Human basophils are peripheral granulocytes involved in immune and inflammatory processes such as hypersensitivity and allergic reactions. The discovery of several basophil activation markers, such as the tetraspanin CD63 (lysosome-associated membrane protein or LAMP-3) and the ectoenzyme CD203c (or ecto-nucleotide pyrophosphatase/phosphodiesterase or E-NPP3), has made it possible to study cellular function by observing the up-regulation or down-regulation of membrane molecules in response to various physiological agonists, antagonists or other soluble regulators [34
], using a standardized cytometric approach.
Among the various soluble factors able to regulate basophil function, a considerable body of literature has been devoted to histamine, a well-known mediator of inflammation produced by basophils and mast cells. Histamine at high doses acts as a downstream regulator by interacting with H2-receptors, thus inhibiting basophil release of mediators and degranulation [36
]; it is therefore very intriguing that the same effect was observed with highly diluted histamine but not with highly diluted histidine [11
], and that it could be inhibited by the H2-antagonist cimetidine [37
]. Multicentre analyses have partially confirmed these findings [10
], but other groups also report some negative results [19
In this work we were able to reproduce evidence for the effects of high histamine dilutions on anti-IgE-triggered basophil activation, specifically evaluated by the CD203 MFI, using a standardized approach with the following characteristics: (a) the method of cell handling prevents spontaneous basophil activation and interference between the anti-IgE-FITC tracer and the agonist used [14
]; (b) the problems of subjective evaluation are avoided through use of flow cytometry and a system of multiple intra-series replication; (c) use of highly pure compounds and parallel comparisons with control pure water dilutions; d) adoption, for the first time in this field, of a mechanical shaking method (succussion in homeopathic terms) with standardized duration and frequency of strokes.
The resultant dose–effect curve displayed a very unusual behaviour, whose underlying mechanism remains to be investigated. It is worth noting that the range of active dilutions is very close to the 16C dilution reported elsewhere [11
], although Brown and Ennis [13
] instead reported inhibitory activities for 10C and 13C, but not for the 12C, 14C and 16C dilutions.
The lack of significant inhibition of CD63 expression by histamine at high dilutions in our experimental conditions (Table ) may apparently be in disagreement with the data of CD203c and with the findings of other authors who detected high-dilution effects also using CD63 marker [11
]. However, this discrepancy may be interpreted as due to the lower sensitivity of this parameter to inhibition by high dilutions or otherwise as a result of the high variability of this response, because of the low percentage of cells expressing CD63 in our test system. In this study we chose to use low doses of agonist, a condition in which cells were consistently activated, as noted by the increase of CD203c MFI, but very low percentage (8–15%) of cells expressed CD63, which is less responsive to low doses of anti-IgE (Fig. a). As a consequence, the high-dilution effects were much more clearly and consistently observable by evaluating the CD203c marker. We have previously shown that the chemotactic peptide fMLP, at very low dose, causes up-regulation of CD203c but not of CD63 [26
], and this observation is consistent with the finding that CD203c reflects the priming events triggered by interleukin-3, which occur previously to degranulation events [28
]. Moreover, CD203c expression in not inhibited by phosphatidylinositol-3-kinase inhibitors, as is CD63 [29
]. Thus, small changes in the experimental conditions used or subtle distinction of the signal transduction mechanisms of the two markers expression could differentiate the results of high dilution experiments obtained by different authors in this model.
Although a variety of gating protocols and different experimental conditions—in terms of the type of dilution/succussion or shaking procedures and solvent used—have been explored [13
], most of the studies done so far did not include succussed water controls. Research on extremely sensitive systems and very high dilutions of substances suggests that trace elements, as well as container materials, storage durations and shaking methods, may influence the results [39
]. Therefore, suitable water controls prepared in an identical manner and subjected to the same storage time should be used. These considerations, coupled with the highly controversial nature of the phenomenon, which would have significant pharmacological implications but is often judged to be improbable and implausible from a conventional scientific perspective [24
], make it all the more important that more replications should be done independently to establish models that are stable across laboratories and teams [22
The possible biological mechanism(s) underlying the regulatory processes affected by high histamine dilutions remain to be elucidated. These findings suggest that the high dilutions of histamine, which are better observed through CD203c expression, might affect some subtle and early level of signal transduction, similar to the priming effects of very low doses, instead of causing a general inhibition of cell responses. Using a surface plasmon resonance biosensor, it has recently been shown that basophil activation by anti-IgE, even in the absence of exocytosis, is associated with changes in the refractive index of the evanescent field [42
]; such changes reflect any changes in molecular mass distribution in the field a few 100 nm from the surface of the cells. It is thus tempting to speculate that changes in the water structure produced by the dilution/succussion procedure in the presence of histamine might influence this crucial layer of water surrounding the cell and hence the processes of signal detection and transduction. Further studies are needed to confirm whether this hypothesis is applicable to the high-dilution effects observed on human basophils.
Our results confirm and build upon the hypothesis that high dilutions of biologically active compounds may indeed have an effect which mimics that of lower dilutions/higher doses. So far there is no satisfactory or uniting theoretical explanation for these observations, but recent evidence seems to point to organization of the solvent water on a mesoscopic scale: the nano-heterogenous structure of water can be determined by interactive phenomena such as coherence [43
], epitaxy [45
], temperature–pressure processes during strong agitation, and formation of colloidal nanobubbles containing gaseous inclusions of oxygen, nitrogen, carbon dioxide, silica, and possibly the remedy source material [46
]. So far, nuclear magnetic resonance (NMR) studies of homeopathic solutions have yielded controversial data, but recently high histamine dilutions (beyond 10−20
mol/l) were distinguished from water solvents on the basis of their NMR relaxation rates [49
]; moreover, after heating of the histamine samples, all the relaxation observed as a function of dilution vanished. It was suggested that histamine molecules might act as nucleation centres, amplifying the formation of stable supramolecular structures, involving nanobubbles of atmospheric gases and highly ordered water around them. In the future, the possible existence and the nature of clathrate-like hydrate nanostructures formed during the dilution and succussion process might be explained by cluster science, in which different geometrical structures of clusters composed of the same chemical species may differ in their chemical reactivity [50
]. These unusual properties of high dilutions, which merit further investigation, are potentially relevant not just to homeopathic pharmaceutical practice, but also to basic research into cell sensitivity to regulation.