[Omeo33] Art 1069 - Homeopathy, 2009, 98 (4), 186-197

[Gino Santini]

Inhibition of basophil activation by histamine: a sensitive and  
reproducible model for the study of the biological activity of high  
dilutions
J. Sainte-Laudy and Ph Belon

Background
At the beginning of this series of experiments we were looking for a  
model based on the use of purified commercially available compounds  
based on a fully described and accepted pharmacological model to study  
of the biological effect of high dilutions. Negative feedback induced  
by histamine, a major pro-inflammatory mediator, on basophils and mast  
cells activation via an H2 receptor me these criteria. The simplest  
way of measuring basophil activation in the early 1980's was the human  
basophil activation test (HBDT).

Objectives
Our major goal was first to study the biological effect of centesimal  
histamine dilutions beyond the Avogadro limit, on the staining  
properties of human basophils activated by an allergen extract  
initially house dust mite, then an anti-IgE and N-formyl-Met-Leu-Phe  
(fMLP). Technical development over the 25 years of our work led us to  
replace the manual basophil counting by flow cytometry. The main  
advantages were automation and observer independence. Using this  
latter protocol our aim was to confirm the existence of this  
phenomenon and to check its specificity by testing, under the same  
conditions, inactive analogues of histamine and histamine antagonists.  
More recently, we developed an animal model (mouse basophils) to study  
the effect of histamine on histamine release.

Methods and results
For the HBDT model basophils were obtained by sedimentation of human  
blood taken on EDTA and stained with Alcian blue. Results were  
expressed in percentage activation. Histamine dilutions tested were  
freshly prepared in the lab by successive centesimal dilutions and  
vortexing. Water controls were prepared in the same way. For the flow  
cytometric protocol basophils were first labeled by an anti-IgE FITC  
(basophil marker) and an anti-CD63 (basophil activation marker).  
Results were expressed in percentage of CD63 positive basophils.  
Another flow cytometric protocol has been developed more recently,  
based on basophil labeling by anti-IgE FITC (fluorescein  
isothiocyanate) and anti-CD203 PE (another human basophil activation  
marker). Results were expressed in mean fluorescence intensity of the  
CD203c positive population (MFI-CD203c) and an activation index  
calculated by an algorithm. For the mouse basophil model, histamine  
was measured spectrofluorimetrically.
The main results obtained over 28 years of work was the demonstration  
of a reproducible inhibition of human basophil activation by high  
dilutions of histamine, the effect peaks in the range of 15–17CH. The  
effect was not significant when histamine was replaced by histidine (a  
histamine precursor) or cimetidine (histamine H2 receptor antagonist)  
was added to the incubation medium. These results were confirmed by  
flow cytometry. Using the latter technique, we also showed that 4- 
Methyl histamine (H2 agonist) induced a similar effect, in contrast to  
1-Methyl histamine, an inactive histamine metabolite. Using the mouse  
model, we showed that histamine high dilutions, in the same range of  
dilutions, inhibited histamine release.

Conclusions
Successively, using different models to study of human and murine  
basophil activation, we demonstrated that high dilutions of histamine,  
in the range of 15–17CH induce a reproducible biological effect. This  
phenomenon has been confirmed by a multi-center study using the HBDT  
model and by at least three independent laboratories by flow  
cytometry. The specificity of the observed effect was confirmed,  
versus the water controls at the same dilution level by the absence of  
biological activity of inactive compounds such as histidine and 1- 
Methyl histamine and by the reversibility of this effect in the  
presence of a histamine receptor H2 antagonist.

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