Sainte-Laudy J, Belon Ph
CHU, Limoges 87042, France.
Homeopathy. 2009 Oct;98(4):186-97. doi: 10.1016/j.homp.2009.09.009.
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).
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.
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.
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.
在这一系列实验开始时，我们正在寻找一种基于使用纯化的市售化合物的模型，该模型基于一个充分描述和被接受的药理学模型来研究高稀释度的生物学效应。组胺是一种主要的促炎介质，通过H2受体对嗜碱性粒细胞和肥大细胞的激活产生负反馈符合这些标准。在20世纪80年代早期，测量嗜碱性粒细胞激活的最简单方法是人类嗜碱性粒细胞激活试验（HBDT）。
我们的主要目标首先是研究超过阿伏伽德罗极限的百分之一组胺稀释液对最初由屋尘螨变应原提取物、然后是抗IgE和N-甲酰甲硫氨酸-亮氨酸-苯丙氨酸（fMLP）激活的人类嗜碱性粒细胞染色特性的生物学效应。在我们25年的工作中，技术发展使我们用流式细胞术取代了手动嗜碱性粒细胞计数。主要优点是自动化和不受观察者影响。使用后一种方案，我们的目的是确认这种现象的存在，并通过在相同条件下测试组胺的无活性类似物和组胺拮抗剂来检查其特异性。最近，我们开发了一种动物模型（小鼠嗜碱性粒细胞）来研究组胺对组胺释放的影响。
对于HBDT模型，通过对采集于乙二胺四乙酸（EDTA）的人血进行沉降获得嗜碱性粒细胞，并用阿尔辛蓝染色。结果以激活百分比表示。测试的组胺稀释液在实验室中通过连续的百分之一稀释和涡旋新鲜制备。水对照以相同方式制备。对于流式细胞术方案，嗜碱性粒细胞首先用抗IgE异硫氰酸荧光素（FITC）（嗜碱性粒细胞标记物）和抗CD63（嗜碱性粒细胞激活标记物）进行标记。结果以CD63阳性嗜碱性粒细胞的百分比表示。最近还开发了另一种流式细胞术方案，基于用抗IgE FITC（异硫氰酸荧光素）和抗CD203藻红蛋白（PE）（另一种人类嗜碱性粒细胞激活标记物）对嗜碱性粒细胞进行标记。结果以CD203c阳性群体的平均荧光强度（MFI-CD203c）和通过算法计算的激活指数表示。对于小鼠嗜碱性粒细胞模型，用荧光分光光度法测量组胺。28年工作中获得的主要结果是证明了高稀释度的组胺可重复性地抑制人类嗜碱性粒细胞激活，效应峰值在15 - 17CH范围内。当组胺被组氨酸（一种组胺前体）取代或在孵育培养基中加入西咪替丁（组胺H2受体拮抗剂）时，效应不显著。这些结果通过流式细胞术得到证实。使用后一种技术，我们还表明4-甲基组胺（H2激动剂）诱导了类似的效应，与无活性的组胺代谢物1-甲基组胺形成对比。使用小鼠模型，我们表明在相同稀释范围内的高稀释度组胺抑制组胺释放。
相继地，使用不同模型研究人类和小鼠嗜碱性粒细胞激活，我们证明了在15 - 17CH范围内的高稀释度组胺诱导了可重复的生物学效应。这种现象已通过使用HBDT模型的多中心研究以及至少三个独立实验室通过流式细胞术得到证实。通过无活性化合物如组氨酸和1-甲基组胺的无生物学活性以及在组胺受体H2拮抗剂存在下这种效应的可逆性，与相同稀释水平的水对照相比，证实了观察到的效应的特异性。
