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original paper

THE SIMILIA PRINCIPLE AS A THERAPEUTIC


STRATEGY: A RESEARCH PROGRAM
ON STIMULATION OF SELF-DEFENSE
IN DISORDERED MAMMALIAN CELLS
Roeland Van Wijk, PhD and Fred AC Wiegant, PhD

Roeland Van Wijk and Fred AC Wiegant are from the he essence of homeopathy is the stimulation of bio-
Department of Molecular Cell Biology, Faculty of Biology,
Utrecht University, Utrecht, the Netherlands.

The similia principle is considered to be the essence of homeopa-


thy. This article describes a research program for study of the similia
principle in cultured mammalian cells. This systematic program with
T logical defense and recovery mechanisms by the use
of compounds according to the similia principle.
The similia principle suggests that any state of dis-
turbance that is not corrected spontaneously (and
leads to a state of “disease”) can be corrected by minute doses of
compounds that at a higher dose can produce effects closely
resembling the symptoms of the disease being treated or by
minute amounts of the compounds that actually caused the dis-
its rather simple research model was set up ultimately to contribute to
the design of studies of the similia principle with more complex organ- ease.
isms such as humans. With respect to application of the similia prin- When cells are damaged, a mechanism is switched on to
ciple, the concepts of self-defense and self-recovery are central. At the withstand the disturbing effects and to stimulate recovery.
cellular level, self-defense and recovery largely depend on the avail- Increased understanding of such a mechanism would strengthen
ability of proteins with a cell-protective function, most notably, stress the foundation of the similia principle.1,2 The basis of this mecha-
or heat shock proteins. nism that regulates the transition between life and death has
To study the similia principle, we use four lines of research to been the subject of much research for many decades. As a tenta-
examine the processes of self-defense. First, stimulation of self-defense tive approach to characterizing the essence of the living state, it
in disturbed and disordered cells is studied by using low doses of an was proposed that life is a process of being an “organizing enti-
agent homologous or identical to the disturbing agent. The second line ty,” which means an open system that (re)structures and
of research deals with the specificity of this stimulation: Is cellular self-
(re)organizes itself. Defense and recovery are biological phenom-
defense after exposure to toxicant A also effectively stimulated in an
analogous or heterologous way by low doses of other toxicants such as ena that encompass the way in which components of the system
B or C? The third line of research involves the duration of low-dose sen- are temporally, spatially, and hierarchically rearranged. To study
sitivity of disordered cells for homologous stimulations, in particular, the similia principle, one must understand how to intervene in,
the desensitization of cells toward these homologous stimulations. The adjust, or stimulate these recovery processes. This approach
fourth line of research deals with whether—according to the similia requires quantitative experimentation and evaluation methods
principle—the state of desensitization can be overruled by heterologous (eg, mathematical modeling) and thus requires parameters to
condition(s) that induce an analogous pattern of protector proteins study the damage-induced recovery mechanisms experimentally.
(ie, a pattern closely resembling the damage-induced pattern) and This article describes the phases of a research program to study
thus effectively stimulate cellular defense and recovery. (Alternative the similia principle in cultured mammalian cells.
Therapies in Health and Medicine. 1997;3(2):33-38) Understanding of the mechanisms of cellular protection
and recovery from damage in non–lethally injured cells at the
molecular level has increased greatly in recent years. These
processes appear to be largely dependent on the availability of
proteins with a cell-protective function, most notably stress or
heat shock proteins.3-5 When working with cells as a model sys-
tem, it is crucial to determine what actually occurs in cases of
Reprint requests: Innovision Communications, 101 Columbia, Aliso Viejo, CA 92656. Tel, (800) disorder and especially the way the defense and recovery mecha-
899-1712 or (714) 362-2050 (ext 532); fax, (714) 362-2022; e-mail, ivcReprint@aol.com. nisms are regulated to prevent further infliction of injury or cell

The Similia Principle as a Therapeutic Strategy ALTERNATIVE THERAPIES, MARCH 1997, VOL. 3, NO. 2 33
death. This article thus focuses on the research strategies for Major stress protein families and their functions
determining which cellular components play a role in defense
and on the integration of this information. The chosen research Hsp Stress protein Functions in cell metabolism
strategy is on complex systems, which requires the analysis of family and cellular stress defense
nonlinear interactions in a network. The task of discovering the
hsp100 hsp100 Heat and ethanol tolerance
nature of the defense process during interventions according to
the similia principle is the next topic of research. Next, the pos- hsp90 hsp90 (hsp84) Stabilization of proteins;
tulated, precise kinetic and interactive behavior of defense pro- maintenance of the inactive
teins must be tested and it must be shown that the results of the protein form during trans-
port
various experiments, when fitted into a quantitative model, are
in accord with the observed changes. Essential for cell viability

DAMAGE AND DEFENSE hsp70 grp94 (grp95) Role in protein folding?


Proteotoxicity hsp70 (hsp73/hsc70) Molecular chaperone;
The so-called stress response, which started as a molecular required for protein assem-
curiosity in fruit flies in the early 1960s, now constitutes an bly, protein translocation,
active area of research in molecular cell biology.3 The heat shock secretion and import into
organelles
proteins, one of the most highly conserved group of proteins
characterized so far, are implicated as being essential compo- hsp68(hsp72/hspi70) (Thermo)tolerance
nents in a number of diverse biological processes, in particular
cellular protection. Following the nomenclature first used for Promotes survival in extreme
temperatures
fruit flies, the various heat shock proteins in animal cells are
named on the basis of their mode of induction and apparent grp78 (BiP) Maturation of proteins for
molecular mass. Hence their designation as hsp70 or grp78, for excretion protein transloca-
example, refers to heat shock proteins of 70 kD and glucose-reg- tion in mitochondria
ulated proteins of 78 kD, respectively. A survey of these proteins hsp60 hsp60 Molecular chaperone that
is presented in the Table. facilitates folding of
Earlier work was perplexing in that many different agents monomeric proteins and
were able to lead to similar changes in gene expression. assembly of oligomeric pro-
tein complexes; mainly in
However, in the past 15 years, a variety of observations have pro- mitochondrial matrix
vided support for the so-called abnormal protein hypothesis sug-
gested to explain the induction of the heat shock response.6 This small hsps hsp32 Metabolism of haem
aspect of toxicity at the level of proteins has been termed pro- (haem oxygenase)
hsp27 Contributes to thermotoler-
teotoxicity.7 When cells have been exposed to heat shock or to ance and cytoskeletal stabi-
toxic substances such as cadmium and arsenite, or to oxidative lization
stresses, many proteins exhibit deleterious changes in structure.
These changes then interfere with the functional interactive ubiquitin Protein denuration
capabilities of these proteins. The risk is also high that these
Adapted from Parsell and Lindquist4 (1993)
abnormal protein molecules aggregate not only with other dam-
aged proteins but also with still functional proteinaceous cellular
structures. The damage at the level of proteins lies at the basis of
cellular damage and eventually cellular death. of stress proteins, collectively called chaperones, ensure that
polypeptides fold and assemble properly in the cell. The stress
Stress Proteins in Cell Protection proteins are also named according to their function. Examples
To understand cellular recovery, one must study the mecha- include chaperone proteins (which form complexes with proteina-
nisms that are activated when proteins are damaged and deter- ceous and other, more intricate, cellular structures in order to
mine how the cell copes with these damaged, life-threatening prevent premature or deleterious interactions between pro-
molecules. teins), binding proteins (which form complexes with receptor pro-
Some work on in vitro refolding of a small number of pro- teins to regulate their activity, ie, a receptor function), or
teins led to the long-lasting impression that folding of newly syn- protector proteins (which have crucial functions in the mechanism
thesized polypeptides is an intrinsic feature of their primary of cell defense and recovery). In this respect, the protector pro-
structure, independent of other factors. However, the relatively teins seem crucial in many ways in the normal functioning of
high protein concentration in the cytosol gives rise to misfolding proteins under adverse conditions and in the refolding of struc-
followed by aggregation. To deal with this kind of problem, a set turally damaged proteins.4

34 ALTERNATIVE THERAPIES, MARCH 1997, VOL. 3, NO. 2 The Similia Principle as a Therapeutic Strategy
Pattern of Protector Proteins is Damage Specific replenishment of these protector proteins starts with activation
A variety of different protector proteins have been found. of associated protector protein gene promoters on the cell’s
Interestingly, the pattern of protector proteins that is induced DNA. This highly specific activation occurs by binding of specif-
appears to be damage specific.8-10 Under different deleterious ic DNA-binding factors, called heat shock transcription factors
conditions, qualitatively different patterns of protector proteins (HSFs), on these DNA sites.11 The binding of the HSF to the pro-
seem to be induced. Although an explanation for such a complex moter on the cell’s DNA is the signal that triggers transfer of
regulation is lacking, the patterns do suggest some differentia- information from DNA into messenger RNA (mRNA), leading
tion in the induction of the type of damage-induced protection eventually to synthesis of new protector proteins. Whether or
mechanisms. not these DNA-binding factors interact with the DNA depends
Because these protector proteins are essential to survive on the existing quantity of protector proteins in the cell. The
threatening conditions and because their induction occurs at genome is specifically activated to trigger this synthesis of addi-
concentrations of various toxic compounds that are not yet tional protector proteins only when the quantity of protector
lethal for the cells, the damage-specific pattern of induction may proteins falls below a certain threshold. Normally, at least one
be considered as the cellular equivalent of the homeopathic rem- type of protective protein, hsp70, and a DNA-binding factor,
edy picture. HSF, form a complex that provides the basis for this regulation.
If protector proteins are required to neutralize abnormal pro-
The Problem of Regulation teins, this complex then dissociates, releasing HSF, which then
of Availability of Protector Proteins binds to the promoters and induces production of mRNA, with
A simple model for the regulation of protector proteins in the ensuing synthesis of new protector proteins. When sufficient
defense after cell damage is depicted in Figure 1. The quantity of new protector proteins have been produced, that is, when the
free protector proteins available in the cell decreases after cell level of these proteins is raised above the threshold value, hsp70
damage. As long as these protector proteins are available, dam- will again form a complex with HSF molecules, uncoupling the
age is reduced to a minimum. However, when a shortage of pro- HSF from DNA, with a concomitant halt in production of
tector proteins arises in the case of an overload of damage, the mRNA. In terms of systems theory, one might say that this is the
abnormal protein molecules can complex with other cell struc- autoregulation loop that forms the basis of damage-induced
tures. Cell damage and death can then be avoided only by pro- recovery processes.
duction of increasing amounts of new protector proteins. The
Mathematical Modeling as a Future Step
in Understanding the Regulation of Defense
Defense and recovery are not simple and singular phenom-
Proteotoxicity Compensation cycle ena but are determined by the kinetic parameters of the autoreg-
HSF (inactive) ulation loop as illustrated in Figure 2. Insight into the
effectiveness of this loop for cellular defense can therefore be
Pool of free obtained only from studies involving the parameters mentioned
HSPs in this mathematical model. This mathematical model of defense
Stress
Protector protein and recovery processes has only recently started with the devel-
opment of a model of hsp70 regulation in the cell.12 So far, five
main blocks, each describing one or more processes involved in
HSF (active) the functions of hsp70 and its synthesis, have been developed.1,12
Denatured These blocks constitute the basic structure of a mathematical
DNA
protein model for the regulation of hsp70 in cells. The major processes
included in the model so far are (1) denaturation of proteins
HSPmRNA after an increase in temperature and the binding of hsp70 to
denatured or nascent proteins; (2) interaction of hsp70 with HSF
and the activation of HSF not bound to hsp70; (3) interaction of
activated HSF with a specific HSF binding sequence (heat shock
element, HSE) on DNA; (4) the relationship between HSE and
HSP transcriptionally active HSF and the level of hsp70mRNA active-
ly engaged in synthesis of hsp70; and (5) translation of viable
FIGURE 1 Basic processes of damage and defense at the cellular hsp70mRNA into its protein.
level. Proteotoxicity is illustrated as a change in protein conforma-
In the model constructed so far, the output of this regula-
tion. Protector proteins interact with these abnormal proteins.
After depletion of the pool of free HSPs, supplementation of these tion is the level of free, that is, unbound, hsp70 in the cell. Future
protector proteins occurs by way of the compensation cycle. steps in developing this mathematical model concern the inclu-
sion of lethality in order to predict stress-dependent survival and

The Similia Principle as a Therapeutic Strategy ALTERNATIVE THERAPIES, MARCH 1997, VOL. 3, NO. 2 35
SIMILIA PRINCIPLE
2.0 A According to homeopathic insights, recovery can be stimu-
lated by all kinds of substances, provided that they are applied in
a specific way. Of all substances, the one most suitable for stimu-
Relative synthesis of hsp70

1.5 lation of recovery is the one that can produce the artificial situa-
tion of disorder that most closely resembles the disordered state.
In other words, this concept hinges on the resemblance between
1.0 symptoms of the disturbed system and the symptoms caused by
the applied substance in a healthy system.

0.5 Application of Low Doses


of Homologous Damaging Conditions
The similia principle in its most elementary form can easily
be tested at the cellular level by determination of the extent to
0.0
which recovery is stimulated by a small dose of a substance that
0 2 4 6 8
in the first instance—at a higher dose—is responsible for dereg-
Time (hours) ulating the system or leading to sickness. The relevant question
for this overview is whether this stimulation will be translated
60 B into increased synthesis of protector proteins and increased sur-
vival of cells.
This hypothesis, representing homologous sensitization,
Relative survival (%)

40
has been studied by a step-down heating protocol in which the
initial treatment with high heat is immediately followed by a sec-
ond treatment at lower doses. When these step-down heating
conditions were used, an enhanced synthesis of protector pro-
20 teins was indeed seen.13-15 Other studies show an enhanced occur-
rence of thermotolerance when mild step-down heatings were
used.16
The next relevant question is whether this response is a gen-
0
C x X X-x eral occurrence and can thus also be observed after stressor con-
ditions other than heat shocks. To this end, a damage-inducing
treatment of cells with arsenite or cadmium was followed by low
FIGURE 2 Schematic presentation of the effect of a low dose of a doses of arsenite or cadmium, respectively. When this step-down
homologous stressor on the synthesis of hsp70 (A) and on devel- treatment with arsenite or cadmium was used, cells exhibited an
opment of tolerance (B). When the stress condition is followed
enhanced synthesis of various protector proteins and develop-
by a low dose of the same stressor, an enhancement of hsp70 syn-
thesis (filled triangles) and of tolerance development is observed ment of tolerance.17,18 The lower doses had no effect on synthesis
in comparison with the effect of the stress condition alone (open of protector proteins or development of tolerance in cells that
circles). A low dose of the stressor does not influence the control were not pretreated (ie, healthy cells).
cells (open triangles). C=control, x=low-dose stressor, X=stres- Figure 2 shows, schematically, the transient induction of
sor, X-x=stressor followed by low-dose stressor. hsp70. The synthesis of this protein is further enhanced when a
low stressor dose is applied during the so-called posttreatment
period. The level of tolerance that is achieved is also higher when
the low dose is applied after treatment as compared to cells that
changes in cellular sensitivity to stressors. received a pretreatment only.
To sum up, the integrity of the cellular systems, their
defense and recovery, depends not only on the available protec- The Specificity of the Low-Dose Effect
tive proteins but also on the speed of activation of the total res- With respect to the specificity of the defense-enhancing
cue mechanism. Future developments in this research are effect of low doses of an analogous stressor, some recent studies
directed at the damage dependency and differentiation of the examined induction of protector proteins in sensitized cells by
mentioned production process. A quantitative experimental low doses of analogous but potentially damaging conditions. So
analysis and a mathematical evaluation are considered essential far, experiments have examined the effects of pretreatments
for this study and for understanding any interventions in this given with either a heat shock, sodium arsenite, or cadmium
production process, particularly interventions according to the chloride. After these pretreatments, the cultures were exposed to
similia principle. low doses of heat, arsenite, or cadmium or to control conditions.

36 ALTERNATIVE THERAPIES, MARCH 1997, VOL. 3, NO. 2 The Similia Principle as a Therapeutic Strategy
The stimulating effect on the synthesis of various protector pro-
teins was then studied. This application of either heat shock, 6 6
arsenite, or cadmium at subliminal conditions to already disor-

HSP inducibility (arbitrary units)


5 5
dered cells early in their recovery yielded a different degree of
synthesis of protector proteins. Only incubation of damaged

LD50 (arbitrary units)


4 4
cells with low doses of homologous conditions resulted in this
extra synthesis of protector proteins.1 Additionally, the degree of
3 3
stimulation by low doses of stressors depended on the degree of
similarity between the two stressors (Figure 3): The more similar
the induced pattern of protector proteins by two stressors, the 2 2
larger the stimulatory action. These observations confirm the
specificity of the low-dose effect and are now extended by incor- 1 1
poration of a broader range of stress conditions in the experi-
mental protocol. 0 0
0 1 2 3 4 5 6
Specific Desensitization Time (hours)
A particular aspect of the cellular response to stressors is
that the cells show a biphasic change in their sensitivity to a FIGURE 4 Schematic presentation of the change in sensitivity
stressor. An initially enhanced sensitivity (sensitization) toward (filled squares) and HSP inducibility (open squares) at various
a second application of the same stressor is followed by a times after stressor application. Closed circles=control level.
reduced sensitivity (desensitization or tolerance). This phenome-
non is represented schematically in Figure 4. This biphasic
change in sensitivity is observed after heat treatments and after
treatments with cadmium 18 or arsenite. 19 In particular, the ness (or tolerance) is also seen in the induction of synthesis of
change in sensitivity can be seen after the period of stressor- protector proteins by a second heat shock20-22 and by fractionated
induced synthesis of protector proteins. This state of refractori- arsenite17,23 or cadmium treatments.18 The consequence of this
temporal refractoriness is that homologous sensitization is only
transient. These observations led to the conclusion that low
doses of homologous substances are effective only during the
early period of recovery. The next questions in the research pro-
gram are whether this desensitization later in cellular recovery is
2.0
a stressor-specific phenomenon and to what extent a heterolo-
gous reinduction remains possible.
Relative synthesis of hsp70

Heterologous Reinduction
Recently, the specificity of desensitization of protector pro-
1.0 tein induction was tested with heat shock, sodium arsenite, and
cadmium chloride used as primary and secondary inducers of
protector proteins.23 The data suggest that the degree, but not
the pattern, of reinduction of protector proteins is influenced by
the type of stressor used in the pretreatments. Thus, stimulation
of synthesis of protector proteins after cadmium as the sec-
0 ondary stressor is severely inhibited in cadmium-pretreated
After 0 as cd 0 as cd cells, whereas reinductions after arsenite and heat shock are
Before As Cd specifically inhibited in cells pretreated with arsenite and heat
shock, respectively.
In summary, homologous sensitization is transient, and
FIGURE 3 Schematic presentation of the specificity in stimula- homologous desensitization develops specifically. Apparently, a
tion of HSP synthesis by low doses of stressors. A high dose common denominator regulates the coordinate expression of a
induces a certain level of HSPs, which is further enhanced when group of protector proteins. Heterologous reinduction can still
subsequently incubated with a low dose of the same stressor, but
occur but, in that case, the pattern of protector proteins induced
not with a low dose of stressor conditions that were not similar.
A low dose exerts no effects on the level of hsp70 found in con-
by the secondarily applied stressor shows a stressor specificity
trol conditions (black bars). that is dependent only on the second treatment and is indepen-
dent of any pretreatment.

The Similia Principle as a Therapeutic Strategy ALTERNATIVE THERAPIES, MARCH 1997, VOL. 3, NO. 2 37
Future Research Aimed application of low doses of damaging conditions. The selection
at Further Understanding of the Similia Principle of condition can be based on the molecular symptoms of the cel-
The possible implication of the results mentioned so far is lular defense process. In this respect, the application of low
that the increased production of protector proteins at a later doses according to the similia principle warrants further study.
time after an injury, that is, during the period of homologous tol- It should now also be easier to understand the role of these
erance, occurs only after application of a heterologous damaging cellular recovery processes in an organ’s functionality after expo-
compound. When we consider the pattern of synthesized protec- sure of an intact organism to stressful conditions. Therefore, the
tor proteins as the molecular symptoms of recovery, the speci- similia principle can be expected to manifest itself as a general
ficity of the similia principle reveals itself in the degree of biological phenomenon. This offers exciting opportunities for
relatedness between the effects of the disturbing compound on developing new avenues for therapeutic interventions in bio-
the pattern of synthesis of these protector proteins. We must medicine.
search for analogous damaging conditions that induce patterns
of protector proteins similar to those generated by the primary Acknowledgments
C A van der Mast, PhD, is gratefully acknowledged for critical reading of the manuscript. This work
stressor without being identical to this primary stressor. For this was supported by the HomInt organization, Karlsruhe, Germany.
step, different compounds must be tested at the cellular level.
Another possibility is to look for conditions that induce the References
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38 ALTERNATIVE THERAPIES, MARCH 1997, VOL. 3, NO. 2 The Similia Principle as a Therapeutic Strategy