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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

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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.

his truthfulness, warm understanding and spiritual support. Cyclamen (31 January 2006) The last piece of work

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

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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.

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

Akira NAITO

Contents Page Abstract 3 List of Figures List of Tables 6 9 List of Text
Contents
Page
Abstract
3
List of Figures
List of Tables
6
9
List of Text boxes
Abbreviations
14
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

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
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

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

Figure 4:

assessment time points. The number of drop-out students is also indicated. Timing of the Exam and Non-exam assessment time points with respect to recruitment for

Figure 5:

individual students in the Cohorts A and B Timing of training, follow-ups and data-collection sessions (Baseline and Exams assessment

Figure 6:

points) and numbers of students at the sessions Timing of the measurement collection time points (Recruitment and After 4 months) and the

Figure 7:

one-month blocks (Baseline, Term one to Term four) for clinical routine blood collections Timing of the two measurement points (Recruitment and Post-intervention) with regard to the

Figure 8:

period of training and practice of the psychological interventions Time-points (Training time point and the Terms according to the Training). Intervals between

Figure 9:

the doted-lines represent one term (one month) 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 1.5x10 4 , 1.5x10 5 and 1.0x10 6 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

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

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 (CD56 dim CD16 + ) 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

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

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

Table 5:

monthly time periods from the Recruitment to four months after the recruitment (Term 4) Number and month of subjects who started anti-retroviral medication and dropped from the

Table 6:

study (NB: Term X represents that X months after the Recruitment time point) Mean percentages and counts (standard deviation: SD) of NK-cells, CD4 and CD8 T-cells and

Table 7:

total lymphocyte counts using a single volunteer (9 tubes collected and averaged on three separate occasions) 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)

Table 16:

in the Not-stressed and Stressed subgroups based on the PSS scores at the Exam time point Mean (95% C.I.) State anxiety scores in the Self-hypnosis, Johrei and Relaxation control

Table 17:

groups at baseline and the Exam time point 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

Table 19:

groups at baseline and the Exam time point Percentages (numbers) of subjects whose NK-cell counts maintained or increased

Table 20:

(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) Mean (95% C.I.) levels of CD4 T-cell (%) in the Self-hypnosis, Johrei and Relaxation control

Table 21:

groups at baseline and the Exam time point Mean (95% C.I.) levels of CD8 T-cell (%) in the Self-hypnosis, Johrei and Relaxation control

Table 22:

groups at baseline and the Exam time point Medians, means and standard deviations of the PSS of the university students at non-exam

Table 23:

and exams and of the HIV-infected individuals at recruitment and four months later Mean (95% C.I.) levels of the PSS in the Not-stressed and Stressed subgroups at the

Table 24:

recruitment and four months later Median, mean and standard deviation of the State anxiety score of the university students at

Table 25:

non-exam and exams and of the HIV-infected individuals at recruitment and four months later 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

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

Table 27:

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

Table 28:

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

Table 29:

and four months later 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-

Table 34:

infected individuals of the Improved sleep quality and Decreased sleep quality subgroups Mean (95% C.I.) levels of the IES in the HIV-individuals in the Self-hypnosis, Johrei and

Table 35:

Control groups at the Baseline and Post-intervention time points Summary of the numbers (percentages) of the HIV-individuals whose changes in the LoC

Table 36:

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 Summary of the numbers (percentages) of the HIV-individuals whose changes in the MCS

Table 37:

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 Summary of the numbers (percentages) of the HIV-individuals whose changes in the PSQI

Table 38:

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 39:

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 Percentages (numbers) of participants whose CD4 T-cell counts maintained or decreased over

Table 40:

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 41:

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 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

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

Table 43:

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) 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

Table A-3:

time points Mean (95% C.I.) NK cytotoxic activity in the Cohorts A and B at the Non-exam and Exam

Table A-4:

time points Mean (95% C.I.) NK-cell percentages in the Cohorts A and B at the Non-exam and Exam

Table A-5:

time points Mean (95% C.I.) CD4 T-cell percentages in the Cohorts A and B at the Non-exam and

Table A-6:

Exam time points Mean (95% C.I.) CD8 T-cell percentages in the Cohorts A and B at the Non-exam and

Table A-7:

Exam time points 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

Table A-9:

points 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

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)

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

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

Akira NAITO

Abbreviations

ACTH: adrenocorticotrophic hormone ANOVA: analysis of variance AP-1: activation protein 1 ART: anti-retroviral treatment CBG: cortisol binding globulin CD: clusters of differentiation CFSE: carboxyfluorescein succinimidyl ester cpm: count per minute CRF: corticotrophin releasing factor / (hormone) CRP: C-reactive protein CSF: Colony stimulating factor CTL: (cytotoxic / cytolitic T lymphocyte) CV: coefficient of variation DHEA (-S): dehydroepiandrosterone (sulphate) EEG: electroencephalogram E:T ratio: Effector vs. Target ratio FITC: fluorescein isothiocyanate FCS: foetal calf serum GABA: gamma aminobutyric acid GCR: glucocorticoid receptor HIV: human immunodeficiency virus HPA: hypothalamus pituitary adrenal (axis) HSV: herpes simplex virus IES: Impact of Event Scale IL: Interleukin IFN: interferon ITAM / ITIM: immunoreceptor tyrosine-based activation / inhibitory motifs KIR: killer immunoglobulin like receptor KPSS: Kesseler Perceived Social Support LoC: Locus of Control LPS: lipopolysaccharide MCR: mineral corticoid receptor MHC: major histocompatiblity complex NCR: natural cytotoxic receptors

NF B: nuclear factor kappa B

NK cell: Natural Killer cell NKCC: Natural killer cytotoxic activity PBL: peripheral blood lymphocyte PBMCs: peripheral blood mononuclear cells PBS: phosphate buffer saline PEI: Personalised Emotional Index PFA: paraformaldehyde PHA: phytohaemagglutinin A PI: propidium iodide PNI: psycho-neuro-immunology PPD: purified protein derivative PSS: Perceived Stress Scale PSQ: Personality Syndrome Questionnaire PSQI: Pittsburgh Sleep Quality Inventory QoL: quality of life RCT: randomised controlled trial REM: rapid eye movement SAM: sympathetic adreno-medulla SAS: Statistical Analysis Software SD: standard deviation SEB: Staphylococcal Enterotoxin B SIBS: Spiritual Involvement and Beliefs Scale SoC: sense of coherence SOPs: standard operating procedures SPSS: Statistical Package for Social Sciences STAI: State and Trait Anxiety Inventory TCI: Temperament and Character Inventory TCM: tissue culture medium Th.: Helper T lymphocyte TNF: Tumour necrosis factor T-reg: regulatory helper T lymphocyte TUNEL: TdT-mediated dUTP nick end labelling WHO: World Health Organisation

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

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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

Chapter I

Introduction

- 17 -

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

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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

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

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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-

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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.

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

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Text box 1: PROJECT SYNOPSIS

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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.

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

 

HUMORAL

 

CELLULAR

 
   

Natural Killer (NK) cells

 

Cytotoxic NK-cells

Cytokines (Pro-inflammatory) Acute phase proteins Complement Lysozome Natural antibodies

Regulatory NK-cells

 

Macrophages

 

INNATE

Monocytes

Phagocytes

 

Neutrophils

Granulocytes

 

Eosinophils

   

Basophils

   

Th. 1

 

Cytotoxic T-cells (CTL)

(cellular)

   

Cytokines

Th. 2

T-lymphocytes

Helper T-cells

 

Type I (Th. 1) Type II (Th. 2) Regulatory (T-reg)

ADAPTIVE

(humoral)

 
 

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.

of the mononuclear cell fraction in normal peripheral blood. Figure 1: A typical Natural Killer cell

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.

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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

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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.

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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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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 (CD56 Dim ) express CD16 while the regulatory NK-cells (CD56 Bright ) are CD16 negative [see also Table 2].

Table 2: CD expression by NK-cells and lymphocytes

NK-cells

CD3 (-) & CD56 +

Cytotoxic

CD56 Dim & CD16 +

Regulatory

CD56 Bright & 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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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 (Catecholamines & Acetylcholine)

Chemical substances released from neurons in synapses that bind to 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 (Arachidonic acid derivatives)

Biosynthesised molecules from arachidonic acid (poly-unsaturated 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].

axis [Moynihan & Stevens, 2001] [Figure 2]. Figure 2: Schema of the neuro-endocrino-immune interaction.
axis [Moynihan & Stevens, 2001] [Figure 2]. Figure 2: Schema of the neuro-endocrino-immune interaction.
axis [Moynihan & Stevens, 2001] [Figure 2]. Figure 2: Schema of the neuro-endocrino-immune interaction.

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.

To obtain worthy awareness by commitment in and investment of his/her own existing resources.

3. Meaningfulness:

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 [ 3 H]-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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Weeks from the Recruitment

4 weeks 4-8 weeks Recruitment (48) Exams [Cohort A (21)] Non-exam [Group A (11)] Withdrew
4 weeks
4-8 weeks
Recruitment
(48)
Exams
[Cohort A (21)]
Non-exam
[Group A (11)]
Withdrew (10)
Non-exam
[Cohort B (26)]
Exams
[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.

Figure 4:

12

8

4

0

Cohort A

Non-exam Exam Recruitment

Non-exam

Non-exam Exam Recruitment
Non-exam Exam Recruitment

Exam

Recruitment

Non-exam Exam Recruitment

12

8

4

0

Cohort B

Exam Non-exam Recruitment

Exam

Exam Non-exam Recruitment

Non-exam

Recruitment

Exam Non-exam Recruitment

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 PPrraaccttiiccee && ffoollllooww--uuppss 4 weeks 4-8 weeks Baseline Exams (48) (21)
Training-session
PPrraaccttiiccee && ffoollllooww--uuppss
4 weeks
4-8 weeks
Baseline
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]:

-- RReeccrruuiittmmeenntt -- AAfftteerr 44 mmoonntthhss -- Term one Term two Term three Baseline Term
-- RReeccrruuiittmmeenntt
-- AAfftteerr 44 mmoonntthhss --
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.

-- RReeccrruuiittmmeenntt -- Baseline Term one -- PPoosstt--iinntteerrvveennttiioonn -- Term four Training
-- RReeccrruuiittmmeenntt --
Baseline
Term one
-- PPoosstt--iinntteerrvveennttiioonn --
Term four
Training
PPrraaccttiiccee && FFoollllooww--uuppss
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:

Johrei:

Self-hypnosis:

34 subjects consisting of seven training cohorts

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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TTrraaiinniinngg Baseline Term four Training Term one PPrraaccttiiccee && FFoollllooww--uuppss 1 month 3
TTrraaiinniinngg
Baseline
Term four
Training
Term one
PPrraaccttiiccee && FFoollllooww--uuppss
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.

TTrraaiinniinngg

Pre-intervention

Post-intervention

r a a i i n n i i n n g g Pre-intervention Post-intervention 4
r a a i i n n i i n n g g Pre-intervention Post-intervention 4
r a a i i n n i i n n g g Pre-intervention Post-intervention 4
4 months
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 [ 3 H] 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 56 Cr-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)

GGrraannuullooccyytteess DDeebbrriiss MMoonnooccyytteess LLyymmpphhooccyytteess FSC
GGrraannuullooccyytteess
DDeebbrriiss
MMoonnooccyytteess
LLyymmpphhooccyytteess
FSC

Figure 10: FSC vs. SSC dot plot

Fluorescence intensities at several wavelengths (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 cell debris from the fluorescence data. It is sufficient to distinguish lymphocytes from monocytes and granulocytes in blood leukocyte samples.

SSC

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].