Experimental Studies on

Basophils Exploring Fundamental

Laws of Homoeopathy:
Controversies and Pitfalls
Dr. Mohit Mathur. B.H.M.S., M.D
Reader, Dept. of Practice of Medicine. Nehru Homoeopathic Medical Collage & Hospital, Bblock, Defence colony, New Delhi (India) PIN-110024. Phone-0129-4133988
E-mail: drmohitmathur@gmail.com

Fundamental concepts:
a) Basophils:are circulating white blood cells. They have
important role in allergic reactions. On their surface they
express the high affinity receptor for IgE FcRI. Cross-linking
of IgE bound to its receptor results in the release of
histamine, cytokines and other biologically active molecules
from basophils.
b) Arndt-Schultz law: it states that small doses stimulates,
medium doses paralyze and large doses kill. In other words
the action of small and very large doses of the same
substance on living matter is opposite.
c) Law of quantity: the quality of the action of homoeopathic
remedy is determined by its quantity, in inverse ratio
(Fincke).

Introduction:

one of the most controversial points in the

history of homoeopathy is the hypothesis that highly diluted
substances-homoeopathic potencies- can have remedial
effects. So far several in vitro studies are carried out to explore
whether highly diluted substances, having theoretically no
molecule of the parent substance from which they are derived,
also have a biological activity just as the parent substance do.
The Basophils degranulation model was introduced into
homoeopathic research by Murrieta et. al. and has been the

most extensively studied model for studying the effects of high

dilutions (homoeopathic potencies). Below is the update on
some famous studies carried out on basophils to explore the
effects of homoeopathic potencies:
1. Study by Poitevin & Colleagues1-3- Two groups of French
researchers (Sainte-Laudy with Belon and Poitevin with
Aubin) conducted experimental studies on Basophils
Degranulation using homeopathic dilutions of Apis mellifica
and Lung histaminum. Apis and lung histamine were chosen
because of their frequent use in the treatment of allergic
syndromes and partially on the known ability of some of their
molecular components (mellitin and histamine, respectively)
to activate basophils or to have regulatory feed- back effects
on them.Histamine in material doses inhibits Basophils
degranulation via negative feedback mechanism. These
studies were based on the hypothesis that, even in ultra-high
dilutions, such as used in homoeopathy, these substances
can inhibit basophils Degranulation.The first publications
describing the effect of these homeopathic dilutions on
basophils reported that the in vitro degranulation induced
by various allergens (domestic dust, house mites) was
stimulated by low dilutions (5c) (centesimal and decimal
homeopathic dilutions) of bee venom (Apis mellifica),
whereas higher dilutions (9c and 15c) had an opposite effect
(>50% inhibition). These effects were obtained by including
the medicines in the incubation mixture and were
statistically significant.
In order to explore the question further, and with the support of
the team of Benveniste, Poitevin carried out another series of
blind experiments using 1c15c dilutions of Apis mellifica and
Lung histaminum on basophils activated by anti-IgE antibodies.
The dilution-effect curves of these products showed an
alternation of inhibition, inactivity and stimulation with an
unusual and complex trend: Apis significantly inhibited basophil

activation at the dilutions of 8c, 9c and 10c when the basophils

were activated with high and low anti-IgE doses, and caused
significant inhibition also at the dilutions of 5c, 7c, 13c and 20c
when the basophils were activated with low anti-IgE doses.
With Lung histaminum, significant inhibitions were observed at
dilutions of about 5c and about 15c. In the case of basophils
activated using small anti-IgE doses, Apis 10c and Lung
histaminum 18c caused 100% inhibition.
These experiments were said to illustrate application of the
principle of similarity in an experimental model: a substance
known to stimulate inflammation at conventional doses can, at
different doses, inhibit cells responsible for many of the
phenomena of the inflammatory process. Although on closer
analysis the above experiments confirms the Arndt-Schultz
law or the Law of quantity (Fincke) rather than the Law of
Similar. Law of Similar takes onto consideration two entities
having similarity in their manifestations in terms of symptoms.
The above experiments demonstrated opposite action by the
same entity (Apis and Lung histamine) albeit in different forms
(low and high dilutions).
2. The Benveniste study4-5: the study was conducted with
the view to explore the effects of dilutions of anti-IgE ranging
from 1x102 to 1 X 10120 on degranulation of human
polymorphnuclear
basophils.
Human
basophils
are
specifically stimulated by immunological stimuli such as
allergen or anti-IgE antiserum that can bridge IgE molecules
in basophil membrane. This process triggers transmembrane
and intracellular signals followed by granule exocytosis with
the release of histamine and loss of metachromatic staining
of basophil granules by a basic dye such as toluidine blue.
Benveniste
reported
that
human
basophils
undergo
degranulation (metachromasia) not only at usual anti-IgE
antibody doses (10-3 mg/ml) but also at extremely high
dilutions (1060 or 10120 times lower than the concentrations
usually leading to a molecular interaction). Using the molecular

weight of immunoglobulins and Avogadros number Benveniste

& his associates calculated that less than one molecule of
antibody was present in the assay when anti-IgE antiserum was
diluted to 1X 1014 but in their experiments they detected
significant basophil degranulation down to the 2.2 X 10 120
dilution. The doseresponse curves at decreasing doses first
showed the disappearance of activity and then its reappearance followed by various peaks of alternating activity
and inactivity up to extremely high dilutions corresponding to
practically zero antibody concentrations. This type of a dose
response curve is also called pseudo-sinusoidal. In addition to
anti-IgE antibodies, significant results were also obtained using
high dilutions of substances such as calcium ionophores and
phospholipase A2, known to have stimulatory effect at
significant doses. The specificity of action was proved by the
lack of effect from highly diluted anti-IgG antibodies (basophils
are only activated by anti-IgE antibodies), which was used as
control and have a different bio- chemical specificity on
membranes. What was more interesting, from homoeopathic
perspective, was the fact that the high-dilution effect was
observed only when the serial dilution was followed by strong
succussion of the solutions. The work of Benvenistes group
was published in the authoritative scientific journal Nature and
had considerable resonance as the presumed demonstration of
the memory of water, but it was heavily criticized on
theoretical grounds (there was no physical basis for such an
activity). Sensing the incredulity of many referees who
commented on the experiments Nature arranged for
independent investigators to observe repetition of the
experiments. The team of investigators visited the French
laboratory and eventually published a report describing the
work
as
pseudo-science.
The
Benvenistes
group
subsequently repeated the tests using more reliable methods
and more complete evaluations made by expert statisticians,
and confirmed many times the existence of an effect at high
dilutions, although it was not as marked as that described in
the first paper. These further experiments were not welcomed

by the scientific community possibly because they were

published in less renowned journals or presented in conference
proceedings.
3. Sainte-Lady and colleagues6 also performed experiments on
basophils degranulation using histamine degranulation
assays. It was found that histamine at both conventional
pharmacological concentrations and at higher dilutions
inhibited allergen and anti-IgE induced Basophils activation.
4. Studies testing basophils activation using flow
cytometry method7, 8: Basophil membrane expresses many
proteins such as adhesion molecules and high-affinity
receptors for IgE, aggregated IgG, CD26, CD33, CD40, CD45
and CD63. The CD63 is expressed on cytoplasmic granules
and on the external membrane after activation, and can thus
be used as a marker of the functional status of the cell. Flow
cytometry utilize the changes in expression of CD63 on
basophil surface as a marker of their activation. Flow
cytometry has the further advantage of being objective
because it does not rely on judgment of the observer,
whereas the degranulation test requires a visual evaluation
as to whether the cells are light or dark after staining. This
technique has been used to demonstrate that basophil
activation by anti-IgE antibodies, observed as an increased
expression of CD63, is blocked by conventional histamine
doses of between 10-2 and 10-4 M, as well as by (theoretical)
ultra-low doses of 10-22 and 10-34 M. Brown and Ennis used
two color method (anti-IgE FITC (Fluorescein Isothiocynate)
and anti-CD63 R-PE) to examine the effect of high dilutions
of histamine on basophil activation. Statistically significant
inhibition was seen both with histamine at pharmacological
concentrations as well as with histamine in high dilutions.
Guggisberg and co-workers used cells from the same donor
for their seven main replicate experiments and two color
method of flow cytometry was used. All samples were coded
and tested in triplicate. There were considerable variations in
the basophil activation with anti-IgE in the seven repeated

experiments. Their study clearly demonstrates that the

histamine dilutions causing significant inhibition vary
between the reported studies. This may be due to
susceptibility of basophils from different donors to histamine.

Discussion: out of several experiments on basophil activation

so far being conducted there appears to be some evidence of
biological activity of high dilutions/homoeopathic potencies,
especially in flow cytometry studies, but a convincing evidence
of a positive effect is still lacking. Several reasons are
attributed to it. First and foremost is the biological
implausibility of the phenomena. So far there is no scientific
explanation for how a highly diluted substance which is
essentially water can modulate basophil stimulation. Second
there were inconsistencies among the experiments conducted
which made it difficult to reach to a concrete decision. The
inhibiting and non-inhibiting dilution levels differed in most
studies. This concerned not only the inhibiting dilution levels of
histamine but also the anti-IgE concentration used for basophil
stimulation. The subjectivity associated with degranulation
tests was also an issue. The degranulation tests require a
visual evaluation as to whether the cells are light or dark after
staining. This subjective analysis can lead to variations in
results. Finally it can be said that a definite answer to the
question of whether high dilutions have specific effect on
basophils can only be found when all above mentioned
limitations are resolved.

References:

1. Poitevin B, Aubin M, Benveniste J. Effect dApis Mellifica sur

la degranulation des basophiles humains in vitro.
Homeopathie Franc 1985; 73: 1938.
2. Poitevin B, Aubin M, Benveniste J. Approcheduneanalyse
quantitative deleffetdapismellifica sur la degranulation des
basophiles humains in vitro. Innov Tech Biol Med 1986; 7:
648.
3. Poitevin B, Davenas E, Benveniste J. In vitro immunological
degranulation of human basophils is modulated by lung
histamine and Apis mellifica. Br J ClinPharmacol 1988; 25:
43944.
4. Benveniste J. et al. human basophil degranulation triggered
by very dilute antiserum against IgE. Nature june 1988; 333:
816-818.
5. High-dilution experiments a delusion. Nature 1988; 334: 287290.
6. Sainte-Laudy J, Sambucy JL, Belon P. Biological activity of
ultra low doses I. Effect of ultra low doses of histamine on
human basophil degranulation triggered by D. pteronissinus
extract. In: Doutremepuich C (ed). Ultra Low Doses. London:
Taylor and Francis, 1991, 12738.
7. Brown V, Ennis M. Flow cytometric analysis of basophil
activation inhibition by histamine at conventional and
homeopathic concentrations. Inflamm Res 2001; 50(suppl 2):
S47-S48.
8. Guggisberg AG, Baumgartner SM, Tschopp CM, Heusser P.
Replication study concerning the effects of homeopathic
dilutions of histamine on human basophil degranulation in
vitro. Complement Ther Med 2005; 13: 91-100.