Differential effects of Zincum metallicum on cell models.

INTRODUCTION: Zinc is an essential trace element necessary for life. Traditional and complementary medicines use zinc-based formulations to treat different classes of diseases. Basic research on homeopathic preparations of zinc are rare and there are a few published clinical cases describing its effects on patients. The use of cell-based models in drug screening is a reliable source of evidence. METHODS: We sought to investigate experimental end-points using cell-based models to determine the effects of dilutions of Zincum metallicum prepared according to the Brazilian Homeopathic Pharmacopoeia. Murine RAW 264.7 macrophages and melanoma B16-F10 cell lines were cultured according to standard procedures. Cells were treated with either 5c, 6c or 30c Zincum metallicum and control cells with its respective vehicle (5c, 6c, or 30c Lactose). Macrophage activation by CD54 immunolabeling and intracellular reactive oxygen species (ROS) using DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) were detected by flow cytometry. Phagocytic capacity (endocytic index) was quantified by light microscopy. Features of melanoma cells were analyzed by colorimetric assays to determine melanin content and cell proliferation rate. All obtained data were submitted to normality test followed by statistical analysis. RESULTS: Zincum metallicum 6c shifted high ROS-producing macrophages to a low ROS-producing phenotype. Macrophage CD54 expression was increased by Zincum metallicum 5c. No changes in endocytic index were observed. Melanoma cells were not affected by any treatment we tested. CONCLUSIONS: Differing responses and non-linearity were found on macrophages challenged with Zincum metallicum at high dilutions. No changes in melanoma cells were observed. Customised assays using target cells can be useful to investigate high-dilution effects. Other cell types and conditions should be explored.

DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by Ruta graveolens in human colon cancer cells.

In the present study, we investigated the anti-cancer effect of various potencies of Ruta graveolens (Ruta) on COLO-205 cell line, as evidenced by cytotoxicity, migration, clonogenecity, morphological and biochemical changes and modification in the levels of genes associated with apoptosis and cell cycle. On treatment of COLO-205 cells maximal effects were seen with mother tincture (MT) and 30C potencies, wherein decrease in cell viability along with reduced clonogenecity and migration capabilities were noted. In addition morphological and biochemical alterations such as nuclear changes (fragmented nuclei with condensed chromatin) and DNA ladder-like pattern (increased amount of fragmented DNA) in COLO-205 cells indicating apoptotic related cell death were seen. The expression of apoptosis and cell-cycle related regulatory genes assessed by reverse transcriptase-PCR revealed an up-regulation of caspase 9, caspase-3, Bax, p21 and p27 expression and down-regulation of Bcl-2 expression in treated cells. The mode of cell death was suggestive of intrinsic apoptotic pathway along with cell cycle arrest at the G2/M of the cell cycle. Our findings indicate that phytochemicals present in Ruta showed potential for natural therapeutic product development for colon carcinoma.

Lymphocyte proliferation stimulated by activated Cebus apella macrophages treated with a complex homeopathic immune response modifiers.

INTRODUCTION: Canova is a complex homeopathic medicine that enhances a specific immunologic responses against several exogenous and endogenous conditions. Canova activates macrophages both in vivo and in vitro. AIM AND METHOD: We evaluated the effects of macrophages activated by Canova in vivo and ex vitro in the proliferation of lymphocytes. Canova was used to activate Cebus apella macrophages in vivo or ex vitro with Canova. Lymphocytes were cultured with the macrophage culture medium. The analysis of Canova effects in cultured lymphocytes was performed according to the cell cycle phase using flow cytometry. The Interferon gamma and Interleukin-5 cytokines quantification in these lymphocyte culture media was performed by Enzyme-linked immunosorbent assay (ELISA). RESULTS: We observed that Canova actives macrophages in vivo and ex vitro. The lymphocytes cultured in a supplemented medium with macrophages activated by Canova treatment presented a higher number of proliferation cells than lymphocytes not exposed to macrophages activated by Canova. The Interferon gamma and Interleukin-5 cytokines were only observed in the medium of lymphocytes exposed to macrophages activated by Canova. Thus, Canova has potential as a new adjuvant therapy.

Bufalin inhibits platelet-derived growth factor-BB-induced mesangial cell proliferation through mediating cell cycle progression.

Bufalin, a traditional Chinese medicine, has been reported as a protective factor in many tumors. We therefore investigated the effect of bufalin on platelet-derived growth factor (PDGF)-BB-induced proliferation of cultured rat mesangial cells. The effect of bufalin on cell proliferation and its underlying mechanisms were investigated in cultured rat mesangial cells (MCs) by the methylthiazoletetrazolium (MTT) assay, flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and cyclin-dependent kinases (CDK)2 and CDK4 kinase assays. Bufalin inhibited 20 ng/ml PDGF-BB-induced MC proliferation in a dose-dependent manner. Similar results were observed in different concentrations of bufalin, which blocked PDGF-BB-induced progression through G0/G1 to S phase of the cell cycle. Furthermore, bufalin not only inhibited upregulation of cyclin D1 and CDK4, but also downregulation of p21 in both mRNA and protein levels. Although bufalin did not affect p27 and CDK2 mRNA expression, it reversed downregulation of p27 and upregulation of CDK2 in protein level. Activity of CDK2 and CDK4 was also inhibited by bufalin. However, both bufalin and PDGF-BB did not affect cyclin E mRNA or protein expression. These results suggest that bufalin could inhibit MC proliferation by modulating cell cycle progress, indicating that bufalin could be a potential therapeutic agent for the prevention of mesangial proliferative glomerulonephritis.

[Effects and mechanism of Plastrum testudinis extracts on PC12 apoptosis].

OBJECTIVE: To observe the inhibitive effects of Plastrum testudinis Extracts (PTE) on 6-Hydroxydopamine (6-OHDA) induced PC12 cells apoptosis and explore its mechanism. METHODS: PC12 apoptosis model was established by serum starvation and damaged for 24 hours. The cells were randomly divided into four groups:control group, 6-OHDA group, PTE 3, 30 microg/mL group. Cell optical density was determined by MTT; Ratio of cell apoptosis was examined by Annexin V/PI double stain flow cytometry (FCM), and Western blot was applied to detect the BCL-X/L expression. RESULTS: MTT and FCM analysis demonstrated that PTE can elevate PC12 cells viability and reduce their apoptotic ratio in a dose dependent manner. Western blot showed that PTE promoted the expression of BCL-X/L. CONCLUSION: PTE can inhibit the apoptosis of PC12 induced by 6-OHDA in a dose dependent manner, and its mechanism maybe associated partially with up-regulating BCL-X/L signaling pathway.

[Inhibitory effects of Gecko alcohol extract on human esophageal squamous carcinoma cell line EC-109 proliferation and associated mechanism].

OBJECTIVE: To explore the proliferation inhibition effects of Gecko alcohol extract (GAE) on human esophageal squamous carcinoma cell line EC-109 and its mechanism. METHODS: The inhibitory effects of GAE on proliferation of EC-109 cells were measured by MTT. Nucleolus change of apoptotic cells was observed by Hoechest33342 fluorescence staining. Apoptosis rate of EC-109 cells was detected by flow cytometry. The expressions of apoptosis protein Caspase-3 and FAS in EC-109 cells were investigated by immunohistochemistry. RESULTS: GAE had the inhibition effects on the proliferation of esophageal carcinoma cell EC-109. The apoptosis rate of EC-109 cell treated with GAE(3.0 mg/mL, 4.0 mg/mL) for 48h was 20.63% and 39.73%, respectively. Compared with control group,the expression of Fas and Caspase-3 was significantly up-regulated in GAE treated group. CONCLUSION: GAE can inhibit the proliferation of esophageal carcinoma EC-109 cells and induce them apoptosis which may be correlated with increasing expression of protein Fas and Caspase-3.

Basophil models of homeopathy: a sceptical view.

This paper examines the activation and inhibition of activation of human basophils. After a brief description of human basophils, different methods to determine basophil activation are discussed with a special emphasis on the use of flow cytometric methods, as these circumvent the potential problems of assays based on the loss of colour by activated basophils. The activation of human basophils by ultra-high dilutions of anti-IgE is discussed. The majority of the paper describes the inhibition of basophil activation by ultra-high dilutions of histamine. The results from published papers are described and discussed. After over 20 years research trying to find out if high dilutions of histamine have a negative feedback effect on the activation of basophils by anti-IgE, what do we know? The methods are poorly standardized between laboratories - although the same is true for conventional studies. Certainly there appears to be some evidence for an effect - albeit small in some cases - with the high dilutions in several different laboratories using the flow cytometric methodologies. After standardization of a number of parameters, it is recommended that a multi-centre trial be performed to hopefully put an end to this "never-ending story".

Mice as a model for homeopathy research.

Mice (Mus musculus) have been used as a model for homeopathy research in relation to cytotoxicity, genotoxicity and carcinogenesis in our laboratory for the last three decades. Initially, anti-radiation activities of several potentized homeopathic drugs were tested against suitable controls by taking into consideration several cytogenetic endpoints. Subsequently, anti-cytotoxic, anti-genotoxic and anti-oxidative stress effects of some homeopathic drugs were tested against several chemical toxic metalloids and metal compounds. Modern techniques including Western blot, immunofluorescence, electron microscopy, UV-spectroscopy, HPLC, FTIR, NMR, RT-PCR etc were deployed to understand the possible mechanisms and pathways of action of potentized homeopathic drugs. We hypothesise that one way by which potentized homeopathic drugs act is through regulatory action on gene expression.

Inhibition of basophil activation by histamine: a sensitive and reproducible model for the study of the biological activity of high dilutions.

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.

[Effects of Yi Fu Ning soft gelatin capsules on reproductive endocrine-immune function of ovariectomized rats].

OBJECTIVE: To investigate the effect of Yi Fu Ning Soft Gelatin Capsules (YFN) on reproductive endocrine-immune function of ovariectomized rats. METHODS: 60 3-month old female Sprague-Dawley rats were used, 50 of them were ovariectomized and randomly divided into 5 groups: ovariectomizy (OVX) group, OVX with diethylstilbestrol tablets (DT) group, OVX with YFN (high dose, middle dose and low dose) group. The others were sham-operated group. The rats were administrated initially in the 4th week after the operation. After drugs had been given for 12 weeks the rats were sacrificed, blood serum hormone, IL-2 content and T lymphocyte subpopulation were detected with methods radioimmunoassay and flow cytometry (FCM). RESULTS: (1) Compared with sham group, the level of serum E2, Te and P significantly decreased (P < 0.01), FSH, LH content significantly increased; Blood T lymphocyte subpopulation CD3+ cells, CD4+ cells and CD4+/CD8+ ratio significantly decreased, serum IL-2 content also significantly decreased (P < 0.01). (2) Compared with model group, after treated by YFN, the level of serum E2 and P significantly increased (P < 0.01), serum FSH and LH content significantly decreased; T lymphocyte subpopulation CD3+ cells, CD4+ cells and CD4+/CD8+ ratio were improved significantly and serum interleukin-2 (IL-2) content increased significantly. CONCLUSION: YFN can increase serum sexual hormone content,reduce the level of FSH and LH, and improve imbalanced T lymphocyte subpopulation, stimulate IL-2 excretion, which means YFN can regulate inordinate reproductive endocrine-immune network in ovariectomized rats.

[Immunologische Ex-vivo-Untersuchung zur Wirkung potenzierter Substanzen bei parodontaler Entzündung unter Verwendung der Durchflusszytometrie]. / Immunological ex vivo Study on the Effects of Potentiated Substances in Periodontal Inflammation Using Flow Cytometry.

Hintergrund: Bei der Behandlung parodontaler Entzündungen werden in der Versorgungspraxis auch homöopathische Mittel eingesetzt. Noch ist weniger über deren grundlegende Wirkprinzipien bekannt. Ziel dieser Arbeit war es daher, die Auswirkungen potenzierter Substanzen bei parodonta-ler Entzündung mittels Durchflusszytometrie zu untersuchen. Material und Methoden: Lymphozyten aus Blutproben von drei Parodontitis-Patienten und drei gematchten gesunden Probanden wurden extrahiert und mit stark verdünnten wässrigen Extrakten (D12 und C200) aus Mercurius solubilis, Silicea, Sulphur, Tuberculinum oder Placebo inkubiert. Um die Lymphozytenexpression zu untersuchen, wurde die Durchflusszytometrie für CD45R0- und CD25-Antikörper angewandt. Die statistische Analyse wurde unter Verwendung von Histogramm- und bivariaten Dot-Plot-Analysen durchgeführt. Ergebnisse: Veränderungen der Expression von CD25 und CD45R0 wurden bei Mercurius C200, Mercurius D12, Silicea D12 und Sulphur D12 beobachtet. Mit 36,47% zeigte Sulphur D12 die höchsten Veränderungen in der CD45R0-Expression zwischen Verum und Placebo bei den Parodontitis-Patienten. Die CD25-Expression war in Mercurius D12 mit 18,68% am höchsten. Aufgrund der hohen Variabilität konnten die Ergebnisse jedoch nicht durch statistische Analysen untermauert werden. Diskussion: Diese Studie konnte zeigen, wie Effekte hoch verdünnter Substanzen mit modernen immunologischen Methoden analysiert werden können. Obwohl die Schlussfolgerungen aufgrund der hohen Variabilität der Lymphozytenexpression begrenzt sind, könnten die Ergebnisse dieser Pilotstu-die weitere Untersuchungen anregen. BACKGROUND: Several homeopathic remedies are applied in the treatment of periodontal inflammation. Still, little is known about their basic working principles. We therefore aimed at investigating the effects of homeopathic drugs in periodontal inflammation by flow cytometry. MATERIAL AND METHODS: Lymphocytes from blood samples of three periodontitis patients and three matched healthy volunteers were extracted and incubated with highly diluted (D12 and C200) aqueous extracts from Mercurius solubilis, Silicea, Sulphur, Tuberculinum, or placebo. To investigate lymphocyte expression, flow cytometry was applied for CD45R0 and CD25 antibodies. Statistical analysis was performed using histogram and bivariate dot-plot analysis. RESULTS: Changes in CD25 and CD45R0 expression were observed in Mercurius C200, Mercurius D12, Silicea D12, and Sulfur D12. With 36.47%, Sulfur D12 showed the highest differences in CD45R0 expression in periodontitis patients between verum and placebo. CD25 expression was highest in Mercurius D12 with 18.68%. Due to high variability, the results could, however, not be underpinned by statistical analyses. CONCLUSION: This study demonstrated how effects of highly diluted substances can be analyzed using modern immunological methods. Although conclusions are limited due to high variability in lymphocyte expression, results from our pilot study might encourage further investigations.

[The primary evaluation of Sal-Ammoniac extract on the therapy action in mice bearing Lewis lung cancer].

OBJECTIVE: To evaluate the therapy action of Sal-Ammoniac extract on Lewis lung cancer and its toxicity to immunity. METHODS: The proliferation and cell cycle of Lewis lung cancer cells were determined by MTT assay and flow cytometry respectively. The antitumor effect of Sal-Ammoniac extract was observed by tumor injected subcutaneously in mice and its toxicity to immunity was examined by clearance rate of charcoal particles and delayed type hypersensitivity. RESULTS: Sal-Ammoniac extract could inhibit the proliferation of Lewis lung cancer cells with S cell cycle arrest in a dose-dependent manner in vitro. Sal-Ammoniac extract solution injected in tumor for eight days had 46.7% inhibition on Lewis lung cancer, if taken orally had only 15.7% inhibition on Lewis lung cancer in mice. Sal-Ammoniac extract solution injected subcutaneously or taken orally had no effect on the clearance rate of charcoal particles and delayed type hypersensitivity in mice. CONCLUSION: The antitumor action of Sal-Ammoniac extract has relation to its recipe and has no influence on immunity.

Treatment of Chronic Immune Thrombocytopenic Purpura with Homeopathic Dilutions of Patient Blood.

BACKGROUND: Conventional or homeopathic treatment of chronic immune thrombocytopenic purpura (ITP) is often difficult. The use of homeopathic dilutions of patient blood (HPB) for immunomodulation has been described, which inspired us to try the method in an ITP case. CASE REPORT: A 2-year-old girl with chronic ITP was treated with homeopathic dilutions of her own capillary blood, given orally over 5 months. Immediately after treatment onset there was a rapid normalization of the thrombocyte counts. Within 6 weeks, they rose from 15,000/µl to 254,000/µl. After treatment stop, they decreased to 155,000/µl, increased again spontaneously to 270,000/µl and remained within normal range for over 3 years. CONCLUSIONS: Oral administration of homeopathic dilutions of capillary patient blood may possibly be an effective treatment in chronic ITP. If our results can be reproduced, this will revolutionize the treatment of ITP.

[A technique for investigating the component of cantharidin blisters by flow cytometry].

OBJECTIVE: Cantharidimide cause blister. The effect of blister on immunoregulation was investigated. METHODS: Cantharidimide was placed on the skin, 48h later, the blister was analyzed by flow cytometry. RESULTS: The blister contained 1 x 10(6) - 1 x 10(7) cells per ml, most of which were neutrophils, macrophages, dendritic cells (DC), and IL-12 secreted by Thl cells. CONCLUSION: There are high concent of DC in the blister, which is differential and induce the secretion of Th1, the activation of T cell. The blister modulate the biological response of patients and is helpful for treatment with infective disease.

Replication study concerning the effects of homeopathic dilutions of histamine on human basophil degranulation in vitro.

BACKGROUND: Various investigators have observed significant effects of highly diluted histamine on human basophil degranulation in vitro, compared to corresponding water controls. However, active and inactive dilution levels differed in most studies. OBJECTIVE: We aimed to reproduce former studies with flow-cytometry using rigorously controlled experimental conditions to minimise confounding factors. METHODS: In seven independent experiments, basophils of the same human donor were incubated with diluted histamine (up to 10(-34)M) or water controls and activated with anti-IgE antibodies. Basophil activation was determined by using bi-colour flow-cytometry. Experiments were blinded and performed with a randomised arrangement of the solutions on microtiter-plates. RESULTS: Histamine at the dilutions 10(-2)M and 10(-22)M was associated with a significant inhibition of basophil degranulation (p=0.018, Wilcoxon signed rank test) of 23.1% and 5.7%, respectively, if compared to "diluted" water treated in an identical manner. However, if all controls were pooled, only histamine 10(-2)M had a significant effect. Significant effects were seen for row numbers of the microtiter plates. CONCLUSION: We were not able to confirm the previously reported large effects of homeopathic histamine dilutions on basophil function of the examined donor. Seemingly, minor variables of the experimental set up can lead to significant differences of the results if not properly controlled.

[Effect of taurochenodeoxycholic acid on immune function in mice].

OBJECTIVE: To observe the influence of Taurochenodeoxycholic Acid (TCDCA) on immun, function in mice. METHODS: T-Cell subgroups were determined by using Flow cytometry; The content of anti-body in serum was assayed by using Spectrophotometry; The phagocytosis of mononuclear phagocyte system was determined by using Carbon particle clearance test and anti-sheep blood cell hemolysin was determined by using Turbidimetric method. RESULTS: TCDCA signifeantly enhanced the percentage of CD and CD19+ lymphocytes and CD4+/CD8+ value in peripheral blood and the content of serum hemolysin and lysozymem in mice. Moreover, TCDCA markedly improved the phagocytosis functions of mononuclear phagocyte system and observably inhibited delayed type hypersensitivity. CONCLUSION: TCDCA can significantly enhance the immune function in mice.

Ruta 6 selectively induces cell death in brain cancer cells but proliferation in normal peripheral blood lymphocytes: A novel treatment for human brain cancer.

Although conventional chemotherapies are used to treat patients with malignancies, damage to normal cells is problematic. Blood-forming bone marrow cells are the most adversely affected. It is therefore necessary to find alternative agents that can kill cancer cells but have minimal effects on normal cells. We investigated the brain cancer cell-killing activity of a homeopathic medicine, Ruta, isolated from a plant, Ruta graveolens. We treated human brain cancer and HL-60 leukemia cells, normal B-lymphoid cells, and murine melanoma cells in vitro with different concentrations of Ruta in combination with Ca3(PO4)2. Fifteen patients diagnosed with intracranial tumors were treated with Ruta 6 and Ca3(PO4)2. Of these 15 patients, 6 of the 7 glioma patients showed complete regression of tumors. Normal human blood lymphocytes, B-lymphoid cells, and brain cancer cells treated with Ruta in vitro were examined for telomere dynamics, mitotic catastrophe, and apoptosis to understand the possible mechanism of cell-killing, using conventional and molecular cytogenetic techniques. Both in vivo and in vitro results showed induction of survival-signaling pathways in normal lymphocytes and induction of death-signaling pathways in brain cancer cells. Cancer cell death was initiated by telomere erosion and completed through mitotic catastrophe events. We propose that Ruta in combination with Ca3(PO4)2 could be used for effective treatment of brain cancers, particularly glioma.

Results of five randomized studies on the immunomodulatory activity of preparations of Echinacea.

This article describes and discusses five placebo-controlled randomized studies investigating the immunomodulatory activity of preparations containing extracts of Echinacea in healthy volunteers. A total of 134 (18 female and 116 male) healthy volunteers between 18 and 40 years of age were studied. Two studies tested intravenous homeopathic complex preparations containing Echinacea angustifolia D1 (study 1) and D4 (study 5). Two studies (2 and 3a) tested oral alcoholic extracts of roots of E. purpurea, one study an extract of E. pallida roots (study 3b), and one study an extract of E. purpurea herb (study 4). Test and placebo preparations were applied for four (study 5) or five (studies 1-4) consecutive days. The primary outcome measure for immunomodulatory activity was the relative phagocytic activity of polymorphonuclear neutrophil granulocytes (PNG), measured in studies 1 and 2 with a microscopic method and in studies 3, 4, and 5 with two different cytometric methods. The secondary outcome measure was the number of leukocytes in peripheral venous blood. Safety was assessed by a screening program of blood and other objective parameters as well as by documentation of all subjective side effects. In studies 1 and 2 the phagocytic activity of PNG was significantly enhanced compared with placebo [maximal stimulation 22.7% (95% confidence interval 17.5-27.9%) and 54.0% (8.4-99.6%), respectively], while in the other studies no significant effects were observed. Analysis of intragroup differences revealed significant changes in phagocytic activity during the observation periods in five test and three control groups. Leukocyte number was not influenced significantly in any study. Side effects due to the test preparations could not be detected. Our studies provide evidence for immunomodulatory activity of the homeopathic combination tested in study 1 and the E. purpureae radix extract tested in study 2. The negative results of the other three studies are difficult to interpret due to the different methods for measuring phagocytosis, the relevant changes in phagocytic activity within most placebo and treatment groups during the observation period, and the small sample sizes. Future studies should be performed on patients rather than healthy volunteers and use standardized or chemically defined monopreparations of Echinacea.

Sensitive flow cytometric method to test basophil activation influenced by homeopathic histamine dilutions.

OBJECTIVE: In an experimental setting, human basophil degranulation was triggered by anti-IgE to measure the effects from homeopathic solutions in an in-vitro cell system. A 3-color flow cytometric method with enhanced accuracy was established. As an example we looked at the influence of histamine on anti-IgE activation of basophils. METHODS: Basophils were identified in the flow cytometer by their physical properties in the forward and side scatter light depiction and by gating on CD2(-), CD14(-), CD16(-), CD19(-), HLA-DR(-) negative and CD123-positive cells. CD63 expression on the cell surface of the anti-IgE-activated basophils served as an activation marker. RESULTS: With this method we were able to study basophil function of the 0.6-3.9% basophils out of the mononuclear blood cell fraction and to document their activation status upon anti-IgE activation. Optimal activation occurs at 0.6 microg/ml final anti-IgE concentration; not less than 10,000 basophils have to be counted per batch to reduce the variation of the measurement. The fixation method was able to stabilize activation for two days. After investigation and reduction of the source of measurement variability, an unequivocally inhibited basophil activation was documented in a partly optimized system with homeopathic dilutions of histamine (10(-22)M, 10(-23)M, 10(-24)M, and 10(-25)M histamine). Dilutions greater than 10(-20)M histamine (Avogadro's number 6.02 x 10(23)) account for less than 1.36 molecules of histamine in the test sample, indicating a true homeopathic effect. CONCLUSIONS: This test system is adequate for studying the effects of highly diluted mediators on basophil activation by anti-IgE. The systematic application of this experimental arrangement is recommended to study the effects of homeopathic dilutions on basophils.