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39Sleep and Hypnosis, 10:2, 2008
INTRODUCTION
In the past decade DNA microarray technology
has made it possible to measure the expression
levels of many thousands of genes
simultaneously. This novel experimental
approach has revolutionized research in
molecular biology and become a new standard
in personalized medicine (Eisen et al. 1998).
Recent research has documented the use of DNA
microarrays for assessing therapeutic responses
to psychological relaxation and meditative
practices on the molecular-genomic level (Dusek
et al., 2008; Rossi, 2002, 2004, 2007; Rossi,
2005/2006). This has lead to calls for further
research on the pathways of psychotherapeutic
A Pilot Study of Positive Expectations
and Focused Attention via a New
Protocol for Optimizing Therapeutic
Hypnosis and Psychotherapy Assessed
with DNA Microarrays: The Creative
Psychosocial Genomic Healing Experience
Ernest Rossi1, Salvatore Iannotti1, Mauro Cozzolino2, Stefano Castiglione2,
Angela Cicatelli3, Kathryn Rossi1
We extend the use of DNA microarrays to explore a new psychotherapeutic therapeutic
protocol, The Creative Psychosocial Genomic Healing Experience, an easy-to-learn
approach to facilitating therapeutic hypnosis, psychotherapy, rehabilitation, meditation,
and pastoral counseling. This pilot study assessed the hypothesis that a top-down creatively
oriented positive human experience can modulate gene expression on the molecular level.
A DNA microarray data analysis of the white blood cells of three human subjects was
performed immediately before, one hour after, and 24 hours after The Creative
Psychosocial Genomic Healing Experience. We documented changes in the expression of
15 early response genes within one hour that apparently initiated a further cascade of 77
genes 24 hours later. This could provide the mind/molecular genomic foundation of new
therapeutic models for optimizing human consciousness, health, and well being via
therapeutic hypnosis, psychotherapy, pastoral counseling, and psychiatry. This proof-of-
principle pilot study now requires cross validation with more subjects with a variety of
diagnostic classifications to document the validity and reliability of using DNA microarrays
to assess our new creative psychosocial genomic therapeutic protocol in a variety of
cultures. (Sleep and Hypnosis 2008;10(2):39-44)
Key words: Creative Experience, DNA microarray, gene expression, therapeutic hypnosis.
ORIGINAL ARTICLE
Acknowledgement: Microarray experiments have been performed by
the CRIBI Microarray Service at University of Padova, Italy
(http://microcribi.cribi.unipd.it).
1La Nuova Scuola Di Neuroscienze Ipnosi Therapeutica (The New
Neuroscience School of Therapeutic Hypnosis), San Lorenzo Maggiore, Italy.
2The University of Salerno, Italy.
3Angela Cicatelli is at the University of Naples, Italy.
Address reprint requests to: Ernest Rossi,
125 Howard Ave. Los Osos, California, 93402, USA
Ernest@ErnestRossi.com, Tel. 805-528-0200 Fax 805-528-0700
Accepted August 8, 2008
processes on all levels from mind to gene
(Abbott, 2008; Nestler, 2008; Rossi et al., 2006).
In this pilot study we use DNA microarrays to
assess a new therapeutic protocol, The Creative
Psychosocial Genomic Healing Experience, is a
relatively brief, easy-to-learn process for
facilitating a wide range of therapeutic
approaches such as therapeutic hypnosis,
psychotherapy, rehabilitation, meditation, and
pastoral counseling (Rossi, 2005/2006; Rossi
and Rossi, 2008a and 2008b).
MATERIAL and METHODS
Three highly susceptible hypnotic
subjects, one male and two females,
experienced therapeutic hypnosis following
the new protocol called, “The Creative
Psychosocial Genomic Healing Experience”
established by Rossi (2004).
Peripheral blood, about 10 ml, was
collected immediately before, within one
hour of therapeutic treatment (the length of
each treatment depended by the subject, but
never went more than one hour). A total of 6
blood samples were employed to purify total
RNA from leukocytes (the nucleated part of
peripheral white blood cells) using the kit
LeukoLOCK(TM) Total RNA system
following the instruction supplied by the
manufacture (Ambion, USA).
The amount of total RNA extracted from each
blood sample was quantified using the
NanoDrop ND-1000 photometer (Wilmington,
DE). The purity of RNA samples was determined
based on the ratio of spectrophotometric
absorbance of the sample at 260 nm to that of
280 nm (A260/A280). However not all the RNA
samples resulted pure enough (A260/A280 ratio
of 1.8) to go through the microarray procedure,
in fact protein contamination was still present in
some of the samples. A further purification step
by means of one phenol chloroform and one
chloroform treatment was necessary to eliminate
the contaminants.
Total RNA of each purified sample was
further quantify using the NanoDrop
apparatus and its integrity was ascertained by
means of electrophoresis in agarose gels
followed by ethidium bromide staining on
the ribonucleic acid. Around 2.5 µg of total
RNA was delivered to the MicroCRIBI Service
(University of Padova, Italy) for microarray
analysis. MicroCRIBI Service performed the
microarray analysis on 21,329 - 70mer
oligonucleotides (Operon version 2.0)
designed on Human Unigene clusters.
For each sample, 1.0 µg of total RNA was
reverse transcribed and labelled using Amino
Allyl cDNA Labeling Kit (Ambion, USA)
following the manufacture instruction.
Cy3/Cy5 was from Amersham Biosciences
(Amersham, United Kingdom). Cy3/Cy5 dye
incorporation into aRNA yielded
incorporation rates of 30 to 60 dye molecules
per 1000 nucleotides by spectrophotometric
analysis, as requested by the manufacturer.
The microarrays were scanned with a two-
channel confocal microarray scanner
(ScanArray# Lite, Perkin Elmer,USA) using its
dedicated software (ScanArray Express
3.0.0.,Perkin Elmer). The laser power and the
photomultiplier tube (PMT) were set between
70% and 80% of maximum. The excitation/
emission settings were 543/570 nm for Cy3
and 633/670 nm for Cy5. After laser focusing
and balancing of the two channels, scans were
conducted at a resolution of 5 µm. For any
scan, two separate 16-bit TIFF images were
produced. Data were normalized by
ScanArray Express using the LOWESS
(Locally Weighted Regression Scatter Plot
Smoothing; Cleveland, 1979) algorithm
After normalization, data from each slide
were split in two, by using Microsoft Excel, since
each probe is spotted twice. Thereafter, each
spot value was considered to be independent
and subjected to SAM (Significance Analysis of
Microarrays; Tusher et al., 2001) analyses.
Since each comparison (S1/S1-1h, S2/S2-
1h, S3/S3-1h,) was repeated at least twice,
there were at least four values for each gene
to be used in the SAM analyses.
Lists of genes with significant changes in
40
A Pilot Study of Positive Expectations and Focused Attention via a New Protocol for Optimizing Therapeutic...
Sleep and Hypnosis, 10:2, 2008
expression among at least two experimental
samples were identified at delta values that
gave a false discovery rate (FDR) of 0%.
The possible role played by therapeutic
hypnosis via “The Creative Psychosocial
Genomic Healing Experience” on three
subjects in up-regulating gene expression in
leukocytes in the peripheral blood was
investigated. To accomplish this,
transcriptome changes were first monitored
in untreated subject, just after the treatment.
The transcriptome variations were analysed
by means of the Operon Human Genome
Oligo Set Version 2.0 platforms. The effect of
positive and creatively oriented therapeutic
hypnosis immediately after the session was
investigated using a direct comparison
experimental design, with four repetitions of
which one was a dye swap. As each probe was
spotted twice on human array, the following
SAM (Significance Analysis of Microarrays,
Tusher et al., 2001) analysis was performed
on a dataset of eight values for each gene.
Experimental Design
The effect of positively oriented therapeutic
hypnosis via the administration of our
Creative Psychosocial Genomics Healing
Experience on gene transcription was
monitored and the subject’s measurements
before the treatment were the common
references. Each comparison (S1/S1-1h,
S2/S2-1h; S3/S3-1h) was repeated at least
twice, so that at least four values for each gene
were used in the subsequent SAM analyses.
The dataset was tested with SAM using a ‘one
class’ study design. This analysis was carried
out on the following three subgroups of data:
S1/S1-1h, S2/S2-1h; S3/S3-1h. Using a delta of
0.2 and a median false discovery rate (FDR) of
0.00%, the analysis yielded 3207 genes as
differentially expressed, six groups of genes
were selected (the up- and down-regulated for
each of the three subgroups of data). By
crossing these data, it was possible to show the
different up- and down-regulating effects.
RESULTS
DNA microarray results on the three
subjects in response to the therapeutic
protocol within one hour after the treatment
indicated that expression of 15 early
response genes were up-regulated between
1.2 and 1.8 folds and no single gene was
down-regulated. The list of the up-regulated
genes is presented in Table 1.
41
Ernest Rossi et al.
Sleep and Hypnosis, 10:2, 2008
Table 1. The Gene Bank Accession, Gene Symbol, Gene Description and results in fold changes in response to therapeutic hypnosis.
GB_accession Gene_Symbol Description Fold changes
AK057104 Homo sapiens cDNA FLJ32542 fis, clone SMINT2000537
Sodium-coupled neutral amino acid transporter 2 1,777714817
NM_000329 RPE65 Retinal pigment epithelium-specific protein (65kD) 1,664647867
AK055997 Homo sapiens cDNA FLJ31435 fis, clone NT2NE2000612
Ring Finger protein 165 1,617968537
AK056729 Homo sapiens cDNA FLJ32167 fis, clone PLACE6000450
Serpin B Proteinase Inibitor 1,596523872
NM_001074 UGT2B7 UDP glycosyltransferase 2 family, polypeptide B7 1,578875081
BC018130 F2RL1 Coagulation factor II (thrombin) receptor-like 1 1,506199199
NM_030824 FLJ14356 Hypothetical protein FLJ14356 zinc finger protein 442 1,469687506
NM_021122 FACL2 Fatty-acid-Coenzyme A ligase, long-chain 2 1,380622376
NM_004126 GNG11 Guanine nucleotide binding protein 11 1,372082479
NM_020980 AQP9 Aquaporin 9 1,366899043
NM_001186 BACH1 BTB and CNC homology 1, basic leucine zipper transcription factor 1 1,330834867
NM_002921 RGR Retinal G protein coupled receptor 1,312291611
NM_024911 FLJ23091 Hypothetical protein FLJ23091
G protein-coupled receptor 177 Isoform 1 and Isoform 2 1,274787709
NM_000860 HPGD Hydroxyprostaglandin dehydrogenase 15-(NAD) 1,224585804
NM_002110 HCK Hemopoietic cell kinase 1,190732546
DISCUSSION
The major limitation of this pilot study
was the small number of subjects and the
lack of appropriate controls. The lack of
statistical power in this pilot study, due to the
limited number of treated subjects, for
example, does not enable us to assess the
degree to which our results coincides with
previous research supporting the hypothesis
that therapeutic hypnosis could modulate
gene expression (Lichtenberg et al., 2000,
2004). Well funded major research studies in
psychiatric genetics utilizing DNA
microarrays typically include as many as
2,000 to 20,000 subjects (Abbott, 2008). Our
results, however, suggest that this pilot study
provides documentation consistent with the
hypothesis that our new therapeutic
protocol, The Creative Psychosocial Genomic
Healing Experience, may modulate gene
expression in human white blood cells.
These preliminary findings suggest that
positive expectation via The Creative
Psychosocial Genomic Healing Experience, a
new protocol for facilitating therapeutic
hypnosis, generated the up-regulation of 15
early response genes within one hour.
The expression of these early response
genes apparently initiated a larger cascade of
gene expression 24 hours later. This
unexpected finding may have important
implications for the role of time and post-
hypnotic suggestion in therapeutic hypnosis
and many other psychological experiences.
We are currently investigating the
unexpected finding that a gene associated
with bipolar disorder may be over expressed
in response to our new protocol of
therapeutic hypnosis.
We propose that the genes expressed in
response to this new protocol may be related
to a variety of functions associated with stress
(Dusek et al. 2008), cognition and dreaming
(Riberio et al., 2007), and psychiatric
conditions (Tsankova et al., 2007).
Suggestions for further research in this area
have been recommended previously (Kustra
et al., 2006; Nestler, 2008; Nuzzo, 2008).
We introduced a new therapeutic protocol,
The Creative Psychosocial Genomic Healing
Experience, for assessing the contribution of
positive expectation, focused attention,
therapeutic hypnosis, and psychotherapy to
stress reduction and mind-body healing
(Rossi, 1986/1993, 2002, 2004, 2007; Rossi
and Rossi, 2008b). A salient feature of this
new protocol is that it was found to be more
acceptable, with high face validity for the
subjects who experienced it and the
psychiatrist who administered it, as a positive
therapeutic process in contrast to the more
research oriented classical scales of measuring
hypnotic susceptibility, which have been
questioned regarding their appropriateness
for therapeutic applications (Fromm & Shor,
1972 Wester and Sugarman, 2007).
Our new protocol, The Creative
Psychosocial Genomic Healing Experience,
however, now requires standardization in
relation to the classical assessments of
therapeutic hypnosis such as the Stanford
Hypnotic Susceptibility Scale (Hilgard, 1965)
and the Harvard Group Scale of Hypnotic
Susceptibility (Shor and Orne, 1978) as well
as the more general evaluation of
consciousness and focused attention with
objective measures such as the Tellegen
Absorption Scale (Tellegen, 1981, 1982,
1992; Tellegen & Atkinson, 1974).
The successful utilization of this new
Creative Psychosocial Genomic Healing
Experience protocol for assessing research
on humans with DNA microarrays may
foreshadow a new psychosocial genomic
paradigm to facilitate a “top-down”
therapeutic approaches from mind to gene
(Rossi, 1986/1993, 2007; Rossi and Rossi,
2008a & b). This could provide the
mind/molecular genomic foundation of new
therapeutic models for optimizing human
consciousness, health, and well being via
therapeutic hypnosis, psychotherapy,
pastoral counseling, and psychiatry.
42
A Pilot Study of Positive Expectations and Focused Attention via a New Protocol for Optimizing Therapeutic...
Sleep and Hypnosis, 10:2, 2008
CONCLUSIONS
This pilot study assessed the hypothesis
that a creatively oriented positive human
experience of therapeutic hypnosis could
modulate gene expression on the molecular
level. We documented changes in the
expression of 15 early response genes
within one hour that apparently initiated a
further a further cascade of 77 genes 24
hours later. This proof-of-principle pilot
study now requires cross validation with
more subjects to document the validity and
reliability of using DNA microarrays to
assess our therapeutic protocol, The
Creative Psychosocial Genomic Healing
Experience, as a new approach for
facilitating therapeutic hypnosis,
psychotherapy, rehabilitation, meditation,
and pastoral counselling.
43
Ernest Rossi et al.
Sleep and Hypnosis, 10:2, 2008
REFERENCES
1. Abbott A. The brains of the Family. Nature 2008; 454,
7201:154-157.
2. Dusek J, Hasan H, Wohlhueter A, Bhasin M, Zerbini L, Joseph
M, Benson H, Libermann T. Genomic Counter-Stress Changes
Induced by the Relaxation Response. PLoS ONE 2008; 3(7):
e2576. doi:10.1371/journal.pone.0002576.
3. Eisen M, Spellman P, Brown P, Botstein D. Cluster analysis and
display of genome-wide expression patterns. Proceedings of the
National Academy of Sciences, 1998;95:14863-14868.
4. Fromm E, Shor R. Hypnosis: Research Developments and
Perspectives. Chicago:Aldine Publishing, 1974.
5. Hilgard, ER. Hypnotic Susceptibility. NewYork: Harcourt, Brace
& World, 1965.
6. Kustra R, Shioda R, Zhu M. A factor analysis model for
functional genomics. BMC Bioinformatics, 2006; 7:216doi:
10.1186/1471-2105-7-216.
7. Lichtenberg P, Bachner-Melman R, Gritsenko I, Ebstein R.
Exploratory association study between catechol-O-
methyltransferase (COMT) high/low enzyme activity
polymorphism and hypnotizability. American J. Medical
Genetics 2000; 96:771-774.
8. Lichtenberg P, Bachner-Melman R, Ebstein R, Crawford H. Hypnotic
susceptibility: multidimensional relationships with Cloninger's
Tridimensional Personality Questionnaire, COMT polymorphisms,
absorption, and attentional characteristics. International Journal
Clinical of Experimental Hypnosis 2004;52:47-72.
9. Nestler E. A psychiatrist talks about finding answers that add up
across all levels. Nature 2008; 451:1033.
10. Nuzzo R. Nabbing suspicious SNPs. Scientists search the whole
genome for clues to common diseases. Science News 2008;173:20-24.
11. Ribeiro S, Xinwu S, Engelhard M, Zhou Y, Zhang H, Gervasoni D,
Lin S, Wada K, Lemos N, Nicolelis M. Novel Experience Induces
Persistent Sleep-dependent Plasticity in the Cortex but not in the
Hippocampus. Frontiers in Neuroscience 2007;1:43–55.
12. Rossi ER. The Psychobiology of Mind-Body Healing: New
Concepts of Therapeutic Hypnosis. 2nd ed. New York: W. W.
Norton, 1986.
13. Rossi ER. The Psychobiology of Gene Expression: Neuroscience
and Neurogenesis in Therapeutic Hypnosis and the Healing Arts.
NewYork: W. W. Norton Professional Books, 2002.
14. Rossi ER. (Translator & Editor, Salvador Iannotti:
Saiannot@tin.it) Discorso Tra Geni [Italian]. A Discourse with
Our Genes: The Psychosocial and Cultural Genomics of
Therapeutic Hypnosis and Psychotherapy. Benevento, Italy:
Editris SAS Press. [Available in Italian and English editions]
New York: Zeig, Tucker, Theisen, 2004.
15. Rossi ER. Prospects for Exploring the Molecular-Genomic
Foundations of Therapeutic Hypnosis with DNA Microarrays.
American Journal of Clinical Hypnosis 2005/2006;48:165-182.
16. Rossi ER The Breakout Heuristic: The New Neuroscience of
Mirror Neurons, Consciousness and Creativity in Human
Relationships: Selected Papers of Ernest Lawrence Rossi.
Phoenix, Arizona: The Milton H. Erickson Foundation Press,
2007.
17. Rossi ER, Rossi, KL, Yount G, Cozzolino M, Iannotti S. The
Bioinformatics of Integrative Medical Insights: Proposals for an
International PsychoSocial and Cultural Bioinformatics Project.
Integrative Medicine Insights 2006. http://www.la-press.com/
integmed.htm.
18. Rossi ER, Rossi, KL. Open Questions on Mind, Genes,
Consciousness, and Behavior: The Circadian and Ultradian
Rhythms of Art, Beauty, and Truth in Creativity. In Lloyd &
Rossi (eds.) Ultradian rhythms from molecule to mind: A new
vision of life. New York: Springer, 2008a; 391-412.
19. Rossi ER, Rossi KL. The New Neuroscience of Psychotherapy,
Therapeutic Hypnosis & Rehabilitation: A Creative Dialogue
with Our Genes. A Free Book on Line at www.ErnestRossi.com,
2008b.
20. Shor R, Orne, E. Harvard Group Scale of Hypnotic Susceptibility.
Palo Alto, California: Consulting Psychologists Press, 1978.
21. Tellegen A. Practicing the two disciplines for relaxation and
enlightenment: Comment on Qualls & Sheehan. Journal of
Experimental Psychology: General, 1981;110:217-226.
22. Tellegen A. Content categories: Absorption Items (Revised).
Unpublished Manuscript, University of Minnesota, 1982,
October 10.
23. Tellegen, A. Note on structure and meaning of the MPQ
Absorption scale. Unpublished Manuscript, University of
Minnesota, 1992, August.
24. Tellegen A, Atkinson G. Openness to absorbing and self-altering
experiences ("absorption"), a trait related to hypnotic
susceptibility. Journal of Abnormal Psychology 1974;83:268-277.
25. Tsankova N, Renthal W, Kumar A, Nestler E. Epigenetic
regulation in psychiatric disorders. Nature Reviews Neuroscience
2007;8:355-367.
26. Tusher V, Tibshirani R, Chu G. Significance analysis of
microarrays applied to the ionizing radiation response.
Procedings of the Natational Acadamy of Science, U S A,
2001;98(9):5116-5121.
27. Wester W, Sugarman L. Therapeutic Hypnosis with Children
and Adolescents. Bethel, CT: Crown House Publishing, 2007.
44
A Pilot Study of Positive Expectations and Focused Attention via a New Protocol for Optimizing Therapeutic...
Sleep and Hypnosis, 10:2, 2008
