endogenous
levels of stress-associated hormones (cortisol, DHEA-S and melatonin) and NKCA levels were
used to test the hypothesis.

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3.3.4.1 Endogenous hormone levels and NKCA at Time 0 point


At Time 0 point, there was no significant correlation between endogenous cortisol levels
and NKCA levels at both analyses ratios of 50:1 (r = .09, n = 31, ns) and 25:1 (r = - .01, n = 31
ns). DHEA-S levels, on the other hand, had a significant positive correlation with NKCA levels
but only at the ratio of 25:1 (r = .38, n = 31, p = 0.03), and not with the levels at the ratio of 50:1
(r = .19, n = 31, ns). No significant correlation between endogenous melatonin levels and
NKCA levels at the ratio of 50:1 (r = - .09, n = 31, ns) and 25:1 (r = - .03, n = 31, ns) was found
at Time 0 point.

Conclusion:
These results suggest that prior in vivo exposure to cortisol or melatonin did not affect
NKCA levels at the Time 0 point, although there is still a possibility that prior in vivo exposures
to DHEA-S may be positively associated with NKCA levels at Time 0 point.

3.3.4.2 Endogenous hormones levels and the increased levels of NKCA after 24 hours in vitro
incubation of PBMCs without exogenous cortisol
The results from previous experiment [3.3.4] showed that there were individual
differences in the levels of NKCA after 24 hours incubation without exogenous cortisol, i.e.
some of them had high levels of NKCA and the others had low levels. Individuals were divided
into three groups (high, middle and low NKCA levels) according to averaged NKCA levels at
the ratio of 50:1 and 25:1 after 24 hours incubation without cortisol [Figure 64].

NKCA after 24hrs incubation


45
Group of individuals with high levels of the NKCA
(High NKCA: mean = 34.2, SD = 6.8, n = 11)
30

Group of individuals with middle levels of the NKCA


(Mid NKCA: mean = 20.6, SD = 3.3, n = 9)
15

Group of individuals with low levels of the NKCA


(Low NKCA: mean = 7.1, SD = 4.8, n = 11)
0

Figure 64: Groups defined by tertiary split of the NKCA levels after 24 hours incubation

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The group of people with high levels of the NKCA in the tertiary splits (High NKCA:
mean = 34.2, SD = 6.8, n = 11/31) and the group of people who have low levels (Low NKCA:
mean = 7.1, SD = 4.8, n = 11/31) were compared with regard to the levels of their endogenous
hormones by independent t-tests. In addition, correlation analyses were performed to examine
the associations between endogenous hormones levels and the increased levels of NKCA after
24 hours incubation.

Cortisol levels
Endogenous plasma levels of cortisol had no significant correlation with the levels of the
NKCA both at the ratio of 50:1 (r = .09, n = 31, ns) and 25:1 (r = .02, n = 31, ns), and no
statistically significant difference in means between the High NKCA group and the Low NKCA
group was found [Figure 65].

200
Mean (95% C.I.) plasma cortisol levels (nM)

150

100

Not significant
50

Low NKCA Mid NKCA High NKCA


individuals individuals individuals

Figure 65: Mean (95% C.I.) plasma levels of cortisol in the High, intermediate (Mid) and Low NKCA
level groups after 24 hours incubation

DHEA-S levels
Endogenous plasma levels of DHEA-S appeared to be positively associated with NKCA
levels both at the ratio of 50:1 (r = .38, n = 31, p = 0.037) and 25:1 (r = .37, n = 31, p = 0.038),
and the High NKCA group had significantly higher levels of DHEA-S compared with the levels
of the Low NKCA group [Figure 66] (mean difference = 1.6, t = 2.1, df = 20, p = 0.045).

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5 p = 0.045

Mean (95% C.I.) plasma DHEA-S levels (mg/dl)


4

Low NKCA Mid NKCA High NKCA


individuals individuals individuals

Figure 66: Mean (95% C.I.) plasma levels of DHEA-S in the High, intermediate (Mid) and Low NKCA
level groups after 24 hours incubation

Melatonin levels
Endogenous plasma levels of melatonin appeared to be positively associated with NKCA
levels both at the ratio of 50:1 (r = .42, n = 31, p = 0.020) and 25:1 (r = .38, n = 31, p = 0.038).
Moreover, the High NKCA group had significantly higher levels of melatonin compared with
the levels of the Low NKCA group [Figure 67] (mean difference = 15.2, t = 3.2, df = 20, p =
0.010).

In addition, endogenous plasma levels of melatonin showed significant correlations with


the increased levels of the NKCA after 24 hours from Time 0 time point at the ratio of 50:1 (r
= .47, n = 31, p = 0.008) and 25:1 (r = .42, n = 31, p = 0.019).

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p = 0.037
40

Mean (95% C.I.) plasma melatonin levels (pg/mL)


30

20

10

Low NKCA Mid NKCA High NKCA


individuals individuals individuals

Figure 67: Mean (95% C.I.) plasma levels of melatonin in the High, intermediate (Mid) and Low NKCA
level groups after 24 hours incubation

Conclusion:
These results suggest that prior in vivo exposure to cortisol did not affect NKCA levels,
but that prior in vivo exposures to DHEA-S and melatonin were associated with the increased
levels of NKCA after 24 hours in vitro incubation.

Summary:
These results confirm, by the flow-cytometry and colorimetric methods that the levels of
the NKCA after incubation of PBMCs for 24 hours increased. There were no changes in NK-
cell and cytotoxic NK-cell percentages after the 24 hours incubation of PBMCs, but the
expression of the Nkp46 receptor was shown to increase while the expression of the Nkp30 was
not affected significantly. The increase of NKCA levels after 24 hours incubation was shown to
be associated with the increase in Nkp46 expression.

Upper physiological levels of cortisol (250nM), in vitro stress model, were shown to
inhibit the increase of NKCA seen after 24hrs incubation. The degree of inhibition in the NKCA
by the 250nM cortisol was almost to the same degree of increase in the levels of NKCA after 24
hours incubation of PBMCs. This inhibition in the NKCA level was not associated with a
numerical change in the levels of NK-cells and the cytotoxic NK-cell sub-population, but the

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inhibition was shown to be associated with inhibition of Nkp46 expression (m.f.i.) on cytotoxic
NK-cell subsets. However, the degree of inhibition of Nkp46 expression did not fully match the
suppression of the increase after 24 hours incubation. This finding implies that there are other
contributing factors apart from the Nkp46 expression.

The analyses in associations between endogenous levels of stress-hormones (cortisol,


dehydroepiandrosterone sulphate (DHEA-S) and melatonin) and NKCA levels at Time 0, i.e.
before the incubation of PBMCs, demonstrated that there were no significant relationships.
However, there appeared to be positive correlations between endogenous levels of DHEA-S and
melatonin, but not cortisol, and the NKCA levels after 24 hours of in vitro incubation, i.e. after
incubation without the presence of endogenous hormones. These results suggest that the levels
of prior in vivo exposure to DHEA-S and melatonin, but not cortisol, may have been positively
associated with the increased levels of NKCA after 24 hours in vitro incubation. Together with
the findings that the increased levels of NKCA were inhibited by the presence of upper
physiological levels of cortisol [3.3.1.3 and 3.3.3.4] and in the literature [1.2.2.3 and 1.2.4.2], it
was suggested that DHEA-S and melatonin may have affected to NK-cells in vivo as a counter
regulatory hormone against cortisol in NKCA levels.

These findings support the hypotheses that physiological levels of cortisol can suppress
NK cytotoxic activity directly; and that DHEA-S and melatonin may have counteracting effects
against cortisol upon the NK cytotoxic activity. This conclusion supports the premise that there
is a direct hormone-immunity linkage as an endocrino-immune arm of the proposed integrated
psycho-neuro-endocrino-immune network; and partially supports the hypothesis that stress may
have a detrimental effect upon health. This leads onto the next in vitro experiments examining
the effect of cortisol upon T-lymphocytes as an investigation of another direct hormone-
immunity linkage, i.e. linkage between cortisol and the adaptive immune system.

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3.4 In vitro investigation into the effect of exposure to stress hormone upon T-
lymphocytes

Background
T-lymphocytes are important cellular components of the adaptive immune system, and
one of their major functions, proliferation (an increase of the specific cell types against specific
antigens) [see also 1.2.1.1] is known to be affected by the stress hormone, cortisol.
Pharmacological levels of exogenous glucocorticoids are known to suppress T-lymphocyte
proliferative activity in vitro. In this project, the effects of physiological levels of cortisol upon
T-lymphocytes were investigated by assessing proliferative responses against antigens and a
mitogen. The proliferation rate was assessed by using [3H]-thymidine incorporation method, and
later the expression of various cell surface receptors was examined by using flow-cytometry to
explore the profiles of T-lymphocyte sub-populations.

Aims

To confirm the suppressive effect of a major stress hormone, cortisol, upon T-


lymphocyte proliferative responses, and to explore the underlying mechanisms at the
cellular level

Working hypothesis
“An in vitro model of sustained stress impairs T-lymphocyte proliferative activity.”
In vitro model of sustained stress is defined as “more than 24 hours of exposure to upper
physiological levels of cortisol”.

3.4.1 T-lymphocyte proliferative responses against common antigens


This series of experiments was designed to confirm the suppressive effect of cortisol upon
T-lymphocyte proliferation, and to explore the effect of cortisol at upper physiological levels.

When peripheral blood mononuclear cells (PBMCs) are incubated with previously
encountered antigens, memory T-lymphocytes are stimulated to divide leading to increased
incorporation of [3H]-thymidine. Under the culture conditions used, such cultures should give
radioactive counts in excess of 3,000 counts per minute (cpm).

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PBMCs from ten healthy subjects were incubated with or without antigens (purified
protein derivative (PPD) or Herpes zoster antigen) in various concentrations of cortisol (0, 25,
250 and 2500nM). All data were collected on the 6th day of incubation, and proliferative
responses were measured by the [3H]-thymidine incorporation method.

3.4.1.1 T-lymphocyte proliferation after incubation of PBMCs for six days with and without
exogenous cortisol
As expected, in vitro incubation of peripheral blood mononuclear cells (PBMCs) in tissue
culture medium (TCM) alone for six days did not cause lymphocyte proliferative responses
[Figure 68a, b]. Not surprisingly, the addition of various amounts of cortisol (upper
physiological levels: 250nM, 25nM and 2500nM) had no effect upon this result.

5000
6 day culture without antigen
Individual 3H-thymidine incorporation per PBMCs culture well (cpm)

4000
4000
Mean of 3H-thymidine incorporation per PBMCs culture well (cpm)

3000
3000

2000 2000

1000 1000

0
0
N= 10 10 10 10
0 nM 25 nM 250 nM 2500 nM
0 nM 25 nM 250 nM 2500 nM

㪚㫆㫉㫋㫀㫊㫆㫃㩷㪺㫆㫅㪺㪼㫅㫋㫉㪸㫋㫀㫆㫅

Figure 68a: individual background proliferations of Figure 68b: mean background proliferation
PBMCs cultured without antigen of PBMCs cultured without antigen

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3.4.1.2 T-lymphocyte proliferative response against purified protein derivative (PPD) after
incubation of PBMCs for six days with or without exogenous cortisol
PBMCs from nine volunteers out of ten responded satisfactorily to in vitro stimulation
with mycobacterium antigen (purified protein derivative: PPD). Figure 69a shows the individual
responses from nine volunteers whose PBMCs responded satisfactorily to in vitro stimulation
with the PPD antigen and that all responses but one were reduced by the addition of 250nM
cortisol and all responses were suppressed by the addition of 2500nM cortisol. The addition of
cortisol induced a dose-dependent decrease in the amount of [3H]-thymidine incorporated (F =
34.95, p < 0.001) [Figure 69b]. Paired t-tests showed significant reductions from 0nM to 250nM
(t = 8604, p = 0.001), and from 0nM to 2500nM (t = 12017, p = 0.001)
Individual 3H-thymidine incorporation per PBMCs culture well (cpm)

Lymphocyte proliferative responses to PPD


Individual proliferation response to PPD 20000

p = 0.001 p = 0.001
30000
15000

25000
10000

20000
5000
15000

0
10000

-5000
5000
N= 9 9 9 9
No drug 250nM
0 25nM 2500nMcortisol
0 nM 25 nM 250 nM 2500 nM

Figure 69a: individual proliferation response Figure 69b: Mean (95% C.I.) proliferation
of PBMCs to the PPD antigen responses of PBMCs to the PPD antigen

Conclusion:
Exogenous cortisol suppresses PPD-dependent T-lymphocyte proliferative responses, and
this can be seen at the upper physiological levels of cortisol, 250nM as well as 10 times higher
(2500nM).

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3.4.1.3 T-lymphocyte proliferative response against Herpes antigen after incubation of PBMCs
for six days with and without exogenous cortisol
Similar findings to those with PPD were obtained when a Herpes zoster antigen was used.
PBMCs from ten volunteers responded satisfactorily to in vitro stimulation with Herpes antigen.
Figure 70a shows individual responses from ten volunteers. All responses but one were reduced
by the addition of cortisol. The addition of cortisol induced a dose-dependent decrease in
proliferative responses measured by [3H]-thymidine incorporation (F = 15.96, p = 0.002)
[Figure 70b]. Post hoc paired t-test showed significant reductions from 0nM to 250nM (t = 9578,
p = 0.004), and from 0nM to 2500nM (t = 15386, p = 0.001).

Lymp hoc yte proliferative resp on ses to He rp es


60000
Individual proliferation response to Herpes
p = 0.004 p = 0.001
Mean 3H-thymidine incorporation per PBMCs culture well (cpm)

80000 50000

70000
40000
60000

50000
30000

40000
20000
30000
10000
20000

10000 0
N= 10 10 10 10

0 No drug 250nM
0 nM 25 nM 250 nM 2500 nM 25nM 2500nMcortisol

Figure 70a: individual proliferation response of Figure 70b: Mean (95% C.I.) proliferation
PBMCs to Herpes antigen responses of PBMCs to Herpes antigen

Conclusion:
Exogenous cortisol suppresses T-lymphocyte proliferative responses to memory antigens,
and this can be seen at the upper physiological levels of cortisol, 250nM as well as 10 times
higher (2500nM).

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3.4.2 T-lymphocyte proliferative responses against super-antigen and mitogen

Having shown that the addition of cortisol induced a dose dependent decrease in in vitro
antigen specific T-lymphocyte proliferative responses, this series of experiments was designed
to determine if concurrent exposure to cortisol (250nM) had similar effects following in vitro
stimulation with powerful pan T-cell stimulants, i.e. super-antigen - staphylococcal enterotoxin
B (SEB) and mitogen - phytohaemagglutinin A (PHA).

The number of cells particularly in the proliferative response to these SEB and PHA is
greater and the maximum response occurs earlier than for memory recall antigens. These
responses should give radioactive counts in excess of 10,000 counts per minute (cpm) on Day 3
of culture. With these more powerful stimuli, the assay was modified to use a whole blood
method rather than PBMCs which are time consuming to prepare.

Blood from twelve healthy subjects were diluted, one in four, with TCM and incubated
for three days with and without SEB and PHA, in the presence or absence of 250nM cortisol.
All data were collected on the third day of incubation.

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3.4.2.1 T-lymphocyte proliferative responses against staphylococcal enterotoxin B (SEB) after


incubation of diluted whole blood for three days with or without exogenous cortisol
Lymphocytes in the diluted whole blood samples from twelve volunteers responded
satisfactorily to in vitro stimulation with staphylococcal enterotoxin B (SEB) [Figure 71a]. The
addition of 250nM cortisol induced a decreased response (mean difference = 18235, SD = 8672,
t = 7.3, df = 11, p < 0.001) [Figure 71b].

P ro liferativ e resp o n ses to S E B (9 5 % C . I.)


100000
Lymphocytes proliferative responses

Mean (95% C.I.) proliferative responses to SEB (cpm)


to SEB
Individual proliferative responses to SEB (cpm)

120000
75000
100000

80000
50000
60000

40000
25000 p < 0.001
20000

0 0
N= 12 12
No drug Cortisol No drug Cortisol

Figure 71a: Individual proliferative Figure 71b: Mean (95% C.I.) proliferative
responses of PBMCs to the SEB responses of PBMCs to the SEB
with/out cortisol at Day 3 with/out cortisol at Day 3

Conclusion:
Upper physiological level (250nM) of cortisol can decrease proliferative responses of T-
lymphocytes against a super-antigen, SEB.

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3.4.2.2 T-lymphocyte proliferative responses against phytohaemagglutinin A (PHA) after


incubation of diluted whole blood for three days with or without exogenous cortisol
Lymphocytes in the diluted whole blood samples from twelve volunteers responded
satisfactorily to in vitro stimulation with phytohaemagglutinin A (PHA) [Figure 72a and b]. The
addition of 250nM cortisol induced a decreased response (mean difference = 74514, SD =
22856, t = 11.3, df = 11, p < 0.001).

P roliferative responses to P H A (95% C . I.)


Lymphocytes proliferative responses 200000

to PHA
Individual proliferative responses to PHA (cpm)

250000
150000
200000

150000 100000

100000
50000

50000
0 p < 0.001
0 N= 12 12

No drug Cortisol No drug Cortisol

Figure 72a: Individual proliferative Figure 72b: Mean (95% C.I.) proliferative
responses of PBMCs to the PHA responses of PBMCs to the PHA
with/out cortisol at Day 3 with/out cortisol at Day 3

Conclusion:
T-lymphocyte proliferative responses to in vitro stimulation with a super-antigen and a
mitogen were suppressed by the addition of cortisol (250nM).

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3.4.3 Cell surface markers on proliferating T-lymphocytes following stimulation with


PHA

Having shown that 250nM cortisol decreased T-lymphocyte proliferative responses


against a mitogen, a serious of investigations was performed to identify the underlying
mechanisms for this suppressive effect of cortisol upon lymphocyte proliferation.

3.4.3.1 Expression of an apoptosis (CD95: Fas) cell surface marker by T-lymphocytes in vitro
during PHA-induced proliferation with and without exogenous cortisol
This experiment examined the expression of CD95/Fas which is a marker of apoptosis on
T-lymphocytes during PHA-induced proliferative responses. Diluted whole blood from seven
healthy subjects were analysed by flow cytometry at time points of 0, 24, and 48 hours of
incubation after stimulation with PHA. The expression of CD95-FITC fluorescent conjugated
antibody on each lymphocyte sub-population surface was measured by flow cytometry.

In un-stimulated blood cultures, the expression of CD95/Fas, apoptosis marker, by CD4


and CD8 T-cells was unaltered over the 48 hours period [Figure 73a/b and Figure 74a/b].

80 80
Mean (95% C.I.) CD95 expression (%) on CD8 T-cells
Individual percentages of CD8 T-cells

60 60
expressing CD95 (%)

40
40

20
20

0
0
Time 0 background24hrs background 48hrs background
0h 24h 48h

Figure 73a: Individual background percentages Figure 73b: Mean (95% C.I.) background
of CD8 T-cells expressing CD95 percentages of CD8 T-cells expressing CD95

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80 80

Mean (95% C.I.) CD95 expression on CD4 T-cells


60
Individual percentages of CD4 T-cells

60
expressing CD95 (%)

40 40

20 20

0 0

0h 24h 48h Time 0 24hrs 48hrs

Figure 74a: Individual background percentages Figure 74b: Mean (95% C.I.) background
of CD4 T-cells expressing CD95 percentages of CD4 T-cells expressing CD95

When blood was stimulated with PHA, increasing percentages of CD4 and CD8 T-cells
expressed the CD95 marker at 24 and 48 hours incubation [Figure 75a and Figure 76a]. The
increases at 48 hours were statistically significant [Figure 75b and Figure 76b].
p < 0.001
80 80
Mean (95% C.I.) CD95 expression (%) on CD8 T-cells

70 Not significant
Individual percentages of CD8 T-cells

60 60
expressing CD95 (%)

50
40
40

30
20
20

p = 0.003
0 10

0h 24h 48h Time 0 with PHA 24hrs with PHA no48hrs with PHA no
no cortisol cortisol cortisol

Figure 75a: Individual percentages of CD8 Figure 75b: Mean (95% C.I.) percentages of CD8
T-cells expressing CD95 in PHA stimulated T-cells expressing CD95 in PHA stimulated
whole blood culture in the absence of cortisol whole blood culture in the absence of cortisol

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Mean (95% C.I.) expression of CD95 (%) on CD4 T-cells


80
80 p < 0.001
Individual percentages of CD4 T-cells

60
60 Not significant
expressing CD95 (%)

40
40

20
20
p = 0.001
0
0
0h 24h 48h Time 0 with PHA 24hrs with PHA no48hrs with PHA no
no cortisol cortisol cortisol

Figure 76a: Individual percentages of CD4 Figure 76b: Mean (95% C.I.) percentages of CD4
T-cells expressing CD95 in PHA stimulated T-cells expressing CD95 in PHA stimulated
whole blood culture in the absence of cortisol whole blood culture in the absence of cortisol

Conclusion:
The stimulation by PHA leads to progressively increasing CD95 expression on both CD4
and CD8 T-cells at 24 hours and 48 hours incubation.

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Addition of cortisol in the culture with PHA had little or no effect upon the percentage of
both CD4 and CD8 T-cells expressing CD95 [Figure 77 and Figure 78].
p = 0.001
80 80

Mean (95% C.I.) CD95 expression (%) on CD8 T-cells


Individual percentages of CD8 T-cells

p = 0.053
60 60
expressing CD95 (%)

40
40

20 20

p = 0.003
0
0
0h 24h 48h Time 0 with PHA, 24hrs with PHA, 48hrs with PHA,
cortisol cortisol cortisol

Figure 77a: Individual percentages of CD8 Figure 77b: Mean (95% C.I.) percentages of CD8
T-cells expressing CD95 in PHA stimulated T-cells expressing CD95 in PHA stimulated
whole blood culture in the presence of cortisol whole blood culture in the presence of cortisol
80 80 p < 0.001
Mean (95% C.I.) CD95 expression (%) on CD4 T-cells
Individual percentages of CD4 T-cells

60 60
Not significant
expressing CD95 (%)

40
40

20
20
p = 0.001
0
0
Time 0 with PHA, 24hrs with PHA, 48hrs with PHA,
0h 24h 48h cortisol cortisol cortisol

Figure 78a: Individual percentages of CD4 Figure 78b: Mean (95% C.I.) percentages of CD4
T-cells expressing CD95 in PHA stimulated T-cells expressing CD95 in PHA stimulated
whole blood culture in the presence of cortisol whole blood culture in the presence of cortisol

Collectively, these results showed that the addition of cortisol did not alter the
percentages of the cells expressing CD95 surface markers on CD8 T-lymphocyte [Figure 79]
and CD4 T-lymphocytes [Figure 80] at 24 (a) or 48 (b) hours of incubation.

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80 80

Mean (95% C.I.) CD95 expression (%) on CD8 T-cells

Mean (95% C.I.) CD95 expression (%) on CD8 T-cells


60 60

40 40

20 20

Cortisol (-) Cortisol (+) Cortisol (-) Cortisol (+)


0 0

24hrs with PHA no Cortisol 24hrs with PHA + Cortisol 48hrs with PHA no Cortisol 48hrs with PHA + Cortisol

Figure 79a: Mean (95% C.I.) percentages of Figure 79b: Mean (95% C.I.) percentages of
PHA stimulated CD8 T-cells expressing CD95 PHA stimulated CD8 T-cells expressing CD95
after 24 hours of incubation with/out cortisol after 48 hours of incubation with/out cortisol
80
80
Mean (95% C.I.) CD95 expression (%) on CD4 T-cells
Mean (95% C.I.) CD95 expression on CD4 T-cells

60
60

40 40

20 20

Cortisol (-) Cortisol (+) Cortisol (-) Cortisol (+)


0 0

24hrs with PHA no Cortisol 24hrs with PHA + Cortisol 48hrs with PHA no Cortisol 48hrs with PHA + Cortisol

Figure 80a: Mean (95% C.I.) percentages of Figure 80b: Mean (95% C.I.) percentages of
PHA stimulated CD4 T-cells expressing CD95 PHA stimulated CD4 T-cells expressing CD95
after 24 hours of incubation with/out cortisol after 48 hours of incubation with/out cortisol

Conclusion:
Upper physiological levels of cortisol did not alter the expression of the CD95, the
apoptosis surface marker, on PHA stimulated proliferating T-lymphocyte sub-populations.

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3.4.3.2 Expression of cell-surface Annexin-V and cytoplasmic Propidium iodine (PI) in T-


lymphocytes after incubation with PHA for three days with or without cortisol
Having shown that cortisol did not affect the percentages of cells expressing the CD95
apoptosis marker, this experiment was performed to investigate actual apoptotic changes of
PHA stimulated proliferating T-lymphocytes. Peripheral blood mononuclear cells (PBMCs)
from ten subjects were analysed at 48 hours of incubation by flow cytometry using Annexin-V-
FITC fluorescent conjugated antibody and propidium iodine (PI) to gate and to measure the
apoptotic changes on CD8 and CD4 T-lymphocyte sub-populations. Apoptotic cells were
defined as cells with Annexin-V-FITC positive (i.e. cell surface has changed) and PI negative
(i.e. cell has not been collapsed or necrosis) staining in flow cytometry.

There was no significant difference in the percentages of apoptotic cells (Annexin-V


positive and PI negative) between PHA stimulated PBMCs incubated with and without 250nM
cortisol both on CD3+CD8+ cells [CD8 T-lymphocyte: Figure 81a] and CD3+CD8- cells (CD4 T-
lymphocyte) [Figure 81b].

Annexin V(+) %in Annexin V(+) %in


CD3CD8 cells CD3+CD8- cell
100
100

80 80

60 60

40 40

20 20

0 0
No drug Cortisol No drug Cortisol

Figure 81a: Individual percentages of Figure 81b: Individual percentages of


apoptotic CD8T-cells incubated 3 days apoptotic CD4T-cells incubated 3 days
with and without cortisol with and without cortisol

Conclusion:
Upper physiological levels of cortisol did not alter the rate of apoptosis of PHA
stimulated T-lymphocytes.

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3.4.3.3 Expression of an activation (CD25: IL-2 receptor) cell surface marker by T-


lymphocytes during PHA-induced proliferation with and without exogenous cortisol
This experiment examined the expression of the T-cell activation surface marker, CD25:
IL-2 receptor, on T-lymphocytes following stimulation of whole blood with PHA; and the effect
of 250nM cortisol on this expression.

Diluted whole blood from seven healthy subjects was stimulated with PHA and analysed
by flow cytometry at time points of 0, 24, and 48 hours of incubation. The expression of CD25-
FITC fluorescent conjugated antibody by each lymphocyte sub-population was measured at
each time point.

In un-stimulated blood cultures, the expression of CD25, IL-2 receptor, by CD4 and CD8
T-cells was unaltered over the 48 hours period [Figure 82 and Figure 83].

60 60
Mean (95% C.I.) CD25 expression (%) on CD8 T-cells
CD8 T-cells expressing CD25 (%)
Individual percentages of

40 40

20 20

0 0

0h 24h 48h Time 0 background24hrs background 48hrs background

Figure 82a: Individual background percentages Figure 82b: Mean (95% C.I.) background
Of CD8 T-cells expressing CD25 percentages of CD8 T-cells expressing CD25

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60
60

Mean (95% C.I.) expression of CD25 (%) on CD4 T-cells


CD4 T-cells expressing CD25 (%)
Individual percentages of

40
40

20
20

0 0

0h 24h 48h Time 0 24hrs 48hrs

Figure 83a: Individual background percentages Figure 83b: Mean (95% C.I.) background
of CD4 T-cells expressing CD25 percentages of CD4 T-cells expressing CD25

When blood was stimulated with PHA, increasing percentages of CD4 and CD8 T-cells
expressed the CD95 marker at 24 and 48 hours incubation [Figure 84a and Figure 85a]. The
increases at 48 hours were statistically significant [Figure 84b and Figure 85b].

60 60
p < 0.001
Mean (95% C.I.) CD25 expression (%) on CD8 T-cells

p = 0.001

40 40

p = 0.003
20
20

0
0
Time 0 with PHA 24hrs with PHA no48hrs with PHA no
0h 24h 48h no cortisol cortisol cortisol

Figure 84a: Individual percentages of CD8 Figure 84b: Mean (95% C.I.) percentages of CD8
T-cells expressing CD25 in PHA stimulated T-cells expressing CD25 in PHA stimulated
whole blood culture in the absence of cortisol whole blood culture in the absence of cortisol

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60
60 p = 0.001

Mean (95% C.I.) CD25 expression (%) on CD4 T-cells


40 40
p < 0.001

p = 0.003
20 20

0
0
Time 0 with PHA 24hrs with PHA no48hrs with PHA no
0h 24h 48h no cortisol cortisol cortisol

Figure 85a: Individual percentages of CD4 Figure 85b: Mean (95% C.I.) percentages of CD4
T-cells expressing CD25 in PHA stimulated T-cells expressing CD25 in PHA stimulated
whole blood culture in the absence of cortisol whole blood culture in the absence of cortisol

Conclusion:
PHA stimulation leads to progressively increasing CD25 expression on both CD4 and
CD8 T-cells after 24 hours and 48 hours incubation.

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Addition of cortisol to the culture with PHA had little or no effect upon the percentage of
both CD4 and CD8 T-cells expressing CD25 [Figure 86 and Figure 87].
p = 0.001
60
60

Mean (95% C.I.) CD25 expression (%) on CD8 T-cells


Individual percentages of CD8 T-cells

p = 0.002
expressing CD25 (%)

40
40

p = 0.005
20
20

0
0
Time 0 with PHA, 24hrs with PHA, 48hrs with PHA,
0h 24h 48h cortisol cortisol cortisol

Figure 86a: Individual percentages of CD8 Figure 86b: Mean (95% C.I.) percentages of CD8
T-cells expressing CD25 in PHA stimulated T-cells expressing CD25 in PHA stimulated
whole blood culture in the presence of cortisol whole blood culture in the presence of cortisol
p < 0.001
p = 0.001

60
Mean (95% C.I.) CD25 expression (%) on CD4 T-cells

40

30
40
p = 0.003
20

20
10

0
0
Time 0 with PHA, 24hrs with PHA, 48hrs with PHA,
0h 24h 48h cortisol cortisol cortisol

Figure 87a: Individual percentages of CD4 Figure 87b: Mean (95% C.I.) percentages of CD4
T-cells expressing CD25 in PHA stimulated T-cells expressing CD25 in PHA stimulated
whole blood culture in the presence of cortisol whole blood culture in the presence of cortisol

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Collectively, these results showed that the addition of cortisol did not alter the
percentages of CD8 [Figure 88] and CD4 [Figure 89] T-cells expressing CD25 surface markers
at 24 (a) or 48 (b) hours of incubation.
60 60
Mean (95% C.I.) CD25 expression (%) on CD8 T-cells

Mean (95% C.I.) CD25 expression (%) on CD8 T-cells


Cortisol (-) Cortisol (+) Cortisol (-) Cortisol (+)
40 40

20 20

0 0

24hrs with PHA no Cortisol 24hrs with PHA + Cortisol 48hrs with PHA no Cortisol 48hrs with PHA + Cortisol

Figure 88a: Mean (95% C.I.) percentages of Figure 88b: Mean (95% C.I.) percentages of
PHA stimulated CD8 T-cells expressing CD25 PHA stimulated CD8 T-cells expressing CD25
after 24 hours of incubation with/out cortisol after 48 hours of incubation with/out cortisol

60 60
Mean (95% C.I.) CD25 expression (%) on CD4 T-cells
Mean (95% C.I.) CD25 expression (%) on CD4 T-cells

Cortisol (-) Cortisol (+) Cortisol (-) Cortisol (+)


40 40

20 20

0 0

24hrs with PHA no Cortisol 24hrs with PHA + Cortisol 48hrs with PHA no Cortisol 48hrs with PHA + Cortisol

Figure 89a: Mean (95% C.I.) percentages of Figure 89b: Mean (95% C.I.) percentages of
PHA stimulated CD4 T-cells expressing CD25 PHA stimulated CD4 T-cells expressing CD25
after 24 hours of incubation with/out cortisol after 48 hours of incubation with/out cortisol

Conclusion:
Upper physiological levels of cortisol did not alter the expression of CD25, an activation
surface marker, on PHA stimulated proliferating T-lymphocytes sub-populations.

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Summary:
T-lymphocyte proliferative responses to in vitro stimulation with memory recall antigens
(PPD and Herpes) as well as a super-antigen (SEB) and a mitogen (PHA) were suppressed by
the addition of the upper physiological level (250nM) of cortisol.

Stimulation by PHA leads to progressively increasing surface expressions of both CD95


(Fas-L: apoptosis marker) and CD25 (IL-2 receptor: activation marker) on CD4 and CD8 T-
cells at 24 hours and 48 hours incubation; and the addition of the upper physiological level
(250nM) of cortisol did not alter these expressions. Upper physiological level (250nM) of
cortisol was also shown not to alter percentages of the apoptotic surface changes of the cell
membrane (measured by Annexin V and PI) on PHA stimulated T-lymphocytes.

Although the underlying mechanisms are still to be elucidated, these results show that the
upper physiological level (250nM) of cortisol to suppresses T-lymphocyte proliferative
responses.

Ph.D. at University of London - 169 - Imperial College London


Chapter IV

Discussion

- 170 -
Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

CONTENT OF CHAPTER 4
Discussion

4.1 The influence of psychological intervention upon sustained stress and stress-
associated changes in university students facing exams and in patients with
HIV-infection
4.1.1 The influence of psychological intervention upon stress perception in
university students and HIV-infected adults 173
4.1.1.1 Anticipation of stress - academic examinations by university students
and awareness of carrying HIV-infection by treatment naïve patients 174
4.1.1.2 The influence of psychological intervention upon psychological
well-being 176

4.1.2 The influence of psychological intervention upon immune parameters in


university students facing academic exams 181
4.1.2.1 Stress-associated changes and differences in immune parameters in
university students 181

4.1.2.2 The influence of psychological intervention upon immune


parameters in university students 184

4.1.3 The influence of psychological intervention upon disease progression marker


(CD4 T cell count) and stress-related perception in HIV-infected individuals 186
4.1.3.1 Disease progression marker and stress-related perception in HIV-
infected individuals 187

4.1.3.2 The influence of psychological intervention upon CD4 T-cells as a


disease progression marker in the HIV-infected individuals 188

4.2 Stress hormone associated changes in vitro in immune cells as an exemplar of


the psycho-neuro-endocrino-immune network interaction
4.2.1 Changes in NK-cells after in vitro incubation 191
4.2.1.1 NK cytotoxic activity (NKCA) and NK-cell profiles after incubation
of PBMCs for 24 hours in vitro 192

4.2.1.2 Cortisol-induced changes in NKCA levels and NK-cell profiles 193

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4.2.1.3 Endogenous levels of stress hormones (cortisol and DHEA-S) and


NKCA 194

4.2.2 Changes in T-cells after in vitro incubation 195


4.2.2.1 Expression of CD25 during proliferative response 195

4.2.2.2 Expression of CD95 and Annexin V during proliferative response 196

4.3 Conclusions 198

4.4 Future directions


4.4.1 Psychological interventions 199

4.4.2 Measurements 199


4.4.2.1 Psychological measures 199

4.4.2.2 Immunological measures in vivo 199

4.4.2.3 In vitro investigation on NK-cells 200

4.4.2.4 In vitro investigation on T-lymphocytes 200

4.4.3 Proposal for future in vivo study 200

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4.1 The influence of psychological intervention upon sustained stress and stress-
associated changes in university students facing exams and in patients with
HIV-infection

This project examined the hypothesis that the effects of stress upon psychological well-
being and general health can be alleviated by psychological intervention, acting through the
integrated psycho-neuro-endocrino-immune network.

Two groups of individuals with different life-event associated stresses were selected to
test the hypothesis:
1. University students facing academic examinations were used as an example of
individuals with sustained, but a time-limited, stressful situation, and the primary
outcomes were NK-cell percentages and NKCA levels; and
2. HIV-infected patients living with the ongoing stress of infection were used as an
example of subjects with sustained life-long stress, and the primary outcome was
CD4 T-cell counts.

4.1.1 The influence of psychological intervention upon stress perception in university


students and HIV-infected adults

The first parts of this section discuss the effects of stress (either anticipation of exams for
university students or awareness of carrying on-going life-threatening disease for patients with
HIV-infection) on psychological well-being [4.1.1.1].

Thereafter, the following parts of this section discuss the influence of psychological
intervention upon stress perception and of following the categorisation of the appraisal of the
stressful life event [1.2.3.2] as:
1. Primary appraisal: Perception as a neutral or benign stimulus or a threat; and
2. Secondary appraisal: Judgement of one’s capability of coping with the stressor
subsequent to the primary appraisal.
Finally the results as they relate to perceived quality-of-life, i.e. perception of control
(inner or outer locus of control), psychological functioning, and sleep quality, are discussed
[4.1.1.2].

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4.1.1.1 Anticipation of stress - academic examinations by university students and awareness of


carrying HIV-infection by treatment naïve patients
Anticipation of academic examinations for university students was examined in the
current study to determine if it causes increases in stress levels. The mean PSS level of the
university students both at academic examination and when they were free from examination
were similar to the published mean levels for university students [Cohen et al., 1983]. In line
with previous findings [Maes, Van Bockstaele et al., 1999; Whitehouse et al., 1996], university
students in the current study reported more anxiety and stress when they faced academic
examinations compared to when they were free from academic examinations [3.1.1.1]. This
finding supports the validity of using academic examinations as a time-limited stressful
situation.

One possible criticism for using academic examinations as a stressor is the variability in
student responses to academic examinations. Some students experience more stress than others
in response to this situation, and some confident students may experience very little stress, if
any. Moreover, although expressed anxiety at academic examinations is usually thought of as a
negative experience [Deinzer & Schuller, 1998; Glaser et al., 1985; Gruzelier, Levy et al., 2001;
Gruzelier, Smith et al., 2001; Kiecolt-Glaser et al., 1986; Kiecolt-Glaser et al., 2001], some
students might feel academic examinations are an opportunity to test his/her capability, so that
the academic examinations for them can be perceived as an exciting experience, or in other
words, an impetus in primary appraisal [1.2.3.2]. For those students who perceive academic
examinations as an opportunity to show their capability, the examinations may be perceived as
not a fear but a positive anxious anticipation which can also cause a response towards fighting
or tackling for achievement in the ‘fight or flight’ responses in the psycho-neuro-endocrino-
immune network as can be seen in acute stress responses.

Nevertheless, the perceived stressed scale (PSS) can be used to distinguish those who
perceive the life events as an excitement from those who perceive it as a stressful event since
the PSS were designed to assess the degree to which situations in one’s life are appraised as
stressful [Cohen et al., 1983]. This a priori exposition of the PSS was tested as discussed in the
following section.

Not surprisingly, awareness of carrying life-threatening ongoing disease, HIV-infection,


for patients was found to be more stressful than anticipation of academic examinations for
university students [3.2.1.1]. On the other hand, and perhaps contrary to intuition, the anxiety

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levels expressed by individuals in these two different stressful situations were shown to be the
same [3.2.1.1]. Hence, it can be implied that the measured levels of anxiety by the State anxiety
score may characterise more an acute response of ‘fight or flight’ in the primary appraisal
[1.2.3.2] whereas the measured levels of stress by the PSS may indicate the accumulated levels
of stress over the time period.

Notably, anxiety levels in the HIV-infected individuals did not change over the study
period, although those reporting high levels of stress consistently reported more anxiety than
individuals who reported low levels of stress [3.2.1.1]. This trend between the two types of
individuals (labelled as the Stressed and Not-stressed defined by a midline split of the PSS
scores) was also shown in the Impact Event Scale (IES) measurement of primary appraisal in
event-specific stress perception. The primary appraisal against awareness of carrying HIV-
infection decreased over the study period, but the levels of these decreases were almost
paralleled in the Stressed and Not-stressed subgroups so that Stressed HIV-infected individuals
also reported higher IES sores than the Not-stressed individuals [3.2.1.1].

The finding that the state anxiety did not change, but that the primary appraisal level did
decrease over the study period suggests the possibility that the stressful impact of being
diagnosed as carrying a life-threatening disease, HIV-infection, can decrease over time, (in this
case a four month period with access to psychological care when needed), but that the anxiety
associated with being aware of carrying this disease is less amendable to intervention, possibly
through anticipating future stressful life events related to the disease. This may indicate that
anxiety can be driven or provoked not only from the primary appraisal of the stressor, which has
occurred in the past or the present, but also from thoughts about and anticipation of possible
future negative events associated with the disease.

In contrast, there was little change over the four months period in perceived quality-of-life
levels, i.e. sense of taking control of one’s own life measured by the Locus of Control (LoC)
scale; levels of psychological functioning as measured by the Mental Component Summary of
the SF-36 (MCS); and levels of sleep quality as measured by the scale of Pittsburgh Sleep
Quality Index (PSQI). These findings suggest that these psychological domains may be more
stable over time (i.e. more than the four months used in this study) or a more powerful
intervention may be needed to detect changes. Alternatively, there is a possibility that other
measurements of quality of life domains may be more sensitive for detecting the change than
the above questionnaires. However, as was the case with the measures of anxiety and primary

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appraisal, the Non-stressed HIV-infected individuals were shown to constantly report a greater
sense of taking control of one’s own life, better psychological functioning, and better sleep
quality than the Stressed individuals [3.2.1.1]. Hence, it can be concluded that the method to
compare two Stressed and Not-stressed subgroups based on the midline split of the PSS levels
may be a useful and practical way to identify the stress-related and the quality-of-life-related
differences in the measurements in the psycho-neuro-endocrino-immune network.

4.1.1.2 The influence of psychological intervention upon psychological well-being


As described in the Introduction [1.2.3], the stress perception to be examined in the
project was the participants’ appraisal of stressful life events. The results from both study
populations indicated that the psychological interventions had no clear benefit upon stress
perception and perceived quality-of-life. There are a number of factors that could explain the
generally negative results:
1. Psychological interventions may not impact upon the stress perception measured in
the current studies;
2. The short period of time in training and practice of the psychological interventions
for achieving to show clear effects; and/or
3. The relatively small sample sizes.
Despite these negative results, data from some of the psychological measures, i.e.
perceived quality-of-life (specifically, the LoC, MCS and PSQI), indicate some benefit from the
Johrei intervention on the sense of taking control of one’s own life, psychological functioning,
and sleep quality. Sleep quality may also indicate sense of control in their life, particurly in the
sense of control in relaxation at night-time both cognitively and physically, so change levels in
sleep quality may also represent changes in the sense of control. These benefits may have been
clearer had the study involved a greater number of subjects (e.g. more than 121 for the LoC and
PSQI or 133 for the MCS).

This section discusses the effect of psychological intervention upon these stress
perception (primary and secondary appraisals) and perceived quality-of-life as follows.

Primary appraisal
A major aim of stress management intervention is to alter the primary appraisal of the
stressful life event from a threat to a neutral (or even a benign stimulus) [1.2.3.2: Text box 2] so
that the event is no longer perceived as stressful. This was aimed to achieve by providing
alternative perspectives [1.2.3.3: Text box 4].

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In the project, the university students in the Relaxation control group exhibited increased
levels of the State anxiety scores when they were facing exams, and the psychological
interventions did not show any effects on the increase in the State anxiety scores [3.1.3.1]. This
finding may be interpreted as their perception of the exams was not ‘neutral’ in the primary
appraisal category. As discussed before [4.1.1.1], the State anxiety scores might represent the
summed levels of anticipation of both threat and impetus, so there remains a possibility that
some of the participants might have been changing the way they view the exam as a way to cope
with this stressor, or even a way to up-lift the morale to tackle the exams. Hence, future studies
using university students should investigate this hypothesis by measuring the levels of
excitement as well as anxiety.

Similarly, there was no change in the State anxiety score over the four months period in
the HIV-infected individuals in either the wait-listed control group or the intervention groups.
On the other hand, the IES scores did decrease significantly as described before, but there was
no difference in the decrease between the psychological interventions and the wait-listed control
groups. One possible explanation for the improvement in their IES scores noted over time in the
wait-listed control group might be related to the timing in measuring the questionnaire. Due to
practical difficulties in asking the participants to fill in the questionnaires, the participants in the
wait-listed control group completed the questionnaires shortly before (and for some of
participants, during) the first week of either of the psychological training session. Hence, a
positive anticipation toward the forthcoming psychological intervention after a long waiting
period might explain, at least in part, the observed improvement in this domain.

Nonetheless, the psychological interventions showed no greater improvement or


alleviation in the State anxiety scores or the IES scores, relative to the wait-listed control group
in HIV-patients.

Secondary appraisal
The PSS scores showed no difference between the intervention groups and the Relaxation
control group at academic examinations, so it was concluded that the psychological intervention
did not affect the perceived stress levels in university students facing academic examinations
more than the relaxation might have done.

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The scores of the PSS in the HIV-infected individuals at recruitment were much higher
than the levels in the university students facing exams, and the levels stayed at the same high
levels after four months period in the wait-listed control group participants. Moreover, the four
months of training and practice in the psychological interventions did not change these high
PSS scores. This finding suggests that perceived stress level in HIV-infected individuals was
not affected by the psychological interventions. It is possible that the high stress levels
associated with being HIV-positive may not be amenable to change with the brief interventions
used in this study. It is also possible that the relatively small sample size and short time period
limited the power to detect significant effects that might have been shown had there been more
participants and a longer intervention time period.

Novertheless, it is concluded that the psychological interventions showed no significant


improvement or alleviation in the Perceived Stress Scale scores relative to the relaxation in
university students facing exams or to the wait-listed controls in HIV-patients.

Quality-of-life
The measures of perceived control (locus of control), psychological functioning, and sleep
quality did not change in the wait-listed control group in the HIV-infected individuals over the
four months period. Moreover, neither of the psychological intervention groups showed any
statistical differences in these levels after four months of the intervention period. However,
although a larger sample size would be needed to confirm, there was a trend, in the Johrei group,
for improved scores across all the three outcome domains. Hence, there remains a possibility
that the training and practice of Johrei has improved perceived quality-of-life levels, which can
be associated with improvement in the meaning-focused coping skills [1.2.3.2: Text box 3].

Together with the clear different trends in the effect upon the immune cells (discussed
below [4.1.2.2 and 4.1.3.2]), and the fact that the effect seemed favourable to Johrei for
university students [3.1.3.2] and HIV-infected individuals [3.2.2.2 and 3.2.3.2], it is suggested
that unique psychological features in the Johrei training and practice may be worthwhile to
explore further.

The training and practice of the self-administered psychological intervention used in the
project aimed to achieve two objectives for the participants:
1. Behavioural domain: spending time and effort on oneself for one’s own benefit; and
2. Cognitive domain: acquiring additional concepts and perspectives for stressful events.

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In the behavioural domain, one of the unique features of Johrei practice is that it requires
a partner to practise with while the Self-hypnosis practice requires only one’s own time and
effort. This involvement of others as a partner / receiver for their own Johrei practice comes
from two of its key concepts and principles [Naito, 2003]:
1. “Healing oneself by healing others”: healing is a mutual process and interaction, and
it benefits equally both the Johrei giver and receiver since the power of healing was
taught to arise from the spiritual healing-light which flows through giver and receiver
during the Johrei practice; and
2. ‘Spiritual cord’: where the spiritual healing-light and love comes and travels beyond
time and space, and this Spiritual cord bonds a Johrei giver with all of the people
whom the giver cares about and also with something greater in the spiritual realm, i.e.
the source of the spiritual light itself.
Continued practice, which is a common feature in both Johrei and Self-hypnosis, can also
enhance a trainee’s sense of confidence in his or her own skills, the manageability in coping
strategies [1.2.3.2], which can be rephrased as ‘Self-empowerment.’ The above two concepts
found in the Johrei practice, therefore, can be strengthened synergistically with an increase in
their ‘Self-empowerment’, as they are repeated in practice.

In the cognitive domain, these tangible concepts may also have provided a Johrei giver
with both a sense of support from- and a sense of connectedness to- a receiver, and even
something greater in the spiritual realm, i.e. the source of the light. This may have buffered
against any feelings of loneliness or helplessness, and contributed to an overall sense of well-
being [Fromm, 1956], consistent with the findings from the Whitehall Study II which showed
that social support and self-confidence about one’s own skills are important factors for the
prevention of poor mental health [Stafford & Marmot, 2003; Stansfeld et al., 1997].
Furthermore, a belief and confidence in one self and/or something greater might have increased
the meaningfulness as Fromm [1956], Frankl [1970] and Strang et al. [2001] suggested.
Accordingly, Johrei might have enhanced their abilities in the meaning-focused coping
approach as well as the emotion- or problem- focused approaches [1.2.3.2: Text box 3].

In addition, the above two concepts, given repeatedly in the Johrei practice, have provided
the participants with a chance to recall or to give insight about their own perspectives related to
‘Spirituality’. The word ‘Spirituality’ or ‘Spiritual’ may suggest various concepts according to
each individual and each occasion or situation [Richards & Bergin, 1997]. In the Johrei training
session, this variety of ‘Spiritual’ thoughts and concepts are allowed and shared in a group, and

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could be left in ambiguity if total agreement could not be achieved, as long as they agree to feel
comfortable to practise Johrei [Appendix 2-ii].

The Johrei participants were also encouraged to appreciate their own doubts about the
efficacy of the intervention, or the existence of the healing-light if doubts about these arose
naturally. These doubts can also be left in ambiguity although they were also encouraged to
practise even with doubts so that they can test or challenge the doubts by themselves [Appendix
2-ii] in order to maintain one’s inner coherence. These methods used in the Johrei session are
based on the concept that ambiguity may provide important insights into self-cognition of one’s
own life [Empson, 1930; Toyama, 1973], so that the individuals left in ambiguity may be able to
(1) establish an alternative perspective to cope with stressful life events; (2) achieve a gradual
increase of their inner-personal coherence [Antonovsky, 1993]; and (3) weigh values and goals
to modify the meaning of a stressful transaction [Frankl, 1970; Park & Folkman, 1997] to
increase the sense of meaningfulness [1.2.3.2].

The possible link between the above behavioural and cognitive domains is found in a
concept of ‘Self-cultivation’ [Yuasa & Kasulis, 1987; Yuasa et al., 1993]. This ‘Self-cultivation’
may not be explicitly recognised in Western culture, but it has been emphasised in Eastern
culture including various traditional Japanese practices [Yuasa et al., 1993]. The mottos of this
self-cultivation include “practice makes perfect” and “practice precedes knowledge.” These
mottos mean that practice does not require a full understanding of knowledge in the subject, but
benefits can be obtained, and skills and knowledge can be developed through basic practice of
‘Kata’ - a standardised form or sequence of exercises [Yuasa & Kasulis, 1987]. The idea is that
practice can be used to develop, and also to bring to the fore of conscious awareness, ‘Tacit
knowledge’, i.e. knowledge that would otherwise remain hidden [Nonaka & Takeuchi, 1995].

Collectively, the development of the above domains should result in the following gains:
1. Social support and sense of support;
2. Spiritual awareness; and
3. Inner-personal coherence and meaningfulness.

Hence, future investigations of Johrei should include examination of the effects on


perceived support and these quality-of-life domains using published questionnaires that assess
these constructs, such as the Kesseler Perceived Social Support (KPSS) [Coventry et al., 2004],
the Spiritual Involvement and Beliefs Scale (SIBS) [Hatch et al., 1998] and the Sense of

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Coherence (SoC) [Antonovsky, 1993], as well as the scales of stress perception, used in the
project such as the Perceived Stress Scale (PSS) [Cohen et al., 1983], and sleep quality, the
Pittsburgh Sleep Quality Index (PSQI) [Buysse et al., 1989].

4.1.2 The influence of psychological intervention upon immune parameters in university


students facing academic exams

The changes in percentages of NK-cells and T-cells at academic examination time were
small, and similar in the degree of changes found elsewhere in the literature using university
students [Gruzelier, 2002b; Gruzelier, Smith et al., 2001; Halvorsen & Vassend, 1987; Kiecolt-
Glaser et al., 1986; Maes, Van Bockstaele et al., 1999; Uchakin et al., 2001]. In gender
difference, NK-cells have been reported to be lower in females than males under the age of 50
[Jentsch-Ullrich et al., 2005]. This study showed the same trend, but there was no gender bias
with regard to stress [3.1.2.3]. Similar fluctuations were also reported in studies that used
slightly different immuno-phenotyping: CD2 was used to gate T-cells & NK-cells instead of
CD45 [Maes, Van Bockstaele et al., 1999], and CD16 was combined with CD56 to detect NK-
cells [de Gucht et al., 1999; Uchakin et al., 2001].Hence, changes and differences here may
have no apparent clinical impacts upon health, but the results are discussed in the light of stress
responses.

4.1.2.1 Stress-associated changes and differences in immune parameters in university students


The levels of lymphocyte sub-populations (NK-cells, CD4 and CD8 T-cells) and the NK
cytotoxic activity (NKCA) were analysed by comparing the Stressed and Not-stressed
subgroups in the university students. Although the results of NK-cell percentages from this
study did not support previous findings in the literature in which stressed students had lower
numbers or percentage of NK-cells in their circulation compared with not-stressed students, the
results showed that the Stressed students exhibit low NKCA levels compared with the Not-
stressed students [3.1.2.1] in line with published findings [de Gucht et al., 1999; Deinzer &
Schuller, 1998; Glaser et al., 1985; Gruzelier, Smith et al., 2001; Halvorsen & Vassend, 1987;
Kiecolt-Glaser et al., 1986].

This finding leads to the hypothesis that individuals with high stress levels, from
sustained stress [1.2.3.2], may have low NKCA levels regardless of the situation. This
hypothesis may be specified more such that the high perceived stress level may be associated

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with a low per-NK-cell cytotoxic activity level. NK-cell percentages and NK cytotoxic activity
measured by using PBMCs are often used together or separately as stress benchmarks without
calculating the per-NK-cell cytotoxic activity [Dopp et al., 2000; Levy et al., 1989; Schedlowski
et al., 1993; Whitehouse et al., 1996]. This per-NK-cell cytotoxic activity, calculated as a NK
cell percentage to NK cytotoxic activity ratio, is known to be impaired in certain physical
conditions, such as in cirrhosis [Laso et al., 1997] and endometriosis [Oosterlynck et al., 1994].

In stress research, one meta-analysis paper calculated this per-NK-cell cytotoxic activity
in acute stress settings, and mentioned that acute stress did not alter per-NK-cell cytotoxic
activity, i.e. stress-induced changes in NKCA were due to alteration of NK-cell percentages
[Segerstrom & Miller, 2004]. This may only occur in acute stress situations, given the finding
that acute stress can increase both NK-cell percentage and NKCA levels [Dopp et al., 2000].
Therefore, it is suggested that future studies investigating the effect of stress upon NK-cells
should analyse the per-NK-cell cytotoxic activity.

In this current study, in the Stressed students, the levels of the per-NK-cell cytotoxic
activity and NKCA were found to be lower, although NK-cell percentages were not decreased
[3.1.2.1]. This implies that there may be other factor(s) affecting NKCA than a simple change in
NK-cell numbers / percentages. This was investigated by looking for direct effects of in vitro
exposure of blood to cortisol [3.3] and this will be discussed later [4.2.1].

In the analysis of CD4 T-cells (Helper T-lymphocytes and Regulatory T-cells), the results
showed that there was no significant difference in the levels of CD4 T-cells between the
Stressed and Not-stressed subgroups. This result is in line with previous reports; some studies
report an increase, others a decrease, and still others report no change [de Gucht et al., 1999;
Maes, Van Bockstaele et al., 1999]. The possible changes or no change in the CD4 T-cell
percentages may be due to:
1. A change of distribution in CD4 T-cells and/or in subsets (Th.1, Th.2 and T-reg), i.e.
a release of cells into the circulation or homing of cells into tissues;
2. A change in rate of cell production, i.e. generation or inhibition of new cells; or
3. A change in cell destruction, i.e. preventing or promoting cell death (programmed cell
death in the cell cycle).
These scenarios may occur in a specific time course, starting with a cell distribution
change and then generating new cells and the prevention of cell death. This consecutive course
may reflect a shift from an alarm reaction to a stage of resistance along the lines of Selye’s

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classifications [1.2.4.1]. This alarm reaction means that perceiving a situation as stressful can be
responded to in the immune arm of the psycho-neuro-endocrino-immune network by a release
of CD4 T-cells into the circulation. This possible mechanism in the immune system was
suggested in a report stating that the increase in the blood stream of the CD4 T-cells was due to
an increase of one subset of CD4 T-cells (CD25+CD4+ T-cell subset) in nurses undergoing
sustained stress, i.e. nurses who were diagnosed with ‘burnout’ syndrome [de Gucht et al.,
1999]. This CD25+CD4+ T-cell subset is a main component of the Regulatory T-cells (T-reg)
which play a major role in anergy, tolerance or in switching off the immune responses to
pathogens [Akbar et al., 2003], so this could be an important factor in the suppressive effect of
sustained stress upon the immune responses.

Recently, the direction of stress-induced changes in CD4 T-cell percentages, whether to


increase or to decrease, was demonstrated to depend upon the conditions of the study, i.e. type
of stressor and timing when CD4 T-cell percentages were measured, as shown below
[Segerstrom & Miller, 2004]. The categories of detailed setting of the timing in the reviews
[Miller & Cohen, 2001; Segerstrom & Miller, 2004] are whether there is short lasting time-
limited acute stress or one of the following four categories of sustained stress:
1. Brief naturalistic (predictable self-limited life events, e.g. academic examination),
2. Chronic (on-going or persistent life event, e.g. disease),
3. Event sequence (discrete stressful events occur repeatedly), or
4. Distant (past but traumatic events).
The sustained stress in the current student study falls into the brief naturalistic stress, and
they mentioned that CD4 T-cells seemed not to change in numbers, but functional suppression
or a shift towards humoral immune activation would occur under the category of sustained
stress.

Further studies using university students facing academic examinations should consider
examining the T-reg sub-population and the suppression or shift of immune responses in the
functional measures (by measuring Th.1 and Th.2 cytokine production) to explore the stress-
induced effect of naturalistic sustained stress upon CD4 T-cells.

There was no difference in CD8 T-cells (Cytotoxic T-lymphocytes) percentages between


the Stressed and Not-stressed subgroups, and this is again consistent with the literature findings
showing that stress-induced changes in the levels of CD8 T-cells is inconsistent [Segerstrom &
Miller, 2004]. These inconsistent results may be due to the fact that the CD8 T-cell population

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is not a homogeneous entity. The CD8 T-cell population consists of thousands of specific CD8
T-cells made for a specific antigen, and it takes much longer period to make a specific immune
response than an innate immune response. Hence, inconsistent results may be because the end
results of combinations of various changes in the heterogeneous entity of the CD8 T-cells were
shown and because the time period was shorter than needed to show any changes in numbers.

Future investigations of stress-induced changes in the CD8 T-cell, if any, should take this
heterogeneous feature of the CD8 T-cell population into account, so should use a specific
antigen to stimulate a specific CD8 T-cell subtype, preferably in combination with functional
examinations (proliferative responses or cytokine productions).

4.1.2.2 The influence of psychological intervention upon immune parameters in university


students
Although the subject numbers decreased by the time of the second set of academic
examinations leaving a total of 35 students, fortunately they were distributed almost evenly
across the three groups. The findings indicated that the Relaxation control group showed a
similar trend to the stressed individuals in the literature at exam time, i.e. the trend to decrease
NK-cell percentages; and that this was altered particularly in the Johrei group [3.1.3.2: Figure
26]. Baseline NK cytotoxic activity results were missed due to the development and adjustment
of the Godoy-Ramirez’s assessment method so that analyses of the effects of intervention upon
the NK cytotoxic activity were not possible to assess. Hence the following sections discuss the
results of the NK-cell and lymphocyte distributions only in each group.

Relaxation control group


The relaxation control group at academic exam time showed similar decreases in NK-cell
levels to published reports about high stressed individuals [Borella et al., 1999; Gruzelier, Smith
et al., 2001; Inoue-Sakurai et al., 2000; Maes et al., 1992; Segerstrom & Miller, 2004]. However,
in order to make the obtained changes statistically significant, the sample size calculation shows
that the subject number needs more than 39. The number of subjects was only 11, so definitive
conclusions from these findings cannot be made. This finding may be due to the following
possibilities:
1. Exam stress did not significantly change NK-cell levels;
2. The sample size was too small to detect the change; or

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3. The relaxation, provided by the mock neuro-feedback procedure as shown previously


[Egner et al., 2002], itself only or with attention given to the students (i.e. placebo
effect) might have buffered the stress-induced NK-cell decrease.

In order to determine these hypotheses, as stated previously, future investigation should


have an intervention-free control group as well as the Relaxation control group.

Self-hypnosis group
In the previous reports in the literature, self-hypnosis training and practice were shown to
buffer the stress-induced decline levels in either NK-cell levels (labelled as CD3-CD56+ or/and
CD16+) [Bakke et al., 2002] or CD8 T-cell counts [Gruzelier, Levy et al., 2001], or in both NK
cells and CD8 T-cells [Gruzelier, Smith et al., 2001] whereas non-intervention controls showed
a significant decline. The Self-hypnosis group in the current study showed no changes in the
NK-cells levels at academic exams. However, it is also possible that the number of subjects was
too small to detect differences; or that stress-induced changes in the NK-cells were cancelled as
a result of alteration of the primary appraisal from stressful to ‘neutral.’ In contrast, CD8 T-cell
levels were increased in the Self-hypnosis group as shown previously [Gruzelier, Levy et al.,
2001] although the changed levels of the CD8 T-cells were not related to the stress perception in
this study [3.1.2.2].

Hence, it was concluded that this study partially supports the hypothesis that Self-
hypnosis may have acted as stress-buffering for university students facing academic exams,
therefore, it would be worth examining it further in a study using a greater number of subjects,
and with measurements of functional activity of CD8 T-cells, i.e. cytotoxic T-cell activity to a
specific antigen, using a diseased population in which the antigen(s) can be specified as
proposed above.

Johrei group
Interestingly, the result from the current study showed that the levels of NK-cell
percentage in the Johrei group were increased at academic exams. Although the amplitudes of
this increase were relatively small and all percentages and counts were within the normal range
[Hannet et al., 1992], eight of eleven Johrei participants showed raised percentages of NK-cells
in their peripheral blood. This trend was significantly different from that of the Relaxation
control and the Self-hypnosis groups; and the expected direction in the change of the NK-cells

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based on the observation of stressed individuals both in the current study and in the literature
was opposite to that of the Johrei group in the current study [3.1.3.2].

As described in the introduction [1.2.4.2 and 1.2.4.3], acute stress is known to increase the
levels of NK-cell percentages and NK cytotoxic activity [Dopp et al., 2000] while sustained
stress is known to decrease the levels. Borella et al. [1999] reported that students who are
emotionally stable in personality profile have increased NK cytotoxic activity during their
academic exams. Further, in the other not-diseased population, De Gucht et al. also reported
[1999] that sustained stress increased NK-cell count in the group with high professional stress
(measured by the Nurse Stress Index) but low psychopathology (those who have good adaptive
coping abilities to their symptoms). These findings and the results from the current study
suggested the possibility that Johrei may have increased the emotional stability or decrease
pathological impact of stress. Future research on Johrei using university students facing exams
should employ a larger sample size with measures of per-NK-cell cytotoxic activity levels in
order to confirm this finding.

Despite the limitations, it is, nevertheless, concluded that the psychological interventions,
particularly Johrei, appeared to have possible buffering or even counteracting effects of
examination stress upon the distribution of the NK-cells, and/or CD8 T-cells, in the university
students.

4.1.3 The influence of psychological intervention upon disease progression marker (CD4 T
cell count) and stress-related perception in HIV-infected individuals

The human immunodeficiency virus (HIV), a lentivirus of a subgroup of retro-viruses,


progressively destroys the immune cells, CD4 T-cells in particular, and this leads to the life-
threatening disease, the acquired immunodeficiency syndrome (AIDS), and death. The decline
of CD4 T-cells is known to be associated with a risk of developing opportunistic infections such
as Pneumocystis carinii pneumonia or oesophageal candidiasis as well as the risk of developing
tumours, particularly those related to immune suppression, such as the Kaposi’s sarcoma or
Non-Hodgkins lymphoma.

Hence, HIV infection may be considered to be a life-long biological and psychological


stressor, and it is known that stress levels contribute to detrimental outcomes associated with

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HIV disease progression [Catalan et al., 1995; Cole et al., 2003; Cole et al., 1997; Cole et al.,
1996]. The current project aimed to decrease the level of stress in the HIV-infected individuals
so that their disease progression can be delayed, allowing these individuals to maintain their
health and well-being and delay the requirement to commence antiretroviral drug therapy and its
inherent complications.

4.1.3.1 Disease progression marker and stress-related perception in HIV-infected individuals


CD4 T-cell counts in HIV-infected individuals, a major clinical parameter for monitoring
disease progression, is known to decline over the course of this life-long infection [de Wolf et
al., 1997]. A steady decline in CD4 T-cell counts of 7 ± 3 cells per l per month over a 12
months period prior to their enrolment into the study was observed in the total of 95 HIV-
infected individuals [3.2.3.1]. Similarly, HIV-infected individuals who are not receiving any
anti-retroviral treatments in our wait-listed control group showed a decline in CD4 T-cell counts
of 12 ± 17 cells per l per month over a five-month period [3.2.2.2]. These were within the
average range of published findings (8 to 16 cells per l per month) [de Wolf et al., 1997]. In
the current project, this rate of decline in the number of CD4 T-cells was analysed in order to
determine if stress perception has any associations with this rate of decline.

The results in the HIV-infected individuals showed that observational single time point
measurements of perceived stress, quality-of-life and sleep quality did not correlate with the rate
of change in the CD4 T-cell counts, i.e. scores at one single time point did not predict or
indicate the rate of change in the CD4 T-cell counts. In addition, the change levels over the
study period in the Perceived Stress Scale (PSS) also did not correlate with the rate of decline.
However, the results did show that decreases over a four-month period in the sense of taking
control of one’s own life or inner-personal coping ability (LoC), psychological functioning
(MCS) and sleep quality (PSQI) were strongly correlated with a decline in the CD4 T-cell
counts [3.2.1.2].

These findings may imply the possibility that change levels of autonomy or attitudes of
actively being engaged in their life, as opposed to measures of passively experienced stress
(stress perception), may be associated with the rate of change in the CD4 T-cell count in HIV-
infected individuals. This possibility is consistent with the suggestion in the Whitehall Study II
[Bosma et al., 1998] that ‘self-empowerment’ may have beneficial effects on tackling disease
physiologically. For the purpose of examining this hypothesis that an increase of the sense of
‘self-empowerment’ improves (delays) disease progression in HIV-infection or maintains health,

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the Sense of Coherence (SoC) [Antonovsky, 1987] may be a good domain to assess in future
studies, because the SoC is designed to measure a positive inner-personal coherence which
consists of the levels of comprehensibility, manageability and meaningfulness [1.2.3.2].

In contrast to the analysis of the CD4 T-cell counts, there was no trend in the levels of the
HIV viral load or NK-cell counts in HIV-infected individuals (not receiving anti-retroviral
treatment) across the five months period with regard to either the CD4 gradient or the levels of
the perceived stress, quality-of-life or sleep quality [3.2.1.2]. The levels of HIV viral load are
clinically used to monitor disease progression in combination with the result of the CD4 T-cell
counts, and they are used to examine the response to anti-retroviral treatments, so the viral load
levels might not be a suitable outcome measure for study subjects who are not receiving anti-
retroviral treatment. Similarly, although NK-cells are known to play an important role in
defensive mechanisms against HIV infection and progression [Fauci et al., 2005], no consistent
trend was found between the number of NK-cells and disease progression in HIV-infected
individuals, so NK-cell levels might also not be a suitable outcome measure for study subjects.

Collectively, it can be concluded that the rate of decline in CD4 T-cell count (CD4
gradient) in treatment naïve HIV-infected patients is an important and useful practical outcome
measurement for the investigation of the effect of psychological intervention upon disease
progression. Notably, it is also demonstrated that the change levels of perceived quality-of-life,
which can be interpreted as the change in attitude of actively being engaged in their life and/or
the change in the sense of meaningfulness, may have direct associations with this change in the
CD4 T-cell count in HIV-patients. Accordingly, improvement of the meaning-focused coping
skills [1.2.3.2] may have beneficial effects upon health and well-being, and therefore it can be
of great interest for the effect of psychological intervention.

4.1.3.2 The influence of psychological intervention upon CD4 T-cells as a disease progression
marker in the HIV-infected individuals
The results showed that, in all three groups, there was a steady decline in the CD4 T-cell
counts before the study commenced and the absolute CD4 T-cell counts were similar [3.2.3.2].
After commencement, this decline of CD4 T-cells continued in controls (the wait-listed controls
in the five months study [3.2.2.2: Figure 40] and the Database controls in the 24 months study
[3.2.3.2: Figure 44]). The on-treatment analysis showed that this decline rate of CD4 T-cells
was stopped, and even reversed, in the HIV-infected individuals following Johrei training and
practice over the five months period while there was no change in the Self-hypnosis group. This

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interesting different trend shown by the on-treatment analysis in the five months study, however,
was not confirmed by the Intention-to-treat analysis suggesting that future studies examining
this effect of Johrei will require a larger sample size (more than 84 subjects per each group)
[3.2.2.2: Table 39].

The reduction in the decline of the CD4 T-cells in the individuals in the Johrei group may
have contributed to both psychological and physiological stress reduction. In addition to
improving individual’s well-being, there is both an individual and economic benefit in that any
reduction in the speed of decline of CD4 T-cell counts will effectively delay the time when an
individual patient will commence expensive anti-retroviral treatments. This will only be delayed
rather than avoided, but may help to keep away from a vicious circle of stress-induced
detrimental changes towards acceleration of the disease process towards AIDS and will delay
exposure to the toxic side effects of anti-retroviral drugs.

Limitation should also be mentioned. Recruiting and motivating participants for a


randomised controlled trial (RCT) is known to be challenging. Walker suggested in his review
that performing explanatory and pragmatic RCTs can minimise these problems [Walker &
Anderson, 1999]. In fact, the university student study did not have a high drop-out rate up until
the time the training sessions were completed. This may be due to the fact that the current study
using university students employed the mock neuro-feedback sessions as a control condition,
which is similar to explanatory and pragmatic trial since the same amount of care and attention
was given to the control individuals. On the other hand in the HIV study, the control group
subjects were randomly assigned and had to wait for four months, and had higher drop-out rate
compared with the Self-hypnosis and Johrei groups.

Further, although the study subjects were randomly assigned to the three groups, actual
numbers for the analyses were different and the number in the Johrei group was the smallest in
the HIV-patient study. This was not associated with the drop-out rate but because the number of
subjects who were assigned to the group but did not turn around the first training session was
the largest in the Johrei group. It may have reflected some psychological difficulties in trying a
novel (or even strangely sounding) procedure like Johrei. This may have resulted in a self-
selection within the Johrei group towards a particular personality type, e.g. open mindedness or
novelty seeking in the Temperament and Character Inventory (TCI) [Cloninger et al., 1993].
This personality factor should be examined in future investigation.

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Compliance of using procedure after the training for the study period was similar between
Johrei and Self-hypnosis, but with regard to collection of the data in the HIIV-patient study,
participants in the Self-hypnosis group attended more sample collection time points. The
appointments for the data-collection sessions in both Self-hypnosis and Johrei groups were
made by the same research assistant (Mr. B. Bennett) who had attended both intervention
sessions. Hence, this may indicate that individuals in the Self-hypnosis group were more
committed to the study or benefited more from contacts with the study personnel (Dr. T.
Laidlaw et al.) than the Johrei participants, remembering that Johrei, for the most part, was
performed with a partner. Nonetheless, an extra caution should be given when the psychological
effect from the result was discussed. One procedure to overcome this bias is to use the method
of the Intention-to-treat analysis as applied in the current study.

Although there remain limitations, these in vivo studies demonstrated that:


Academic examinations can induce stress in university students [3.1.1.1];
NK-cell function appeared to be associated with stress perception in university
students [3.1.2.1];
Psychological intervention may counteract, or at least buffer, exam-stress-induced
loss of NK-cell percentage in university students [3.1.3.1: Figure 26];
Psychological intervention, particularly Johrei, may slow the disease progression in
HIV-infected patients [3.2.3.2: Figure 43]; and
Improved perceived quality-of-life scores are negatively associated with disease
progression in HIV-infected patients [3.2.1.2: Table 30], and Johrei appeared to
improve perceived quality-of-life scores [3.2.2.1: Tables 35-37].

These findings support the hypothesis that psychological intervention may alleviate the
detrimental effects of stress upon well-being; and warrant the need of further investigation on
the psycho-neuro-endocrino-immune network, particularly the influence of Johrei upon the
network.

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4.2 Stress hormone associated changes in vitro in immune cells as an exemplar of


the psycho-neuro-endocrino-immune network interaction

The studies using university students and HIV-infected patients demonstrated that
psychological stress affected in vivo distribution of immune cells including NK-cells and T-cells.
There has been a growing body of evidence which suggests that cortisol (particularly interaction
with the glucocorticoid type II receptor) may play an important role in stress-induced changes
[Bauer et al., 2003] in both lymphocyte distribution [Dhabhar et al., 1996] and impaired cell-
mediated response [Bauer et al., 2001].

Hence, the effects of stress hormones (cortisol in particular) upon the immune cells were
investigated in vitro in order to examine if there are any direct interactions between the stress
hormones and immune cells which could functionally demonstrate a part of the psycho-neuro-
endocrino-immune network.

This approach consisted of two series of experiments: (1) an investigation of NK-cells and
(2) an analysis of T-cells. The functional parameters in cellular immunity [Rose, 2002], which
were reported to decrease under sustained stress, include:
1. Cytotoxic activity of NK-cells and/or cytotoxic T-cells against target cells (tumour
cells and virally infected cells) [Borella et al., 1999; Gruzelier, Smith et al., 2001;
Inoue-Sakurai et al., 2000; Maes et al., 1992; Pike et al., 1997]; and
2. T-lymphocyte proliferative responses to a mitogen, a super-antigen or specific
antigens [Silberman et al., 2002].

Hence, in this project, the effect of in vitro exposure to the stress hormone (cortisol), in
the upper physiological levels and for more than 24 hours, upon the NK cytotoxic activity and
the T-lymphocyte proliferative responses were investigated.

4.2.1 Changes in NK-cells after in vitro incubation

Using peripheral blood mononuclear cells (PBMCs), NK cytotoxic activity (NKCA) level
was investigated by using the Godoy-Ramirez’s flow cytometry method [Godoy-Ramirez et al.,
2000] to obtain a pilot result, and then examined by using the CytoTox96® which measures an
amount of released Lactate dehydrogenase (LDH) from the dead cells. These two different sets

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of results from the two alternative methods of measuring NKCA levels provided a more
comprehensive account.

4.2.1.1 NK cytotoxic activity (NKCA) levels and NK-cell profiles after incubation of PBMCs
for 24 hours in vitro
NKCA levels were shown to increase after incubation of PBMCs for 24 hours and this
increased level was associated with an increased levels of the per-NK-cell cytotoxic activity but
not to an increase in cytotoxic NK-cell subset [Cooper, Fehniger & Caligiuri, 2001] numbers.

To minimise the possibility of activation caused by an interaction between antigen


presentation cells (APC) and NK-cells during the 24 hours in vitro incubation, monocytes were
removed from PBMCs by culturing for 1 hour in a plastic flask. Previously, NKCA level was
reported to be higher in the NK-cell subset adhered to plastic flasks than in the non-adherent
NK-cell subset when incubated with IL-2, and these two NK sub-sets express different
chemokine receptor profiles [Inngjerdingen et al., 2001]. The PBMCs prepared with this
monocyte depletion procedure should have removed the NK-cell subset adhered to plastic flasks
as well as monocytes, but per-NK-cell cytotoxic activity was increased in the current study, so
this suggests that there were other factors which contribute to the increase.

NK-cells are rich in their surface receptors which convey inhibition or activation signals
[Middleton et al., 2002]. The Nkp46 is known as a main activation signal for the cytotoxic
lysing activity of NK-cells, namely the Natural Cytotoxic Receptor (NCR) [Spaggiari et al.,
2001], and the expression of this receptor was shown to increase after the incubation although
the expression of the other NCR, the Nkp30, was not affected. Hence, the increased levels of the
per-NK-cell cytotoxic activity after the incubation could be on account of:
1. Increasing this Nkp46 activation signals; and/or
2. Decreasing effects of inhibitory processes of the activation by other unidentified
factors.

One possible decreasing inhibitory effect is the removal of endogenous humoral


substances (such as stress hormones) in the preparation for in vitro incubation. The plasma
separated from the PBMCs was replaced by a culture medium with a one-tenth, low
concentration of Foetal Calf Serum which contains little or no levels of endogenous cortisol.
The hypothesis that this removal of endogenous cortisol causes the activation in the levels of

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per-NK-cell cytotoxic activity and the expression of the Nkp46 (and Nkp30) was investigated in
the following experiments.

4.2.1.2 Cortisol-induced changes in NKCA levels and NK-cell profiles


Cortisol was shown to inhibit the increase of the per-NK-cell cytotoxic activity after 24hrs
incubation of PBMCs and this inhibition of the increase was shown to be associated with an
inhibition of the expression of the Nkp46 (and also with the inhibition of the Nkp30) on the
cytotoxic NK-cell subsets. However, these inhibitions of the increase in the per-NK-cell
cytotoxic activity were not fully achieved in terms of their amplitude of NKCA and Nkp46
expression. This again implies that there may be other contributing factors.

Recently, in vivo studies using mice (c.f. glucocorticoid is the equivalent hormone in mice
to cortisol in human) have shown that glucocorticoid resistance can be induced by social
disruption stress (i.e. repetitive disruptions by inserting intruders into a cage to stress mice)
[Avitsur et al., 2001; Quan et al., 2001]. This glucocorticoid resistance was reported to be
associated with the reduced function in the glucocorticoid receptor in the CD11b positive
macrophages (i.e. one of major antigen presentation cells; CD11b is part of the cells’
intracellular adhesion molecules which are expressed upon activation) in association with their
impaired nuclear translocation of glucocorticoid receptor and the lack of transcriptional
suppression of NF-kB by glucocorticoid when NK-cells interact with the macrophages [Quan et
al., 2003]. It is therefore suggested that monocyte depletion cause a removal of the CD11b
positive macrophages resulting in a decrease in the inhibitory processes of the activation.

It is well-known that pharmacological levels of cortisol affect genomic manifestations in


NK-cells, for example, the levels of the Granzyme A (a major cytotoxic molecule contained in
the cytoplasm of NK-cells) have been reported to be reduced when NK-cells were exposed to
three M of cortisol [Zhou et al., 1997]. However, there is also a growing body of evidence
which has demonstrated that there are also non-genomic cortisol effects such as the direct non-
specific non-genomic steroid action through high-affinity membrane-binding sites, i.e. cortisol
can affect the activity of NF-kB via steroid-membrane interactions without involvement of the
genomic cortisol receptor in the nucleus [Falkenstein et al., 2000]. Although these were based
on the observations when cortisol was given in pharmacological dose, there remains a
possibility that these genomic and non-genomic mechanisms of cortisol may also be involved in
the effect caused by the upper levels of physiological dosage of cortisol shown in the current
study.

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These possible contributing factors remain to be elucidated in the future investigation, but
this series of experiments concluded that upper physiological level of cortisol can suppress per-
NK-cell cytotoxic activity and Nkp46 expression.

4.2.1.3 Endogenous levels of stress hormones (cortisol and DHEA-S) and NKCA
The other proposed stress-associated hormones, i.e. DHEA-S and melatonin, are known to
be antagonistic against the suppressive effects of cortisol upon lymphocytes as described in the
introduction [1.2.2.3 and 1.2.4.2], but the results demonstrated that there was no significant
relationship between endogenous levels of stress-associated hormones and the NKCA levels
before the incubation of PBMCs. However, there appeared to be positive correlations between
endogenous levels of stress-associated hormones (DHEA-S and melatonin) and the NKCA
levels after 24hrs incubation free from endogenous hormones.

This finding was hypothesised to be associated with the long term effects of the DHEA-S
and melatonin upon NK-cells in balance with a suppressive effect of cortisol. In other words,
the difference between these NKCA levels may be associated with the stage of alarm to
resistance and the stage of exhaustion in Selye’s classification [1.2.4.1]:
NK-cells with high NKCA level after 24 hours incubation (with high levels of
DHEA-S and melatonin) may be mixture of NK-cells in a stage of alarm reaction
and resistance where cells are able to respond with both activation and inhibition
from the various factors including hormones, so they can be activated when various
inhibitory factors (including endogenous cortisol) were removed; and
NK-cells with low NKCA level after 24 hours incubation (with low levels of
DHEA-S and melatonin) may be NK-cells in an exhausted stage, so they can not
respond.
This hypothesis can be summarised and paraphrased by stating that the suppressive
effects of cortisol upon NK-cells is counteracted by:
DHEA-S: another major adrenocortical hormone in the HPA axis; and
Melatonin: a major hormone associated with sleep in the circadian rhythm.

In order to test this hypothesis, the diurnal pattern in the levels of these hormones together
with cortisol and diurnal changes in the levels of NKCA both at Time 0 and 24 hours after in
vitro incubation would need to be examined, and then the association between these

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combinations would need to be analysed in future in vitro studies investigating the effect of
stress-associated hormones upon NK-cells.

Nonetheless, the results support the direct interaction between stress hormones and NK-
cells as a part of the psycho-neuro-endocrino-immune network.

4.2.2 Changes in T-cells after in vitro incubation

One of major functions of T-cells in self-defence is the ability to proliferate and generate
appropriate cell phenotypes which have specific affinity against a specific antigen. One method
used to measure this ability has been the [3H]-thymidine incorporation, proliferative T-cell
response assay [Weyts et al., 1997] based on the findings that in vitro stimulation with a
mitogen, super-antigen or specific antigens activates T-cells resulting in proliferation [Antia et
al., 2003; Kaech & Ahmed, 2003; Seder & Ahmed, 2003].

It is well-known that pharmacological levels of cortisol (or glucocorticoids) can suppress


the proliferative responses of human cells [Hettmannsperger et al., 1993]. In the current study,
the upper physiological level of cortisol (250nM) was also shown to suppress T-cells’
proliferative responses against mitogen, super antigen and recall antigens, and expression of
their cell-surface markers were measured concurrently in order to investigate the possible sites
of the suppressive effects of cortisol.

4.2.2.1 Expression of CD25 during proliferative responses


The proliferative response in T-lymphocytes consists of four processes: (1) recognition of
a stimulus; (2) activating cell cycle progression; (3) process of actual cell division; and (4)
apoptosis of excessive cells. Within this process, the following two possibilities for suppressive
effect of cortisol on the proliferative responses were investigated in the current study:
1. Decreasing activation, and
2. Increasing apoptosis.

Flow cytometry technique has been introduced to examine the mechanisms of


proliferative responses using some major cell surface activation markers, i.e. CD69, CD71 and
HLA-DR, but it was shown that the expression of these markers can not be alternatives to

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reflect the levels of proliferation in comparison with the method using the [3H]-thymidine
incorporation [Caruso et al., 1997; Hutchinson et al., 1999; Rutella et al., 1999].

Interleukin-2 (IL2) is known as a major cytokine to induce T-cell proliferation by binding


with the IL-2 receptor, i.e. CD25, and this IL2-CD25 interaction is also known to be essential
for T-cells to proliferate with other cytokine stimuli [Malek et al., 2001]. Hence, one of the
above hypotheses of suppressive effects of cortisol upon proliferative responses, that cortisol
decreases activation process of T-lymphocytes, was tested by examining the levels of CD25
expression on the cell surface.

The expression of CD25 in the current study showed a dramatic increase after 48 hours of
incubation with PHA or PPD, but this increased rate was again not decreased by the
physiological level of cortisol. Hence, although the possibility of impairment in other activation
markers on the cell surface, such as HLA-DR, is still to be examined in future experiments, it
appears that there may be another unidentified mechanism that has a greater impact on the
suppressive effect of cortisol in proliferative responses.

4.2.2.2 Expression of CD95 and Annexin V during proliferative response


Previously in the literature, it was reported that altering the level of cortisol in carp
lymphocytes was associated with an increase in apoptosis during the proliferation response to a
mitogen (PHA) and a super-antigen such as lipopolysaccharide (LPS) [Weyts et al., 1997]. In
their study, the rate of apoptosis was measured by the method using the TdT-mediated dUTP
nick end labelling (TUNEL) assay, so the actual apoptotic process in a nucleus was detected and
the amount of apoptotic changes was measured.

Apoptosis is known to start from either the cell-surface mechanism (e.g. a cascade
induced by CD95/Fas and CD95L/Fas-ligand contacts) or the cytoplasmic mechanism (e.g.
mitochondria releases the cytochrome C; a genomic cascade [Bedner et al., 1999] mediated by
Bax complexes within a nucleus). For the purpose of detecting apoptotic change on the cell
surface, a method using Annexin V which can bind the phosphatidylserine has been developed
[van Engeland et al., 1998]. Phosphatidylserine is one component of the cell-membrane and it is
located inside the membrane in live cells; and it comes outside of the membrane when the cell
dies and looses this tight control.

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In the current study, sharp increases in the levels of the CD95 (Fas) expression on the
surface of CD4 and CD8 T-cells were observed after 24 and 48 hours in vitro incubation with
PHA or PPD, but the physiological level of cortisol did not influence these increases. Further,
the physiological level of cortisol did not affect the levels of Annexin V on the cell surface after
48 hours incubation. Hence, it was concluded that cortisol did not alter apoptotic cell surface
changes on proliferating T-lymphocytes. In order to confirm the apoptotic change by the
physiological level of cortisol further, the TUNEL assay needs to be performed.

On the other hand, in the nucleus, cortisol has been shown to inhibit the NF- B activation
[Barnes, 1997] and to induce an early G1 phase block in cell-cycle (G1-S-G2-M) progression in
a tumour cell line [Sanchez et al., 1993] resulting in delay of the cell division. The process of
actual cell division can be measured by the method using carboxyfluorescein succinimidyl ester
(CFSE) [Lyons, 2000], in which the level of intensity of the CFSE can be measured in the
stained cells of the cytoplasm and this level halves with each cell division. Hence, future
investigation on the effect of cortisol upon T-lymphocytes should include this cell-cycle
progression analysis in addition to the further analyses of apoptotic changes.

Nevertheless, the physiological level of cortisol was shown to suppress human T-cells
proliferative responses. This may or may not increase apoptosis, but this possible increased
apoptotic change by cortisol was not detected through the change on the cell surface.

These in vitro experiments demonstrate the feasibility of the endocrino-immune network.


The in vitro model of stress was shown to suppress:
NK-cell function, i.e. per-NK-cell cytotoxic activity to tumor cells; and
T-cell function, i.e. proliferative response to substances related to infection.
In addition, DHEA-S (in adrenocortical hormones) and melatonin (in hormones
associated with circadian regulation) may have antagonistic associations against cortisol as
shown by their effects on NK-cell function.

These support that there is a direct relationship between hormones (cortisol, in particular,
and DHEA-S and melatonin) and immune cells (NK-cells and T-lymphocytes) and demonstrate
possible interaction within the psycho-neuro-endocrino-immune network.

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

4.3 Conclusions

The in vivo study using university students confirmed that exam preparations provoke
anxiety and increase stress in students. The study showed that stress perception was associated
with NK cytotoxic activity levels; and it demonstrated that psychological intervention may
buffer and/or reverse detrimental effects of stress upon the immune cells, particularly the effects
on NK-cells. The in vivo study using HIV-infected individuals confirmed that there is a steady
decline in CD4 T-cell counts in the patients who are not receiving anti-retroviral treatment; and
it demonstrated that psychological intervention may decrease this decline. These effects were
particularly associated with the training and practice of Johrei.

In addition, the decline in CD4 T-cell counts in HIV-infected patients was found to be
positively associated with declines in the perceived quality-of-life scores. Although a larger
sample size would be needed for statistical significance, Johrei appeared to improve these
quality-of-life scores. These raise a possibility that improvement of the meaning-focused coping
skills may have beneficial effects upon disease progression more than the emotion- and/or
problem-focused coping strategies.

In vitro exposure to the stress hormone, cortisol, suppressed NK-cell and T-lymphocyte
functions. Other stress-associated hormones (DHEA-S and melatonin) appeared to be
antagonistic against the effect of cortisol on NK-cell function. These showed direct interactions
of stress hormones on cells of the immune system, and demonstrate a mechanism whereby
stress could act upon the psycho-neuro-endocrino-immune network to regulate immune function.

These findings support the hypothesis that psychological intervention may counteract the
detrimental effects of stress upon psychological well-being and general health acting through an
integrated psycho-neuro-endocrino-immune network; and clearly warrant the need for further
investigation of interactions in the network, particularly the influence of Johrei upon the
network.

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

4.4 Future directions

In order to investigate the psycho-neuro-endocrino-immune network interactions further,


several suggestions were raised in the preceding report. This section lists those suggestions and
proposes future work.

4.4.1 Psychological interventions

Intervention-free controls as well as relaxation control group for students study


A larger sample size (e.g. more than 84 subjects per group for HIV-patients study)

4.4.2 Measurements

4.4.2.1 Psychological measures


Comparison analysis between two subgroups based on:
1. The midline split of the stress perception scales for the stress-related differences:
The Perceived Stress Scale (PSS) [Cohen et al., 1983]
The Kesseler Perceived Social Support (KPSS) [Coventry et al., 2004]

2. The criterion whether the quality-of-life scores improving or worsening for the
change in disease progression or maintenance of health:
The Sense of Coherence (SoC) [Antonovsky, 1987]
The Spiritual Involvement and Beliefs Scale (SIBS) [Hatch et al., 1998]
The Pittsburgh Sleep Quality Index (PSQI) [Buysse et al., 1989]

3. A covariate factor: personality trait


The Temperament and Character Inventory (TCI) [Cloninger et al., 1993]

4.4.2.2 Immunological measures in vivo


NK-cells: Per-NK-cell cytotoxic activity, NK-cell subset counts and percentages for
individuals with acute or sustained, but limited, stress

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CD4 T-cells: Subset (Regulatory T-cell in particular) counts and percentages, functional
measures e.g. Th.1 and Th.2 cytokine productions for both healthy
individuals and patients with sustained stress

4.4.2.3 In vitro investigation on NK-cells


CD11b positive macrophages analysis concurrently with per-NK-cell cytotoxic
activity
NF-kB analysis with cortisol receptor (GCR) analysis
Diurnal pattern in the endogenous stress hormone levels concurrently with the NKCA
levels both at Time 0 and 24 hours after in vitro incubation

4.4.2.4 In vitro investigation on T-lymphocytes


Cell-cycle progression analysis (e.g. CFSE analysis)
TUNEL assay for proliferative T-lymphocytes

4.4.3 Proposal for future in vivo study

Ethical approval and funding have been granted for a further study into the effects of
Johrei in HIV patients. The proposal aims to investigate the integrated psycho-neuro-endocrino-
immune network interactions; and to confirm and explore the major results from the Ph.D.
project that Johrei may have beneficial effect upon disease progression, (measured in CD4 T-
cell counts), in HIV-infected individuals not receiving anti-retroviral treatment [Appendix 7].

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Appendix 1: Ethics approval, information sheet and consent form
RIVERSIDE RESEARCH ETHICS COMMITTEE
APPLICATION FORM
If you require more space to answer a
question then please adjust the sizing Version 2003.01
of the answer space accordingly.
Name of Lead Investigator: Prof. John H Gruzelier
Department: Cognitive Neuroscience and Behaviour
Address: Charing Cross Campus, Imperial College London, St Dunstan's Road,
London, W6 8RF
[1] Please state project title.
The Effects of Two Psychological Intervention Techniques, Self-Hypnosis and the
Johrei Healing Method, on Quality of Life, Psychological Well-Being, EEG
measures and Various Immunological Measures including CD4+ counts in early
HIV: A Randomly Controlled Pilot Study.

[2] What question is the project seeking to answer, and what will be the value of
obtaining this answer? This should also be clearly explained within the
patient information sheet.
When patients are newly diagnosed with HIV, there is often a substantial period of time when
their CD4+ counts are preserved, and antiretroviral treatment (ART) is not required. Patients are
healthy in all other respects, and jobs and home life carries on. Over time, CD4+ counts fall and
ultimately most patients start to take ART. Our primary question is whether we can prolong this
state.

This project seeks to investigate the possible impact upon Quality of Life, psychological well-
being, various immune parameters and health status of two interventions, self-hypnosis and Johrei
(a Japanese healing method). A third group will be put on a waiting list and act as ‘wait-listed'
controls for the first 4 months of the study. Subjects will be randomly assigned after
stratification on CD4+ levels to one of these three groups.

The purposes of Johrei and self-hypnosis are similar. Each technique will be taught to participants
randomly assigned to the specific intervention with the purposes of aiding in psychological
adjustment to their condition, maintaining their quality of life and bolstering their immunological
defence system through daily practice. In addition, cognitive assessments will be undertaken
using electrophysiological (EEG) measures.

Should participants maintain this state where their CD4+ counts remain high and pharmacological
treatment is still deemed unnecessary, or that their quality of life and psychological well-being is
significantly better than those without the interventions, either of these two interventions could be
easily added to treatment protocols in early HIV.

These two interventions are in addition to best conventional medical care given by the Chelsea &
Westminster Healthcare NHS Trust, and will be administered with the participation of clinicians
involved in the patients’ care.

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[3] Please provide an outline of the project, this should include procedures to be
used, measures to be made, justification of sample size & plans for statistical
analysis. [Paras2.1-2.8]
The two interventions are self-hypnosis and the Johrei healing method from Japan.

Hypnosis is a well-recognised medically accepted psychological intervention. It has been used in


many studies with medical conditions and our laboratory has carried out a series of interventionist
hypnosis studies (Fox et al, 1999, Gruzelier et al, 2001, Laidlaw et al., 2003). It has been used in
symptom control in various medical conditions, including pain control, and in normalising the
immune system when under stress (Gruzelier et al., 2001). It has provided a buffer in the face of
stress (Gruzelier et al, 2001, Laidlaw et al., 2003). In the present study, hypnosis will be taught as
a skill so it can be practised in the form of self-hypnosis in the privacy of the participants' own
homes. On-going support and supervision will be provided throughout the study period. Self-
hypnosis will be taught by an expert in medical hypnosis.
In Japan and many other countries, some 3 million people practise the Johrei lifestyle and healing
method. It is primarily practised in the family setting and involves a healthy life-style and
appreciation of beauty. In the first published Johrei study, medical students illustrated that
practising Johrei provided help in coping with exam stress. An introduction to Johrei will be
taught to participants, and supervision for participant and family member or another support
person will be provided throughout the study period, as in the Hypnosis condition. Johrei will be
taught by a medical doctor who has been trained in Johrei and practised it since childhood.

The four areas of outcome measures are immunological variables, psychological variables,
cognitive variables and, for the term of the study, markers of progression of disease, assessed pre-
intervention and at the finish of the study. At the beginning of the study, all participants will
provide descriptive, which will include demographic details and medical histories.

Study design
This randomised controlled trial will include 150 patients who have been diagnosed with HIV but
are naïve-to-treatment, meaning that their CD4+ counts are sufficiently high that ART is not
indicated. Realistically, this means that the CD4+ count is < 400 but above 200 (irrespective of viral
load). The study duration for each participant is approximately 5 months. The planned monthly
intake is of 6 – 9 patients to each of the three groups (i.e. 18 - 27 new people each month).
The three groups will be recruited through Dr Simon Barton and his clinical colleagues at Chelsea
& Westminster Healthcare NHS Trust. An independent statistician will randomly assign
participants to either one of the two intervention groups or the wait-listed control group using a
stratified randomisation on CD4+ counts.
Outcome measures will include the psychological questionnaires, blood and urine samples for
immune assays, and cognitive data detailed below and illustrated in the diagram following.
Basically, the design involves 8 sessions with three major assessment points over, at the most, six
months. The first four sessions in the first month are intervention intensive, followed by 4
monthly follow-up sessions. Each training session will take about 2 hours and is planned to be
held after working hours at the Charing Cross Campus site. Participants will be recruited
monthly to be in training groups of 6 – 9. The three assessment points include all the
immunological and psychological tests described below and designated as outcome measures.

INTERVENTION GROUPS SCHEDULE

1 2 3 4 5 6 7 Intervention sessions (first 4 are weekly, last 3 monthly)

1 &2 3 Main assessment points


End of Month: 1 2 3 4 Time

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

WAIT-LISTED CONTROL SCHEDULE:


0 1 2 3 4 5 6 7 Intervention sessions

1 &2 3 4 Main assessment points


End of Month: 1 2 3 4 5 6 7 8 Time

Assessment variables:

Psychological variables: Series 1 (those not needing repetition)


Hypnotisability and imaginative abilities: Creative Imagination Scale (Wilson & Barber, 1978),
and Stanford Hypnotic Susceptibility Scale (Form-C) (Weisenhoffer & Hilgard, 1962). It is
hypothesised that hypnotisability or ability to visualise will influence the outcome variables
(Laidlaw et al., 1996)

Personality: To study personality types and coping styles in relation to prognosis. (Watson et al.,
1984). Assessed with the Marlowe-Crowne Social Desirability Scale (Crowne & Marlowe, 1960)
and the Courtold: (Barger, Kircher & Croyle, 1997). Trait anxiety from Spielberger's STAI
(Spielberger, Gorsuch & Lushene, 1970), and Personality Syndrome Questionnaire (Gruzelier,
2000)

Social Support: Social support has been linked to mental health of patients. It can be easily
assessed with the 10-min Significant Others Scale (Powers, Campion & Aris, 1988). Further to
this is a scale to assess spirituality (Hatch et al., 1998).

Psychological variables: Series 2 (dependent variables)


Depression and anxiety: Beck Depression Inventory (BDI, Beck, 1990) arguably the most well-
used instrument to assess depression in clinical studies, as is the State Trait Anxiety Inventory
(STAI, Spielberger, Gorsuch & Lushene, 1970) for anxiety.

PTSD- like symptoms: Impact of Event Scale (IES, Horowitz, Wilner, & Alvarez, 1979) used
with HIV diagnosis as event in Nordin, Bergland, Glimelius & Sjoden, 2001. Can identify
intrusive thoughts (associated with high cortisol which can have negative effects on immuno-
regulation). Perceived Stress Scale (PSS).

Quality of life and activity levels: WHOQOL (World Health Organisation, 1996) internationally
used screen for quality of life changes and the SF36 (Jenkinson et al.,1994), a well-used
generalised instrument for quality of life.

Coping : The COPE, a multidimensional coping inventory (Carver, Scheier & Weintraub, 1989)
includes information about turning to spiritual sources, denial and disengagement among other
sub-scales, which are of interest in this study. Locus of Control.

Psychological variables: Series 3 (continuous data)


Diary data: Personalised Emotional Index (PEI, Laidlaw, 1999). Used with patients (Laidlaw,
2001). Mood parameters both positive and negative: anxious, energetic, tired, confident, unsure,
elated, depressed, agreeable, hostile, clear-headed and ineffective rated on a Likert scale as a 5-
min daily diary. Additional questions include sleep quality, homework practice, health status, as
moderator variables that could influence outcome measures. External factors: space for qualitative
comments after the prompt: 'Anything different happen today?' rated by researchers on a -3 to +3
scale.

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Cognitive variables
Neuropsychophysiological studies would include electroencephalographic (EEG), and
electrodermal activity (EDA). These studies will address the following issues:

Lateralised brain function and its relationship with immune status, mood, coping style, and
response to intervention in patients: We hypothesise a relationship among these variables based
on earlier work with HIV infection (Gruzelier et al., 1996) which showed that hemispheric
asymmetry measures predicted immune competence and were correlated with mood status (also
see Davidson and Tomarken, 1989; Kang et al.,1991; Wheeler et al.,1993; Lane et
al.,1997;.Papousek and Schulter, 2001; Gruzelier et al., 2001)

Immunological markers:
- CD profile including numbers and percentages of CD3, CD4, CD45RA/RO, CD8, CD16 and
CD56 (Van der Pompe et al. 1997). & CD4 absolute count, CD4/CD8 ratio and HIV viral
load as a marker of disease progression.
- NK cell activity as markers of anti-infection in innate immunity (Evans et al. 2000)
- Cortisol (urine) as a marker of chronic stress and stimulator of Th2 (Dhabhar et al. 2001,
Evans et al. 2000, Schedlowski et al.1994)
- DHEA (urine) as a marker of Th1/Th2 balance with Cortisol (Evans et al. 2000)
- Melatonin (urine) correlating with sleep quality and might be a immune stimulating (Th1
predominantly) hormone (Maestroni 2001)
- MAO-AI (as a marker of catecholamine secretion), INF-gamma, IL-2, Soluble IL-2 receptor
(as a Th1 dominant cytokine), IL-4, IL-10 (as a Th2 dominant cytokine) (serum), IL-6, CRP
(as a marker of inflammation).

SAMPLE SIZE CALCULATION: The between group comparisons will take place using
Johrei, hypnosis and control group data from groups sizes of 50 participant each. The within
group data can include those participants who have been in the control groups prior to being
randomly assigned to either Johrei or self-hypnosis. This will provide up to 75 participants in
each of the two intervention groups. These numbers come from a power analysis using

STATISTICAL ANALYSIS: Statistics will be calculated using SPSS v 11.


Multivariate analyses will be used in the between-group comparisons, and repeated measures and
correlational analyses for the within-group changes.
[4] Please specify the source and type of subjects to be recruited and how this will
be done. Also state the expected numbers to be recruited.
150 patient volunteers will be recruited on a continuous basis from the clinics where they receive
their diagnosis under the supervision of investigator and clinician Dr Simon Barton.
Recruitment will be via a Research Nurse or by one of the investigators.

Inclusion criteria include:


1. HIV positive
2. Age 18 – 70
3. CD4+ counts > 200.
Exclusion criteria include:
1. On ART.
2. Abusing drugs or alcohol.
3. In another trial which would conflict.
[5] What will be the duration of the project and where will it be undertaken?
The project will take place at the Department of Cognitive Neuroscience & Behaviour, 10th floor
of the Lab block, Charing Cross Campus of Imperial College London. It will commence when
Ethics approval is gained, and continue until 150 patient volunteers have completed their training.
It is estimated that this will take approximately 18 months.

[6] Within this project, what treatment and procedures will be performed on subjects
that are extra to normal patient care? These details should be clearly
stated in the patient information form.

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

There are no specific treatment strategies for HIV positive patients at this stage of their disease.
Normal care includes the monitoring of their CD4+ levels. ART will commence when
appropriate. In addition to this monitoring, patients will be offered a chance of participating in
this project, which offers training in one of self-hypnosis, or Johrei as described above.
[7] Please state the potential hazards to subjects, if any. What is the probability of
these occurring? What precautions will be taken to meet them? This
information should be clearly stated in the patient information form.
No potential hazards.
[8] What procedures, if any, may cause discomfort or distress to subjects? What
degree of discomfort or distress may this entail? What is the probability of
such discomfort or distress occurring? This information should be clearly
stated in the patient information form.
Anecdotally, initial sessions of Johrei occasionally have been associated with minor symptoms
of headache or congestion. This was not observed in either of the previous Johrei studies done at
Imperial College.
[9] Please state the personal experience of the investigator in the field concerned.
Prof Gruzelier has extensive research experience in the field of HIV (Baldeweg, Catalan et al,
1995, Baldeweg, Riccio et al, 1995, Gruzelier et al., 1996, Baldeweg et al, 1997). He has
published many papers about hypnosis including its use as an intervention agent (Gruzelier et al.,
2001; Fox et al, 1999), as has Dr Laidlaw (Laidlaw & Willett, 2002; Laidlaw, 1999). His
research team has conducted two previous studies using similar designs to the present study
involving both Johrei and self-hypnosis (one completed RR 2883, Laidlaw et al, 2003 and one
ongoing, RR2959.).

[10] Written consent should be standard practice, please enclose copies of the
consent form and information sheet. If non-written consent is proposed then
this must be justified to the Committee. [Paras 3.1-3.3]
If a routine clinical procedure precedes or follows what is proposed to be done
by way of this application, the Information and Consent Forms for those
procedures should be also included with Patient Information and Consent
forms for this application.
n/a
[11a] Have the consultants responsible for the overall care of the patient been
informed and their approval given?
Dr Simon Barton is the consultant responsible and is a current investigator in this
application.
[11b] Have you gone through the appropriate process within the Clinical Directorate
in which you wish to undertake your research?
Yes and received approval from the research committee, November, 2002.
[11c] Will the subjects' General Practitioner be informed of the recruitment of the
subject before the study begins? Is the subjects consent to this information
being passed as a condition of participation?

[12] Please state any interest, i.e., of profit, personal or departmental, financial or
otherwise, relating to the study. [Paras 7.1-7.2]
None.

[13] Does the study involve the administration or application of a medicinal product
or substance? [Para 5.1-5.3]
Yes No

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[15] Please list all staff involved with this project - all staff must sign this
section:

Name & Title Signature Qualification(s) Involvement

Prof JH PhD Principle investigator


Gruzelier

Dr TM PhD Overall supervision of data


Laidlaw collection and patient interaction
with the study.

Training in self-hypnosis

Dr A Naito MD and PhD Training in Johrei and


candidate immunological lab work.

Dr P PhD EEG data collection and analysis.


Dwivedi

Dr D PhD Supervision of immunological lab


Henderson work

Dr Simon MD Clinician responsible for patient


Barton treatment.

Research to be appointed Data input, recruitment, lab


Assistant assistance.

Date of submission: Signature of Investigator:


1 February 2003
Name: Address: Dept of Cognitive Contact Tel & Bleep
Prof JH Gruzelier Neuroscience & Behaviour, Charing No:
Cross Campus, Imperial College 0208 846 7386
London, St Dunstan's Road, London,
W6 8RF
Clinical Team in which Investigator works: n/a
Other Clinical Teams/Dept involved in research: Chelsea & Westminster Hospital
Name and Signature of Consultant, GP or Community Physician in overall charge. (You are
reminded that by signing this form you take responsibility for the contents and
accuracy of this Ethics Committee Application. You must be medically qualified):
Dr Simon Barton

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Dr TM Laidlaw
Department of Cognitive
Neuroscience & Behaviour
Charing Cross Hospital
St Dunstan’s Road
LONDON W6 8RP

Participant Information Sheet


Study Title

The Effects of Two Psychological Intervention Techniques, Self-Hypnosis


and the Johrei Healing Method, on Quality of Life, Psychological Well-Being,
EEG measures and Various Immunological Measures in HIV: A Randomly
Controlled Pilot Study (or ‘The Self-hypnosis and Johrei Study’)

Invitation paragraph

You are being invited to take part in a research study. Before you decide, it is important for
you to understand why the research is being done and what it will involve. Please take time to
read the following information carefully and discuss it with friends, relatives and your GP if
you wish. Ask us if there is anything that is not clear or if you would like more information.
Take time to decide whether or not you wish to take part.
Consumers for Ethics in Research (CERES) publish a leaflet entitled ‘Medical Research and
You’. This leaflet gives more information about medical research and looks at some
questions you may want to ask. A copy may be obtained from CERES, PO Box 1365,
London N16 0BW.

What is the purpose of the study?

We would like to invite you to be a participant in a 5-month long study that is intended to
find out whether the addition of self-hypnosis or a healing method called Johrei to the regular
treatment you receive at Chelsea and Westminster Hospital will help clinically or in your
quality of life.

Why have I been chosen?

We would like to examine the efficacy of psychological interventions on the quality of life in
HIV patients, so you have been invited to participate in the project. It is up to you to decide
whether or not to take part. If you do decide to take part you will be given this information
sheet to keep and be asked to sign a consent form. If you decide to take part you are still free
to withdraw at any time and without giving a reason. Refusal to participate or subsequent
withdrawal will not affect the standard of care you receive.

What will happen to me if I take part?

This research project is designed as a randomised trial.

Sometimes because we do not know which way of treating patients is best, we need to make
comparisons. In a randomised trial, people will be put into groups and then compared. The
groups are selected by a computer that has no information about the individual – i.e. by
chance. Patients in each group then have a different treatment and these are compared.

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

In this research project, you will be randomly assigned to one of three groups: self-hypnosis
or Johrei which is a non-touch healing method from Japan and a waiting list before you are
assigned to either self-hypnosis or Johrei. Please read the descriptions for all three groups.

One-third of the people in the study will be put onto the waiting list to have their training in 4
months time, during which, if you are in this group, you will be asked to complete some
psychological tests. This delay for some participants provides a comparison for the effects of
training over the 4 months, a necessary requirement in a scientific study of this type. The
remainder (2/3rds of people in the study) will have their training more-or-less immediately.
You must feel all right about participating in either the immediate or delayed condition, as
you could be randomly assigned to either.

Meeting schedule:
Sessions 1 to 4 are held weekly (2 hours each). Sessions 5 to 14 are held once a month (1
hour each).

INTERVENTION GROUP’S SCHEDULE

1 2 3 4 5 6 7 Intervention sessions (first 4 are weekly, last 3 monthly)

1 &2 3 Main assessment points


End of Month: 1 2 3 4 Time

WAIT-LISTED CONTROL SCHEDULE:


0 1 2 3 4 5 6 7 Intervention sessions

1 &2 3 4 Main assessment points


End of Month: 1 2 3 4 5 6 7 8 Time

If you are in the wait-listed group, you will have your first EEG and testing session, and after
4 months you will enter into the sequence as detailed above.

The main collections of data will be composed of paper-and-pencil test results about how you
are feeling and coping, blood samples to monitor your stress levels and a brain wave study.
All participants will be asked to give blood samples (20 mls) and fill out questionnaires both
at the beginning of the project, at three months and follow-up (at 6 months) for further
hormone and immune analysis. The measurements will include for some of you an EEG
(electroencephalograph) which records the electrical activity of one’s brain. This is a
completely non-invasive procedure, which involves the attachment of various electrodes to
your head using a cap and some gel. Please note that the electrodes are placed on the surface
of the skin only and there is no danger of electric shock.

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Following attachment of the electrodes, you will be seated in front of a computer monitor and
instructed to do certain tasks such as remembering some words and faces presented on the
computer screen. There will be three EEG recording sessions spread throughout a whole
period of the study each lasting about two hours. All details and data resulting from the study
will remain strictly confidential.

The two sets of training are in Self-Hypnosis and Johrei :

Self-Hypnosis: Hypnosis is an altered state of consciousness that can feel like deep relaxation
during which a person can use their imagination in a powerful way. Several research
studies have shown that many, although not all, people can control their pain and affect
their emotions, and even alter their immune systems, determined by how they use the
hypnotic state. One recent study by Dr Michael Antoni of the University of Miami
showed that patients with HIV had, on average, a better quality of life, and indeed, many
positively affected their immune system with a group programme including self-hypnosis,
similar to the one we plan for those of you randomly assigned to this arm of the study.
We think that such a programme should be properly tested here in London. If you are
assigned to the hypnosis condition, you will first experience hypnosis, then learn how to
use self-hypnosis. Various possible goals will be discussed and you will be taught how
to use self-hypnosis for the goals you choose. You will be asked to practice your self-
hypnosis daily during the project.

Johrei: Johrei is a Japanese healing method. Anyone can learn to do Johrei after
understanding the core principles and basic techniques. Healing methods have many
variations and Johrei is based on the concept of ‘healing oneself by healing others’ using
a non-touching method. Johrei has just begun to be scientifically tested with some
encouraging results. Millions of people around the world use Johrei to improve their
health, including people with HIV. During your time in the project, you will learn these
techniques. Johrei is said to relieve symptoms, even though some people for a day or so
as they learn the techniques can experience brief, minor discomfort such as mild
symptoms of headache, sweating or diarrhoea, although we have seen none of this here in
our previous studies. During the project, you will be asked to practise Johrei every day
with someone (partner, daughter, friend etc.).

If you agree to participate, you will be one of 150 people like yourself with diagnosed HIV
who will be invited to participate in the project. You, along with the others, will complete
various tests, and will have your EEG (brain-waves) measured. You will have these tests at
the beginning, and again at four - five months for a repeat set of tests. However, you will
meet regularly in a group of 6 - 9 other study subjects if you are assigned to learn self-
hypnosis or Johrei. In self-hypnosis, the group co-ordinator will be Dr Tannis Laidlaw, a
psychologist with extensive experience in clinical hypnosis. If you are in the Johrei group,
you will be trained by Dr Akira Naito, a doctor trained in both western medicine and the
Johrei healing method. If you are in the waiting list group, you will be randomly assigned to
either Johrei or self-hypnosis in 3 months time when you will join with others in the training.
All the brain wave studies will be with Dr Prabuddh Dwivedi, who is a clinical psychologist
with specialised knowledge of conducting EEG studies. The Self-hypnosis, Johrei and
EEGs will be performed at the Charing Cross Hospital.

Self-hypnosis or Johrei schedule:


If you are in either of the self-hypnosis or Johrei groups, you will meet together with the
other participants and your group co-ordinator one session a week for four weeks (1 to 4 on

Ph.D. at University of London - 222 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

the diagram above). You will learn self-hypnosis or Johrei in order to begin practice at home.
A small daily diary will be created specifically for you, to be filled out at the end of each day
(taking about 2 minutes) and kept for the duration of the project.

After this month of weekly meetings, the shorter meetings will be spaced at one meeting a
month for the duration of the project, and you are welcome to continue past your final
assessment point. Altogether, you will have three testing sessions for EEG and two for
psychological testing, lasting about 2 hours. The testing is for the purpose of monitoring your
progress and your response to self-hypnosis or Johrei. When you attend our department on
the 10th floor of the medical school for your EEG testing you will have plenty of time to fill
out the psychological tests. They are not onerous and there are no right or wrong answers,
although your opinion will be sought about many different aspects of your experience.
Assessment times involving psychological tests and EEG are indicated by the arrows labelled
1,2 and 3.

What do I have to do?

There are no lifestyle restrictions. No special change is required for this specific study in your
lifestyle other than incorporating into your daily schedule some time to practice your self-
hypnosis or Johrei.

What is the drug or procedure that is being tested?

You will not be given any drugs in addition to your usual medications.

What are the alternatives for diagnosis or treatment?

You should feel free to ask your GP about your diagnosis and any alternative treatment for it.

What are the side effects of taking part?

We expect no side effects.

What are the possible benefits of taking part?

We hope that both interventions will help you. However, this cannot be guaranteed. The
information we get from this study may help us to better treat future patients with HIV.

What if new information becomes available?

Sometimes during the course of a research project new information becomes available about
the treatment/drug that is being studied. If this happens, your research doctor will tell you
about it and discuss with you whether you want to continue in the study. You may decide to
withdraw but if you decide to continue in the study you will be asked to sign an updated
consent form.

Alternatively, on receiving new information you research doctor might consider it to be in


your best interests to withdraw you from the study. He/she will explain the reasons and
arrange for your care to continue.

What happens when the research study stops?

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

The intension is that you will be independent in your self-hypnosis and Johrei skills and will
not need further instruction. However, a list of qualified practitioners in London can be
made available to you on completion of the study.

What if something goes wrong?

If taking part in this research project harms you, there are no special compensation
arrangements. If you are harmed due to someone’s negligence, then you may have grounds
for legal action but you may have to pay for it. Regardless of this, if you wish to complain
about any aspect of the way you have been approached or treated during the course of this
study, the normal NHS complaints mechanisms may be available to you.

Will my taking part in this study be kept confidential?

All information that is collected about you during the course of the research will be kept
strictly confidential. All information about you will be kept securely at Imperial College,
Charing Cross Hospital, London, and under personal supervision of the researchers when
being transported from Chelsea & Westminster Hospital to Charing Cross Hospital.

What will happen to the results of the research study?

The results of this study are intended to provide new information about the treatment of
people with newly diagnosed HIV, and will be published as academic papers in professional
journals. It will be the basis of talks at conferences around the world. No names or other
identifying aspects of the participants will be disclosed during any of these communications.

Who is organising and funding the research?

This research is sponsored by the organisation that teaches Johrei in Japan. It is organised by
the Dept. of Cognitive Neuroscience and Behaviour, Imperial College London.

Who has reviewed this study?

The Riverside Research Ethics Committee reviewed the study.

Contact for further information

Dr Tannis Laidlaw can answer any further questions on 0208 846 7042 or email:
t.laidlaw@ic.ac.uk

Thank you for considering taking part in our research project. Please take this information
sheet home with you.

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Dr TM Laidlaw
Department of Cognitive
Neuroscience & Behaviour
Faculty of Medicine
Imperial College of Science,
Technology and Medicine
St Dunstan’s Road
PARTICIPANT CONSENT FORM LONDON W6 8RP

Title of Project:

The Effects of Two Psychological Intervention Techniques, Self-Hypnosis


and the Johrei Healing Method, on Quality of Life, Psychological Well-Being,
EEG measures and Various Immunological Measures in HIV: A Randomly
Controlled Pilot Study.

(The patient/volunteer should complete the whole of this sheet him/herself)

Have you read the Information Sheet? Yes No

Have you had the opportunity to ask questions and discuss the study? Yes No

Have you received satisfactory answers to all of your questions? Yes No

Have you received enough information about the study? Yes No

Whom have you spoken to? (write name) __________________________________

Do you understand that you are free to withdraw from the study,
at any time, without having to give a reason, and without affecting
the quality of your present or future medical care? Yes No

Do you agree to take part in this study? Yes No

I understand that the Local Ethics Committee may review this form as part of a
monitoring process.

NAME IN BLOCK LETTERS:


___________________________________________

Contacts: Address: _______________________________________________


Telephone number(s): _______________________________________________
Email: _______________________________________________

Signature: __________________________________Date: ____ / _____ / _______

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Appendix 2: Psychological training intervention protocols

1. Self hypnosis training

Session plans 227

Instructor manual (with scrip of self-hypnosis for audio-recording) 230

2. Johrei training

Course description 253

One-hour Introductory Seminar Powerpoint Presentation (with notes:


as an example of, extract from, the explanation in the Johrei training
sessions) 256

Johrei Handbook 272

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Dr. Tannis M. Laidlaw


Imperial College London

Ph.D. at University of London - 227 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Self-hypnosis Group

Session 1: Instant Relaxation


1. Name tags

2. Introductions of people including me.

3. Topic of the Day: Introduction to hypnosis and PNI


- Story of my PhD
- Story of using hypnosis in infections
- Imagery to be used

4. Hypnosis Session 1: Instant Relaxation


Hypnosis Session 1: ‘Introduction’
The Hypnosis Procedure: ‘The Beaker Diagram’

Session 2: Viewing things differently to regain control


5. De-Bugging Negative Thoughts Record

6. Hypnosis Session 2: ‘Symbol’

Session 3: Breathing Away Anxiety and Panic

7. The Feedback Loop of Negative Thoughts


Physiological Reactions to Stress
Muscles becoming tense or weak
Some Catastrophic Thoughts
The Breathing Triangle

8. Hypnosis Session 3: ‘Sharks ’n’ Bats’

Session 4: Getting rid of pesky thoughts


9. Step-by-step Instructions for the Interrupt Distraction Procedure

10. Hypnosis Session 4: ‘Coastline’

Post-training Tracks: To provide some variety…

11. Hypnosis Post Training Sessions:


5: ‘Blue’
6: ‘Beach’
7: ‘Thrugmutton’

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Welcome to the world of self-hypnosis.

Self-hypnosis is a very useful technique that has


been part of self-healing for millennia. In this
course of 4 sessions, you will learn how to use self-
hypnosis to enhance your immune system, to
control acute anxiety and to stop old thoughts
bothering you. Self-hypnosis can be used for many
other types of changes people want to make as well, but this set of sessions is aimed at stress
control and disease control from within.


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Ph.D. at University of London - 229 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

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Ph.D. at University of London - 231 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

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Ph.D. at University of London - 232 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

The Hypnosis Procedure: ‘The Beaker Diagram’

 Relaxatio Stretch

6 0
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Session 2 : Viewing things differently to regain control
7KHRU\
When we have negative thoughts, we have a tendency to focus on them to the exclusion of other
neutral or more positive thoughts. The consequence of this is that before you know it, the
negative thought has grown in intensity and is affecting your sense of control. These negative
thoughts can be debilitating and can make you miserable, affecting your self-esteem and self-
confidence. In addition, when we have negative thoughts we also interpret change in a negative
way, our attention to negative events is heightened and importantly, we forget about or don’t
notice any good things going on.


Ph.D. at University of London - 233 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

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Ph.D. at University of London - 234 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

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Ph.D. at University of London - 235 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

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The problem of repeated negative thoughts is slightly different. A method for overcoming the
habit of persistent negative thinking is through the ‘De-bugging Thoughts Record’, an example
of which is found on page 263. On this record, you write down what the repetitive negative
thought is. Be as accurate as you possibly can. Note down what type of circumstances trigger
off the negative thought in the next column. Then rate on a 10-point scale how distressing the
negative thought is to you, that is give it a score from 0 to 10, where 0 would mean that the
thought causes absolutely no stress whatsoever and 1 is mile stress with 10 standing for a
negative thought that is as stressful as could be.


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Ph.D. at University of London - 236 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

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Figure 2: De-Bugging Negative Thoughts Record
Bothersome Thought Circumstances 0 – 10 Substitute Thought 0 - 10

I am useless at In work, just had argument with 9 I am very good at gathering the 5
everything boss over a report I produced information but could brush up on
that he criticised computer skills

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Ph.D. at University of London - 237 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

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Session 3 : Breathing Away Anxiety and Panic




7KHRU\
Often when we are faced with stress we notice changes in our body’s physiology. These
changes are normal and happen to everyone. We tend to notice only some changes, and
sometimes don’t notice them at all. But the changes are there due to the hormones that are
released when we are under stress. The degree of physiological reaction will depend on how
stressful we perceive the event to be, and on how well we can cope with the stress. Remember,
sometimes people can be faced with the same stress, but their reactions to that same stress are
individual.


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Ph.D. at University of London - 238 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

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When faced with stress we often notice these changes in our body. The table below shows some
of the more obvious changes people experience.
Physiological Reactions to Stress

Muscles becoming tense or weak


Heart pounding

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Ph.D. at University of London - 239 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

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Ph.D. at University of London - 240 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

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Ph.D. at University of London - 241 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

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Ph.D. at University of London - 242 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Diagram 3: The Feedback Loop of Negative Thoughts

 Bad thought

 Adrenalin surge
(‘Fight or Flight’)
 6. We can
experience a
 VERY BAD
THOUGHT
5. We feel it mostly
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Ph.D. at University of London - 243 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Hypnosis Session 3: ‘Sharks’n’Bats’ %DVLF&'7UDFN 

This most popular of our tracks on the CD is based upon solid clinical practice and some
interesting scientific experiments. Here, we are encouraged to use our imagination to actually
influence the workings of the immune system. We do this very simply with self-generated
images that mean something to us for the reason that we make them up to suit ourselves. The
traditional immune-stimulating imagery uses the image of little sharks patrolling the blood
stream cleaning up any cancerous or infected matter that should not be there. You will meet
these little sharks in this track.

The track starts in a familiar way. Your eyes are encouraged to close, and the deepening is the
usual countdown from 6 to 0. You then go to a quiet and peaceful place where you can do some
work on your immune system.

The first images are to visualise a factory with an assembly line that produces t-cells. You will
have to imagine what this is like. Then, you can have the factory speed up production; it can
make immune cells that are strong and healthy and that know what to do in the body.

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Ph.D. at University of London - 244 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Session 4 : Getting rid of pesky thoughts


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Ph.D. at University of London - 245 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

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Ph.D. at University of London - 246 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Hypnosis Session 4: Using The ‘IDP’ %DVLF&'7UDFN 




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Ph.D. at University of London - 247 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

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Ph.D. at University of London - 248 - Imperial College London


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Ph.D. at University of London - 250 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

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Ph.D. at University of London - 251 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

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t.laidlaw@imperial.ac.uk

Ph.D. at University of London - 252 - Imperial College London


Course description: Daily Johrei (Laying-on-of-handslike healing method) Course
Length of course: 4 weekly trainings + 3 month f/u Accommodation: Imperial College Charing Cross Campus
Availability: Wednesdays 2 hours (18:30-20:30) Entry: Those who agreed with a consent form
Book: Handouts Tutor’s certificate: Johrei instructor, Adult teaching, M.D.
Progression:
1. Stress management tools
2. Practising healing method as a daily health maintenance technique
Study details:
Johrei is a healing method with the concept of ‘heal oneself by healing others’ for self-care or maintaining health & happiness.
Studying the subject provides a way for you to balance your mind & body in daily life.
You will find some applications of Johrei for your daily life in this course.
The study-subject can be divided into two parts:
1. The study of Daily Johrei, including techniques how to perform the healing method in a competent manner.
2. A study of the philosophy & principles of Johrei, looking particularly at the way the healing process takes place and the significance of these
meanings in real life.

Those two elements are seen as inter-connecting. It provides you with an opportunity to apply these techniques & philosophy into daily life.

Aims and Objectives of Daily Johrei course

Overall
Aims: To introduce the basic concepts of Johrei philosophy, principles and practice
To provide opportunities, encouragement and inspiration to the participants to apply Johrei Art of Living and Johrei practice to daily life.
To ensure the participants are able to give Daily Johrei competently and in safe mannerin a non-professional capacity.
Outcomes: At the end of the course participants will be able to: -
1. Identify the basic principles and core concepts of Johrei
2. Give daily Johrei competently in a non-professional capacity
3. State the main aspects concerning the safe practice of Johrei
4. Be aware of opportunities in the future for reinforcing and further understanding of Johrei philosophy and practice.

- 253 -
Session 1
Aims: To provide an overview and practical introduction to Johrei
Outcomes: At the end of the session participants will be able to: -
1. State the main elements of Johrei as a holistic way towards healthier life style(3 foundation, Johrei viewpoint of illness)
2. Identify how Johrei may be used in daily life (meaning of ‘Spirituality’, Tuning-in, Practical Johrei)
3. Demonstrate the essential elements of Johrei as a daily practice (Daily Johrei techniques)
Session 2
Aims: To introduce the basic principles and a core philosophy supporting Johrei practice and its application for daily life.
Outcomes: At the end of the session participants will be able to: -
1. Identify the basic principles supporting Johrei practice (spirit precedes physical, toxins and clouds, process of purification, IZUNOME)
2. Be aware of ‘MAKOTO’ (love, sincerity and integrity) as a core concept of Johrei philosophy and practice.
3. State the five finger rules of Johrei
4. Recognise the brief historical background of Johrei and appreciate the works of Mokichi Okada
5. Be aware of the importance of intention so that they can apply visualisation into Johrei practice
6. Demonstrate a basic techniques of Johrei with visualisation
Session 3
Aims: To explore the wider aspects of Johrei philosophy embracing the value of art and beauty for both inspiration and well-being
To explore Johrei philosophy of nutrition and the value of a lifestyle in harmony with Nature
To encourage students to be aware of the importance of spiritual connection with ourselves, others and Nature
Outcomes: At the end of session participants will be able to: -
1. Review sequence of Daily Johrei technique
2. Be aware of the wider aspects of Johrei philosophy embracing the value of art and beauty for both inspiration and well being
3. Outline the Johrei philosophy on nutrition, ‘Nature Farming’ and the nature
4. Be aware of the value of spiritual connection ‘Spiritual cord’ with ourselves, others and Nature
Session 4
Aims: To reinforce the basic principles and core concepts of Johrei, and the safe practice within a non-professional capacity
Outcomes: At the end of session participants will be able to: -
1. Practice Daily Johrei competently within the home and with others in a non-professional capacity
2. Review the sequence of Daily Johrei technique and know the sequence of Spiritual Johrei & Self-Johrei
3. Be aware of opportunities in the future for reinforcing and further understanding of Johrei philosophy and practice.

- 254 -
SCHEME OF WORK: Daily Johrei course
Course: Daily Johrei healing methods with background philosophies Lecturer: Dr. Akira Naito
Unit No/ Title 4 units Level: beginner
Session Topics (Aims) & Teaching methods Learning Assessment Resources Key-concepts
No. Contents (Objectives) activities techniques
1 Daily Johrei Facilitating brainstorming Brain storming Observation Power-point Johrei
Foundations of Johrei philosophy Power-point presentation Pair work Q&A White board “Spirit precedes
Daily Johrei techniques Demonstration Practical Group work “Realia” CD physical”
Q&A Q&A “heal oneself by
Given information Guided reading Handouts healing others”
2 Principles of Johrei practice Facilitating discussion Discussion Discussion Power-point “Process of
Historical background Power-point presentation Pair work Observation White board purification”
History of Johrei Demonstration Practical Group work “Realia” CD “MAKOTO”
Process of Purification Guided reading Intention:
Visualisation Q&A Q&A Q & A SPOC “You are what
Intention Given information Handouts you think”
3 Art and Beauty Power-point presentation Guided reading Observation Power-point “Paradise on
Art of Nature Demonstration Practical Pair work White board earth”
“Realia” CD Appreciations
& gratitude
Nature farming Q & A Q&A Q&A Nutrition
Spiritual Cord Given information Handouts “Spiritual cord”
4 Reviewing Johrei sessions Power-point presentation Pair work Feedback Power-point “IZUNOME”
Spiritual Johrei Demonstration Practical Guided reading Observation White board Love for others
Self-Johrei Quiz Quiz Quiz “Realia” CD
Q&A Q&A Q&A
Certification for attendance Given information Group work Handbook

- 255 -
Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Daily Johrei

Welcome! And thank you for coming.


I am very honoured and happy to be able to have this opportunity to talk to you about Johrei.
Let me introduce my self, I am ……

First thing I would like to start with is a self-introduction session.


Could you state your name, where you come from and what kind of thought has brought you here, in
other words, the objective or expectation to come to today’s talk briefly, please?

What do you want to take away from Today’s session?

Ph.D. at University of London - 256 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

What do you consider ‘Spirit’ or


‘Spirituality’ is ?
&
When do you think about it?

Brain storming!
High spirit, low spirit, mean spirit, team spirit…..

First of all, I would like to start with this brain storming session.
In Brainstorming, there are no right or wrong answers.
Please discuss in the group that what do you consider ‘spirit’ or ‘spirituality’ to be?
By this I mean, what sort of thinking comes to your mind when you hear the word ‘spirit’ or ‘spirituality’.
Do you have any definition about these?
Any difference between ‘spirit’ and ‘spirituality’?

Then next question is when do you think about the concepts of ‘spirit’ or ‘spirituality’, in other words,
what sort of situation makes you think these?
Are you always thinking of these? Or you have never thought of these?
You surely know the word, so at least once you have, haven’t you?

Any ideas please.


Anything you may think of would be very helpful and useful for us.

Ph.D. at University of London - 257 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Three ways in which to attain spiritual awareness

Beauty

Spiritual Practice

Life Challenges

This slide is a summary of what I have heard from previous groups of the situations when they may think
of the spirit or spirituality. These are typical examples of the ways to be aware of spirit in Johrei as well.

Life challenges include when you are suffering something difficult like attengding funerals or emotionally
draining time. Spiritual practice includes worship, meditation, and spiritual healing.

In Johrei this ‘Beauty’ is highly emphasized and it includes a piece of art like masterpieces in which you
may find a spirit of an artist, and somewhere with beautiful scenery in which you may feel your spirit is
uplifted.

As you have raised and heard from others’ thoughts about ‘spirit’ and ‘spirituality’, there are quite a lot of
meanings or concepts you could think when you hear these words. The purpose of having this brain
storming was that I would like you to know that the spirit is not very much far from everyday life nor for
certain special people only.

It is, in a way, for everyone of us in everyday life. It is in ‘anytime and anywhere for anyone’

Ph.D. at University of London - 258 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Healing and
Spirituality
for ALL

Mokichi Okada
1882-1955

This is a picture of Mokichi Okada, a founder of Johrei.


The message he had tried to convey was that as spirituality is for all, healing or ability to heal other
people is also for all. Johrei is one of a practical and effective application of and a tangible example of
realisation of what you may see the future in this empowering message.

It is always emphasised in Johrei philosophy that “EVERY ONE OF YOU CAN APPLY JOHREI to
ANYTHING at ANYTIME and ANYWHERE you want”.

Ph.D. at University of London - 259 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Johrei
Purifying our spirit and uplifting our soul

The word ‘JOHREI’ is in Japanese and consists of two Chinese characters, Joh and Rei.
This literately means purifying the spirit and implies uplifting our soul. Johrei has a lot of facets, so I
would like to introduce them one by one.

Ph.D. at University of London - 260 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Daily Johrei practice


Maintaining health and happiness in a family

Home practice

The Johrei practice is a daily exercise like meditation, jogging or walking.


The purpose of Johrei practice, a main objective in the training workshop i.e. learning why and how to
practise Johrei, is to maintain health and happiness in family and friends, in other words, to create “a
better and beautiful world” from your environment.
By this I mean that to become confident and comfortable to practise Johrei within a non-professional
capacity.

Ph.D. at University of London - 261 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Supporting people who suffer

In addition, since Johrei historically began as a therapeutic act, you can support friends and family with
Johrei when they are suffering from some tough conditions as well.

Ph.D. at University of London - 262 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Mutual Healing
‘Healing oneself by healing others’

This is the central concept on which Johrei philosophy and practice is based.
It is not just giving or receiving healing. It is a mutual process.

‘You can heal your self by healing others’

Hence you can play a role in creating‘a better and beautiful world’.

A couple of people is the smallest unit of the society.


Happiness and health can be realised and spread from this smallest unit within family and friends to larger
units, say community, society and ultimately all the world. This process is called by Johrei creating
‘Paradise on Earth’: a better and beautiful world -- first personally and eventually as a community.

This realisation can be achieved through Johrei practice itself, and also through tangible practice of other
tenets of Johrei philosophy.

In order to practise Johrei effectively and to enjoy Johrei thoroughly, appreciations and
acknowledgements of this Johrei philosophy is very important and also encouraging.

Ph.D. at University of London - 263 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Three foundations of Johrei philosophy㩷


- Appreciation with gratitude in a daily life -

Art of Healing
Art and Beauty
Art of Nature

The Johrei philosophy consists of three foundations as you can see here:
Art of Healing which is Johrei practice itself, and emphasising the importance and appreciation of ‘Art
and Beauty’ and ‘Art of Nature’.

In Johrei, it is emphasised that to be aware of these ideal ultimate goals is very important, but it also is
equally important to make one step towards the goals. In other words, you are encouraged to participate in
tangible activities of the Johrei philosophy in all aspects in everyday life from your own environment,
which means to start creating “a better and beautiful world.”

Details of these tangible activities are covered in the training workshop. In the training workshop, first
Art of healing is the main objective to learn and to apply but the other two foundations will be touched
upon for further understanding of first Art of healing, since these three philosophies are inter-connecting
with each other.

Ph.D. at University of London - 264 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

This is a brief introduction and overview of Johrei.


Do you have any questions about it?

Now, if someone asked you “What is Johrei?”, what would you tell them?

Suppose I was the person who asked you what is Johrei.


Tell me about it.

What would you say to me?

Ph.D. at University of London - 265 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Key objectives

Three foundations
Three requirements
Five principles
(Five fingers rule)

Here are the summarised keys to open the door of your Johrei experience which you will be cover in the
training workshop.

Three foundations of Johrei philosophies, which I have already given the titles.

Three requirements to start practising or to get ready to Johrei.

Five principles in order to practise Johrei properly.

Five finger rules for memorising and reminding what the key thoughts are when you practise Johrei.

Please start exploring these details in the training session, shall we?
Today, I would like to show you just the title names of those and want you to try the first step of Johrei
experience, named as Tuning-in.

Ph.D. at University of London - 266 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Three requirements for practice

• TRUST (although you do not have to believe if you practise)


– in the power of the source of LIGHT
– in the ABILITY of your SELF

• INTENTION to channel the JOHREI


• TUNING-IN with appreciation

In order to practise Johrei, there are only three requirements.


You are encouraged to hold your doubts if you have any, so you do not have to BELIEVE these two, but
please TRUST these as you can trust someone even you can not truly believe them ;-).

The two are 1. Trust the power of the LIGHT, and 2. the ABILITY of your self.

You are quite all right to feel that you can not trust yourself right now, but I am sure that you can trust the
ABILITY of your self if you are wishing to be or making some efforts towards your ideal self.
Do not ignore your negativities but focus more into the other side rather than concentrating on negatives.
This is not ignoring, hiding from or even denying that you have doubts. It is just waiting for the right time
to deal with them.

The next requirement is the intention to channel the Johrei, and the final one is so-called ‘tuning-in’.

Metaphorically speaking, the source of the light keeps sending the LIGHT like a radio station.
Like a radio or a TV, there are always waves but you can not see nor sense.
Mokichi Okada said that every one of us has got an antenna to receive it.
Once you have tuned it in the right channel, then you will be able to practise Johrei as once you tuned a
TV tuner in a right channel then you can get beautiful sound and picture. It constantly flows.

This is it.

When you are comfortable to accept these three requirements, it is time when you can practise Johrei.
It is very important to be comfortable your self to begin with, so make sure that you are comfortable
enough.

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Five principles for Johrei practice

‘Toxins’ and ‘Clouds in the spirit’


The process of ‘Purification’
‘IZUNOME’
‘Spirit’ precede ‘Physical’
‘Spiritual Cord’

Next, I would like to show a summary of core principles for Johrei practice by showing title names.

These are the core five principles of Johrei practice.


Each will be covered in detail in the training session.

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Five Fingers Rule

Thumb (T) TUNING IN the source / your self

Index Finger (I) INTENTION and inspiration

Middle Finger (M) Middle way & balance (IZUNOME)

Ring Finger (R) Relax so that you can ENJOY!

Little Finger (L) Let the LIGHT flow

Open palm and receive gratitude with appreciation

These are five practical concepts in order to memorise and to remind yourself when you are applying

Johrei in each session and also in everyday life.


These are the title names which you will master in the training session.

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We are determined by our intentions


“Gratitude attracts gratitude”

‘Ingratitude attracts ingratitude’

Our life is altered by the way we think


As many psychology or self-realisation text books say nowadays, Mokichi Okada was emphasizing this
concept almost a century ago.

We are what we think and what we wish to be.

It is exactly the same to the Johrei practice, of course!


Are you ready to try one simple exercise, which is the first step for the Johrei practice?

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Tuning in

Feel the ‘Flow of Light’

The first step for the Johrei practice is this TUNING-IN exercise. Shall we begin?

Have you got any experience in meditation, visualisation, or even some spiritual practice like Spiritual
Healing, Therapeutic Touch, Tai Chi or Reiki? Those who have such kind of experience, please use your
technique if you feel comfortable and want to, but please combine this ‘LIGHT’ concept with it.
I think this FLOWING LIGHT is a very powerful metaphor, so bear this in mind, please.

Those who have got no experience, you may want to use one visualisation technique I usually teach.
Imagine a beautiful place you have been before, say like a beach or park on a sunny but not a too hot day.
You are sitting there having the sun shining behind you. The light comes from the Sun and you can feel
warmth on the back of your neck and shoulders. How about this?

Try to FEEL the light, please. By this ‘Feel’ I mean, any five sense you can experience, brightness
(seeing), warmth (touch), or even sounds (hearing), scent (smell), and flavour (taste) of the light if you
can.

It is totally up to you whether you have your eyes open or closed.

Shall we begin? Please make your self comfortable first, and think THE LIGHT.

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Daily Johrei Technique Handbook

for Home Practice

Dr. Akira NAITO

Imperial College London

Johrei Academy ©

Ph.D. at University of London - 272 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

Introduction

Welcome to Johrei practice! Some of you may be new to healing or spirituality, and some of
you may already be an experienced healer, however, either way, we hope you find in Johrei an
inspiration and simple means to accessing the intimate and spiritual space into which you may
bring healing and beauty.

This handbook is to remind you various techniques taught on the course, and practical ways
of applying Johrei philosophy. Once you have mastered them, naturally, you will no longer
require this handbook.

Johrei is a wonderful way of maintaining health and happiness, but also a useful skill for
helping people you care about out of the goodness of your heart. (Remember you can heal
yourself by healing others, and, if you want, you can learn to practice Johrei professionally with
the Johrei practitioner training at the Johrei Academy. Please note that Johrei techniques you
have learned is not a therapeutic technique, and you will not be able to either medically
diagnose or treat people.)

We very much hope that you will continue with the Johrei practice, and in whichever way
you chose to practise it, you will be contributing, little by little from your surroundings, to
making this world better, beautiful and harmonious place to live.

May the light and beauty be always with you!

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

1 Overview of Johrei principles and practice

What is Johrei ?
Johrei is a therapeutic art, and in other words: an Art of Living, founded in 1920’s
by a Japanese artist, healer and spiritual leader, Mokichi Okada.
Johrei literally means purification of spirit. Its primary aim is to facilitate the
elimination of toxins from our body and dispersing of ‘clouds’ from our spirit in order to
revitalise us and uplift our soul, thus restoring harmony and well-being.
Johrei can be practised as a therapy that treats illness from all levels (mind, body
and spirit) or as a wonderful and simple way to relieve stress and provide us with
spiritual oasis within our hectic lives.
What is unique about Johrei is that it has a simplified, easy-to-learn technique for
you to practise at home in order to maintain health and well-being. These handouts
outline these techniques for you to learn and practise.
Johrei also extends beyond its healing practice. It is an Art of Living that covers
the following three aspects:-
1. HEALING (Art of life) - Trust the ability of human being
2. BEAUTY - Appreciation of Beauty, enjoy beauty in life
3. NATURE - Living in harmony with nature, Natural farming, diet and nutrition

Johrei activity over the decades gave rise to its own unique flower arrangement
school, SANGETSU, its own chemical-free farming technique, NATURE FARMING, its own
tea ceremony, BONTEMAE, and various art museums, gardens of exquisite beauty and
sacred grounds. As a practitioner of Johrei you will have access to all of these.

Principles of Johrei
Toxins
These are entities (chemicals, inappropriate medicine, pollution, etc.) that are not
intended for our natural consumption. Problems begin to occur when they exceed
beyond our natural capacity and begin to accumulate in our body as toxins. In this
sense, food in excess can become toxic.

Clouds
Think of these as the gloom that looms over us in our spirit when we are ‘under the
weather’. They manifest when we are under stress, emotionally challenged or
become toxin infested.

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The Process of Purification


The Toxins and Clouds are constantly undergoing a natural process of eliminating.
This is called the process of purification. This is a naturally occurring process of
detoxification and spiritual uplift. Johrei encourages this process in a directed way
and alleviates discomfort.

Spirit precede physical


We are what we think and feel. Our mental, emotional and spiritual well-being is of
utmost importance. The problem begins if we ignore this simple truth and let our
mind and spirit go out of synch with our body. (See also next page)

IZUNOME
IZUNOME is a state of balance, harmony and appropriateness. It is the middle way
which gives us the ability to look at both sides of a situation so that you can
maintain the balance. Sometimes we need to trust and let go, other times we need to
take our destiny into our own hands and act. Knowing when to act or when to let go
is not easy unless you can sense that. You will be able to sense it by being in the
state of Izunome which practices of Johrei lead you to.

The Process of Purification

This is a naturally occurring process that eliminates toxins and clouds from the
body and the spirit.
It occurs in two stages:

(1) Accumulation
Toxins accumulate around different areas of the body, which are used frequently.
This means that the areas of toxin accumulation vary from person to person. Areas
of stiffness, ‘knots’ in the muscles, swelling, and the areas of pain are said to be due
to the toxin accumulation. Toxins also accumulate deep within our body and affect
our internal organs.

(2) Elimination
When we are well, our body naturally eliminates toxins. This happens when we go
to the toilet, sneeze, cough, and sweat or shed skins. When the toxin accumulation
is severe, elimination process may also be pronounced and accompanied with

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discomfort. For example, flu-like symptoms or diarrhoea are symptoms occurring as


the result of toxin elimination. We tend to see this elimination process as an
incident of minor illness, however, they are our body’s way of maintaining our
health.

How Johrei Works

Johrei works by dispersing the clouds in the spirit. This eliminates the negativities
within us, thus giving us greater vitality and encouraging our body’s natural ability
to eliminate toxins.
Furthermore, it is thought that Johrei itself helps to dissolve away the
accumulated toxins or ‘burn away’ the toxins themselves, thus helping to support
the body’s immune function and the function of other organs especially kidneys
which filters out toxins from our blood.

Johrei

Toxins Body Spirit Clouds

Us
Interaction between Body and Spirit

In Johrei it is believed that the spirit and body interact in such way that
accumulated toxins cast ‘clouds’ in our spirit, and the ‘clouds’ themselves cause
impurities in our blood that leads to toxin build up and hence illness.

Spirit precedes Physical

On the other hand, when the ‘clouds’ in the spirit are dispersed this encourages
toxins in the body to be eliminated. Just as our will leads to the action in our body,
it is believed that the state of our spirit (mind and emotion) influences our physical
well-being. This principle is called the Spirit precedes Physical. Based on this
principle, Johrei practitioner works by channelling the Universal Energy (referred to
as “the Light”) with the intention of dispersing the ‘clouds’ in the spirit and
eliminating toxins.

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

2. The Finger Rule for preparing yourself for the Johrei experience

You can use the following rules to help you not only with Johrei practice but to prepare yourself
consciously for anything, from flower arranging, art, music, sports to the life in general.

Visualise these rules as you count them out with the fingers of your hand,
starting with:
Thumb (T) -Tuning in
First, tune in to the source.
Tune into the source of your energy, with love, inspiration and your very being. The source
exists at the centre of the Universe and it is up to you whether you think of it as your higher self,
God, the Spirit of the Sun, Nature or the Cosmic Consciousness.
This process is like a TV or a radio; if you do not tune in, you can not obtain a
beautiful picture or clear sounds although you have an antenna to receive it. You also
empower the antenna by practising Johrei.

Index finger (I) –Inspiration and Intention


Be Inspired and hold healing intention
Let the source of Universal Love inspire you by your loving intention for the healing of yourself
and others.

Middle finger (M) (with Index finger (I))


–Mentality; the Middle way (IZUNOME)
Still the mind & balance and harmony with timing: Try hard first then trust!
If your intentions are always for the good of mankind, there is nothing to be afraid of. Allow
your stress to melt away and clear your mind. Everything that happens to you is enable you to
help you become happier, healthier and stronger. A raw diamond has to be polished to shine.
You will find your soul is purified & enlightened by these processes. Keep a smile on your face
always.

Ring finger (R)


–Relax so that you can ENJOY yourself
Relax your body and take it easy so that you can enjoy yourself!
Rocks can be crushed if the force is strong enough, but free flowing water is adaptable to its
surroundings. Always keep the body relaxed and flowing and you will never be crushed. It is all
about learning to trust and let go. Relaxing your body will also improve your Johrei practice.

Little finger (L) –Let the Light flow!


with Love & MAKOTO (integrity, sincerity, love)
Trust in the universal flow & trust your self. Love for others & MAKOTO to you.
Let go and let the light flow. Once you are relaxed, open and tuned in, you can flow with the
light.

Opening your palm to receive gratitude!


Gratitude attracts gratitude!
Everything that happens leads to your elevation. If you are in the state of gratitude
and appreciation, things you desire, will always come your way.

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Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

3. Points to note before giving Johrei

1. Take note of how you are feeling before you give Johrei.

2. Choose comfortable surrounding where you will not be interrupted or disturbed during
Johrei. Try choosing an area where there is harmony or beauty, possible nears a flower
arrangement.

3. Choose two comfortable seats, if possible stools with swivelling seats. However, seating
must be chosen to suit the person receiving Johrei. If the person cannot remain seated
for more than 10-20 minutes, adapt your Johrei accordingly. It is important that person
who is receiving Johrei is informed that it will take this long before hand.

4. A guide to the positioning of your self and your partner is as follows:-


The Light is said to flow from the northeast so ideally position yourself with your back to the
northeast.
It is more important that you do not have your back to the door. Position your
self so that you are in the opposite part of the room from the door.

5. Take care to note if the person about to receive Johrei is already undergoing some sort of
Purification Process or the person is known to undergo pronounced Purification when
receiving Johrei or other forms of healing. Remember that Johrei is about encouraging
the Process of Purification. Although, Johrei will generally alleviate any discomfort, use
your best judgment to decide whether it is best to proceed with Johrei or allow the
process to take its own course and support it with Johrei at a later stage. If you decide to
give Johrei in such a situation, it may be more advisable to adapt or shorten your Johrei
accordingly, rather than doing the whole sequence. If in any doubt seek professional help
before proceeding.

6. If the person to whom you are intending to give Johrei is suffering from acute pain,
severe discomfort or very high fever, especially if the person is a child or pregnant please
seek professional help without delay.

7. Be prepared for emerging emotions. Even though the person you are giving Johrei may be
suffering from physical discomfort or no obvious discomfort at all, you may find that
there are underlying emotions needing to be expressed, Johrei often allows these
emotions to emerge. It can be the case that such emotions are dispersed by Johrei
however, you should be ready to talk. Talking can sometimes be hard amongst friends or
family. Ask yourself if you are prepared to handle it. If the person wishes to talk or cry,
let the person lead the situation. Humility and sincerity is the key. Be there, listen with
an open heart but do not advise or judge. If in doubt seek professional help.

8. Heal your self by healing others. Johrei can benefit you when you give it to others. This is
because you receive the Light before you channel it, and through the healing in your
partner you are also healed. The feeling of appreciation and gratitude from your partner
will also provide you with an up-lift.

9. Make sure you are comfortable when you give Johrei. If you cannot remain in a seated
position for a long time, arrange your seating or length of Johrei accordingly. If you
cannot raise your arm for a long time support it with the other arm or rest it on an
armrest or a table.

10. You must feel comfortable to give Johrei. If you are too unwell, just receive Johrei from
whoever is offering to give it to you and then rest, don’t feel you are obliged to give Johrei
each time.

11. JUST ENJOY the feeling of intimacy, happiness and joy of returning the love and care
you have received from others. Empower your self to help others and in so doing, help
yourself.

Ph.D. at University of London - 278 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

4. Basic Johrei sequence for family practice

1. Sit facing each other.


2. Take a moment to relax and prepare yourself (with the 5 finger guide)
3. Greet and acknowledge each other.

4. Tune in by visualising the source of the Light. Channeling the Light is like ‘playing the
music of the universe’. Appreciate the occasion and if you can, fill your self with gratitude
and joy. Sense of gratitude will allow yourself to be healed in the process of giving Johrei
and you will feel less tired during Johrei. The effect may be further enhanced if you can
feel and visualise the happiness and the positive outcome for the person you are giving
Johrei.

5. Gently raise your hand, relax your arm, cup your palm and begin by channelling to the
forehead (2 minutes)
6. Gently lower your hand to the area near the heart and give Johrei there. (2 minutes)

7. Ask your partner to turn around. Take a moment to tune in again.


Stand up, and steady your partner. Then gently proceed to channel to the crown of your
partner’s head. (2 minutes)
8. Channel to the both sides of the neck with your hand (left side first and then right).
Remember to cup them.
(2 minutes (1 minute each))
9. Cup your hand and place them on the shoulders (left side first and then right again). (2
minutes (1 minute each))
10. Then proceed by cupping your hands and giving Johrei to the centre of your partner’s
back so as to channel to the liver, pancreas and stomach. (2 minutes)
11. Place your hands on either sides of your partner’s lower back and give Johrei. This
stimulates the kidneys. The Kidneys are important organs for purifying the blood.
General fatigue can also be supported by energising the kidneys.
(4 minutes (2 minutes each))
12. Finish off by raising one hand as if to give a shower of Light. (1-2 minute)

13. Signal your partner that you have finished with a gentle tap on the shoulders and ask
your partner to face you once more. Thank each other. (Remember you have benefited
by giving Johrei too).
14. If your partner also practised Johrei then do not hesitate in proceeding to receive Johrei
because Johrei is something to be shared!

Points to note after Johrei:


It does not matter whether your partner or your self did not feel anything during
Johrei, or even that your partner did not immediately feel any effect of it. The effect of
Johrei can just as well be delayed, subtle or unnoticeable.

Reflect more how it was like for you, how you felt afterwards. And think of how you
might do it differently in future for even better results.

Ph.D. at University of London - 279 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

5. Basic technique of Self Johrei

1. Sit on a comfortable chair. It will be more effective if you sit upright.


2. Close your eyes and take a moment to relax and tune in with the Five-finger guide.
3. Tune into the source of the Light by visualising the spirit of the Sun over and above you
shining down a beautiful Light.

4. When you can feel the flow of the Light reaching down to you, slowly raise one hand and
channel to your forehead. Your arm should be relaxed, as it would be when channelling
Johrei in pairs. (2 minutes)
5. Slowly move your hand down to your chest area passing through the throat. Channel Johrei
to your heart. Enjoy the warmth & any alleviation of tension. (2 minutes)
6. Lower your hand further to your stomach area. This area is related to anger and irritation.
Channel Johrei here in order to calm you and make you feel more positive. (2minutes)
7. With one hand at a time, channel Johrei to your groin (left side first then right). (1 minutes
each side)
8. Cover your tailbone with one hand and channel Johrei to the sacrum. This will help the
womb in women, and also your intestines and digestive system. If you have a particularly
problematic digestive system, you may also combine this with channelling Johrei to the
lower abdomen from the front.
9. If you are suffering from period pains, irregular period cycles, menopause or fertility
problems you can follow this up with channelling Johrei with two hands placed on either
side of your abdomen followed by one hand three finger width below your belly button. (in
this occation1-2 minutes each position)

10. Then from the front, channel to the kidneys (left side first) or cup you hand and place it on
your lower back (left first then right). (2 minutes each) Your lower back is related to your
kidneys. Kidneys are important organs for eliminating toxins and filtering blood. Stiffness
in your lower back indicates that kidneys are weakened and that excessive toxins have
accumulated there. If the kidneys are over worked, it will take extra energy to filter your
blood hence you will feel physically drained. Use your hands to press away the stiffness
and then channel Johrei if you feel comfortable.
11. Cup your hands and gently place them on your shoulders. You may prefer to do this one
shoulder at a time. Your shoulder is a barometer for your general health. Make sure they
are always free of stiffness and tension. Your left shoulder is closely associated to your
heart. Spend more time channelling to your left shoulder if you have a heart condition. (1-2
minutes each side)
12. With one hand at a time, channel Johrei to both sides of your neck (left side first the right).
(1-2 minutes each side) This will help the toxins to move down from your head and support
the alleviation of headache.
13. Finally, channel to the forehead like a shower of Johrei! (1 minute)

14. Please note that Johrei, just like every other healing, is effective both when you give it and
receive it. So treat Self-Johrei as a daily self-maintenance practice for improving your
Johrei. Always try to look out for anyone who will receive Johrei.

Remember, with Johrei,


YOU HEAL YOURSELF BY HEALING OTHERS

Ph.D. at University of London - 280 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

6. Visualisation for Johrei practice

The founder of Johrei, Mokichi Okada said that we are what we think and our
intention alters our life, and also that whatever is created in the spiritual dimension pre-
empts the changes taking place in the physical dimension (Spirit precedes physical).
These concepts parallel the work carried out by the Simontons in the USA. Based on this,
it is obvious that visualization plays an important role in whatever we do. Visualisation
is so powerful such that it can even determine the direction of the flow of Light or the
outcome of Johrei.

Directing the flow


In Johrei, you tune in by visualizing the source of the Light and asking it to help you
by channelling the Light to the receiver. The channeller visualizes the flow of the Light
from the source, through him/her to the receiver.
The channeller can intensify the effect of the Light by visualizing it being focused by
the cupped hand as if it is a magnifying lens and beamed through the body of the
receiver to the other side.
Those who are sensitive to people’s energy and internal emotional states, visualize
your self as a river rather than a bowl of water. When people wash their dirty linen in
your bowl of water, it leaves the water dirty. However, if you are a free flowing river,
the dirt is washed away and the water is always pure and clean.

Visualising the outcome


It is helpful to know beforehand the hope and the wishes of the receiver. As a
channeller, you can visualize the wishes of the receiver while you give Johrei. It may
be helpful to visualize a film screen onto which the films of the future events as wished
by the receiver are screened.
The receiver can be encouraged to visualize the future outcomes in the same way if
you want, but do not compel him/her to do so please.

Visualisation by the receiver


It is often found that the receiver is unsure how to sit during Johrei and also asks
the channeller whether it is necessary for them to make his/her mind blank. It is
undesirable for the receiver as well as the channeller to be struggling to visualize or to
blank their minds.
It is far more important to remain in a natural state. Visualise only if it becomes
natural to you and to the receiver. A useful way of quietening the mind Johrei way is
to allow any thoughts to arise in the head and imagine them to be clouds which are
dispersed by the Light of Johrei. More Johrei you practice, you will find that the mind
quietens naturally without making a conscious effort.

Visualising the past and the future


In Johrei, it is said that spirit precedes physical. By uplifting the past and the future
with Johrei, you can uplift the present.
When you are giving Johrei from the front of the receiver especially at forehead,
visualize a pathway from the past behind the receiver, along which he/she had to
travel. It is his/her passage from the past. Visualise to clear the hurdles in it with
your Johrei. When the receiver turns and you are giving Johrei from behind, visualize
a passage in front of the receiver. It is his/her path into the future. Visualise that you
enlighten, energise and heal the path for your receiver with Johrei.

It is up to you as a Johrei practitioner to choose which visualization to use or not to use


visualization at all.
With practice, you will find these come to you naturally

Ph.D. at University of London - 281 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

7. Sensations felt during Johrei


The following is a guide to understanding the meaning of some of the sensations felt by the channeller
or the receiver during the Johrei process. It must be noted that these are not official teachings as taught by
Mokichi Okada, the founder of Johrei. Use this information as examples.

Heat
In the Johrei principle of the purification process, it is understood that when the accumulated and solidified toxins
begin to ‘melt’, the process is accompanied by a localised temperature rise. The characteristic of this is that you will
feel the heat upon contact as well as away from the body. If you feel the heat from the receiver yet you feel
COLDNESS in your hand when channelling Johrei, it usually indicates that the purification process of melting toxins
is taking place in the area and it is drawing in the Light from your hand to support it (see coldness below).
The heat felt by the receiver is the Light which is being channelled. It is usually felt below the surface. How deep
the receiver feels the heat depends on how far away the channeller's hand is. The further away the hand from the
receiver, the DEEPER the heat of receiver. Try not to confuse the channeller's body heat with the heat sensation of
the Light (it has been known that a person with cold hands can still produce the heat sensation within the receiver).
If the channeller feels the heat coming from the receiver with no resulting to temperature rise, it usually indicates that
the area requires no further channelling.

Coldness
The most well recognised sensation other than the heat is the sensation of coldness. The areas with cold sensation
are the areas in need of healing. If the channeller feels the coldness in his/her hand when giving Johrei, it indicates
that the area is drawing the Light in and healing is taking place. Notice if the area becomes warmer as Johrei is given.
If the receiver feels the coldness in the area being given Johrei, find out if the channeller is feeling the heat in his/her
hand. If so, it usually indicates that the Light is flowing in the wrong direction. Stop, tune in again and channel by
visualising the Light being focused by the channeller's hand and flowing from the channeller to the receiver.
It will help to visualise that the beam of Light piercing through the receiver's body to the other side. Other cold
sensations may exist such as the sensation of coldness at the core of the receiver's body, which melts away like
melting ice. This in the past has indicated a profound healing.

Tingling, magnetic repulsion and the sensation of movement


Tingling sensation in the hand of the channeller can occur to varying degrees. Such tingling sensation indicates that
a healing process in which the Light is drawn through the channeller to the area where the purification process is
taking place. For Johrei, any tingling sensation indicates that toxins are on the move. So the tingling sensation in the
channeller's body may mean that the purification process is taking place there.
The sensation of magnetic repulsion mean that the polarity of the channeller's hand and the area receiving Johrei
may be the same, hence the repulsion. Johrei is about channelling the Light from the Universe and not the magnetic
energy of the channeller's body. Therefore, if the channeller feels the sensation of magnetic repulsion in Johrei, then
stop and tune in again.

Pain
When the elimination takes place and the toxins begin to melt and move away from the area being given Johrei, the
receiver may feel the sensation of toxins flowing away. If the receiver feels such sensation, it can only be a positive
indication. If such sensation is uncomfortable or painful, further Johrei may be given to make the process more
comfortable.
Pain indicates that the toxins in that area are melting and moving away. During Johrei, it may be found that the
area of pain, knots and stiffness progressively moves down towards the kidneys. If it does, the purification process is
proceeding as it should. Encourage the receiver by letting them know that this is a good sign. If the area of pain is not
moving, is becoming intense or is moving in the wrong direction (upwards away from the kidneys), use stroking to
encourage the toxin flow downwards.

Cold draft
If the channeller or the receiver feels a draft of cold wind flowing horizontally across the area receiving Johrei, it is
said to indicate that the area requires no further Johrei at that time.

These are guidelines only and true understanding can only come from your own experience gained
through repeated practice.

Ph.D. at University of London - 282 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

8. Taking Care of Symptoms of Purification


It is likely that through the Johrei practice, toxins within your system start to be eliminated. This
will lead to better health and improvement of your current condition. The symptoms of elimination
(process of purification) vary from cold like symptoms to diarrhoea. The severity depends upon
various factors. The following may be a useful to note.

1. History of illness, (how long have you suffered from your current condition, how severe is it,
what other illness have you had in the past)?
2. Symptomatic medication you have been using (medicine is useful in suppressing the
discomfort, however, later your body will need to eliminate it if it is not appropriate.)
3. Your general well being. As your health improves, you may find that your body will start to
eliminate the toxins that were accumulated in the past.

The above will take place irrespective of whether you give or receive Johrei or not. With Johrei
you will find that the elimination process is quicker, with less discomfort. You will also find that
more toxins are eliminated; the elimination process will be less prolonged and less intense.

How to manage the ‘purification’ symptoms

Fever:
We know that our immune system can be most activated at the body temperature of 38 to 40
degrees Celsius. If you are feverish, just take a rest, relax and sleep well in peace (ideally with
supportive Johrei to the kidneys).
When fever causes you to be unable to drink or sleep, an antipyretic such as aspirin helps to
prevent dehydration or exhaustion.

Diarrhoea:
Warm water is the best thing to take at this time as well as keeping your stomach warm.
Diarrhoea is usually a self-limiting symptom, it should last at most about 1-2 days with or
without a severe period of less than 12 hours. What a doctor worries about is dehydration and
exhaustion. Consequently, if you cannot drink water for 12 hours, you should go and see a
doctor to have fluid supplied intravenously.

Sweating or chilling
Once again, warm water is the best thing to take at these times. Keep yourself comfortable by
changing your clothes, wearing warm clothes etc.

Coughing
Coughing is the way to throw sputum out. In Johrei philosophy, toxins can gather in sputum
or in other discharges as well as into urine. When it hurts, give Johrei from the middle of back
to alleviate it.

Pain
If you or your partner gets acute discomfort, contact a doctor without delay.
In Johrei philosophy, it is thought that accumulating or melting toxins cause some discomfort.
This can be a sign that the elimination process is taking place. Johrei, however, is a wonderful
and simple way to alleviate discomfort and help your body to detoxificate.

9. The key areas


1. Forehead
2. Crown
3. Temples
4. Back of the neck and the base of skull (medulla oblongata)
5. Both sides of the neck from the dip behind the ears down to the collarbone or
shirt collar (lymphatic nodes)
6. Kidneys: The motion is always down from the head towards the kidneys.
7. And lymph areas (such as heart area, arm pits, groin).

Ph.D. at University of London - 283 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

10. Brief historical background of Johrei

Johrei is a philosophy and practice, which has its foundation in a Japanese therapeutic art
developed by Mokichi Okada (1882-1955) in the early part of the last century.
Mokichi Okada was born into a poor family Okada was not a healthy youth and indeed his
various afflictions and life threatening diseases kept him from fulfilling his potential of becoming
a professional artist. Following a vegetarian diet, he gradually recovered and with the death of
his father in 1905, he became the sole supporter of his family.
He opened a small retail store in Tokyo where his artistic ability contributed to his success,
however, he was again forced to abandon this business due to illness.
During the next twenty years, the periods of success alternated with times of abysmal tragedy
and disappointment. He lost his wife and their baby due to complications during childbirth.
At one point, the failure of his main bank plunged his firm into bankruptcy, and the Great
Kanto earthquake of 1923, in addition to destroying much of Tokyo, ruined Okada financially,
along with thousands of others.
Around this time, Okada, who had been an atheist, began to search for a spiritual meaning
to life. He became acquainted with various religions and beliefs that were popular at the time,
and in this vein, in 1920, he attended a lecture on Omoto at a movie theatre, which he regularly
visited. Omoto was one of the New Religions making the headlines at the time. It is based on
Shinto, which is the oldest national religion of Japan, and much of what the lecturer said
seemed to be refreshingly straight to the point. Okada later joined Omoto and began studying
its teachings. One thing unique about Omoto was that it had its own healing ministry called
Chikon kishinho, Okada made it his own and began practicing it in 1929. However, by 1934,
dissatisfied with the limitations of religious activities and desperately wanting to reach out to all
those who needs help, he left Omoto in order to develop his gift as a healer and a therapeutic
artist.
The major influence Omoto had on Okada, was to make him realise the importance of the
Spiritual. During his time in Omoto, he also discovered that he had a special ability to heal the
sick.
He had closely studied the way in which people became ill and the way in which people
recovered from illness. He also closely studied medical writings of the day including those on
traditional medicine, and had collaborated closely with the leading medical practitioners of the
day. Through this process, he developed and perfected the unique spiritual therapy, which he
called JOHREI, meaning purification (joh) of spirit (rei)
Okada was passionate about promoting a life style, free from disease, through the use of
natural methods of preventing ourselves from becoming seriously ill. Based on his own
experience, he knew our food played a crucial role in our health.
In 1935 he began research into the possibility of realising a chemical free farming technique,
which he called Nature Farming. By the 1950’s he was able to set up experimental farms and the
Nature Farming Society.
During that period, he also created the basis for the Sangetsu School of Flower
Arrangement, which emphasised the freedom and the natural beauty of flowers. As a way of
bringing beauty into daily life, Sangetsu Flower Arrangement is also practiced alongside the
therapy of Johrei healing.
As an artist he knew that both artistic and natural beauty could inspire us and uplift our
soul. He looked for a way to bring beauty into our daily lives, and he devoted the later part of his
life to creating art museums and inspiringly beautiful gardens. The museums and the sacred
gardens he created near Mount Fuji in Hakone and the seaside resort of Atami are enjoyed by
millions of visitors today. His vision was to inspire and encourage everyone, in making this
world an Earthly paradise.
Back in 1936, Okada set up the Health-care Society of Japan and invited medical
professionals of the day, to contribute, to its promotion of a natural form of health maintenance.
This was based on a way of living that respects our body’s natural ability, to heal itself and
stresses the importance of truth, virtue and beauty within health care.
The Authorities of the day did not appreciate his vision, and he was discouraged from
practicing Johrei as a therapy and healthcare measure. In order to be allowed to promote Johrei
and both the medicinal and the spiritual aspects of its teaching, he decided, in 1950, to set it up,
as a religious organisation, Sekai-Kyusei-Kyo in a style that everyone can practise Johrei.
Following his death in 1955, his teachings spread around the world, and, today, there are 3

Ph.D. at University of London - 284 - Imperial College London


Psychological intervention and psycho-neuro-endocrino-immune network. Akira NAITO

million people practicing Johrei within families, as a natural method of maintaining health and
happiness.
This also helped the development of Nature Farming technique in countries such as Japan,
the USA, Brazil, Thailand and encouraged further research into the possibility of totally
fertiliser- and chemical - free farming techniques.
In 1996, Dr Rosy Daniel, former Medical Director of Bristol Cancer Help Centre and a
leading proponent of the holistic approach to health creation in the UK, encountered Johrei in
Japan and on her return to England, called for the formation of a Johrei society in the UK that
would re-focus its attention on the original integrity of the Johrei philosophy and teach and
promote Johrei practice, not as religion but as an individual’s truly holistic journey towards
healing and health creation.
The British Johrei Society and the Johrei Academy was formed by Dr. Rosy Daniel, Sue Boyd,
Koichi Sakakibara and Junichi Imura, as a secular organization, and on 15th May 2001, the
British Johrei Society and the Johrei Academy were officially launched, commemorating 65th
Anniversary of the launch of Mokichi Okada’s Health-care Society of Japan.
The British Johrei Society is currently helping to oversee scientific research, taking place
around the world, on the efficacy of Johrei. The main research is being directed by Prof. John
Gruzelier of Imperial College, University of London.

11. Johrei practice

It is useful to be aware that their area three distinct styles in which Johrei is practiced:

(1) Johrei as daily practice

This is the style of Johrei, which you w