Abstract: Background: Nanoparticles (NP), because of their size ( 〈 1μm) and high relative surface area, are highly reactive forms of their source material with biological, chemical, optical, electromagnetic, and thermal properties different from larger bulk forms of the same material. It has been speculated that NPs can occur in homeopathic products as a function of trituration and shaking into glass containers. Moreover, the presence of sugars additives like lactose and of silicates leaked from the glassware are reported to stabilize the nanoparticles.1 Up to now some authors observed nanoparticles in highly diluted samples2, 3, but further studies are needed to know the nature of the NPs. Actually, nanostructures in the solutions may derive from the source materials but also from stuff contaminations and also may be constituted by nano-bubbles.4, 5 Moreover, the mechanism by which the nanostructures can be formed and the effect of serial dilution/succussions of the NPs suspension should be studied.6 Many tools are available to analyze the nanostructures both in solutions or in dried samples and these give complementary information about the concentration, stability, structure and chemical nature of the NPs. In the present report we describe preliminary observations obtained by the nanoparticle tracking analysis (NTA) in Zincum metallicum (Zinc met.) solutions at low-grade dilution/dynamization and their lactose controls. This study is part of a formal Brazilian-Italian inter-university collaboration.

Abstract: Background: Immunomodulatory properties are evoked by many highly diluted drugs and may be the base of complex systemic effects of the remedies in the organism. Monocyte/macrophages exert a pivotal role in innate immunity regulation and in inflammation. THP-1 cells have become one of most widely used cell lines to investigate the function and regulation of monocytes and macrophages. These cells resemble primary monocytes–macrophages isolated from donors in the vasculature, with the advantage of homogeneous genetic background that minimizes the degree of variability in the cell phenotype; this trait is particularly important when studying the biological function of chemicals with high dilutions [1]. Using this model we tested different doses/dilutions of homeopathic and allopathic drugs. Methodology: The cell line THP-1 derived from the blood of a patient with acute monocytic leukemia, resemble primary monocytes and macrophages in morphology and differentiation property. The cell line was provided by DSMZ Culture Collection and cultured in RPMI medium (Lonza) added with 10% (w/v) fetal bovine serum (FBS, Lonza) and 2mM ultraglutammine, at 37°C in controlled atmosphere (5% CO2, 95% air) and complete humidity. In all the experiments the cells were seeded in microplates (4ï‚´103/well) in RMPI with 2% fetal bovine serum and exposed to phorbol-12-myristate-13-acetate (PMA, 20 ng/ml) for 48h. In this period, the THP-1 cells adhered to culture plates and differentiated into a macrophage phenotype with marked morphological changes (Figure 1). Concomitantly with the addition of immunoactive drugs, cells were activated with different doses of bacterial lipopolysaccharide (LPS, from 0 to 100 ng/ml) and incubated for 24h. Figure 1: Pics of THP-1 cells without (left) and with PMA treatment (right). Betamethasone (Bentelan) was diluted/succussed in sterile water and added to cells at concentrations ranging from 0.01 to 100 ng/ml. Gelsemium sempervirens centesimal dilutions (c), 2c, 3c, 4c, 5c, 9c and 30c, and the corresponding controls were freshly prepared in glass tubes starting from 30% ethanol solutions (provided by Laboratoires Boiron and prepared following the French Pharmacopoeia) by 100x dilution in pure water and agitated in automatic dynamizer (see detail in [2]). The drugs were kept in the LPS-activated cells for 24 h. The dosage of pro-inflammatory (TNF-a, IL-6 and IL-1b) and anti-inflammatory (IL-10) cytok ines was monitored by immune detection with ELISA assays (eBioscience) starting from the culture supernatants. The variation of viability and cell density were examined by WST assay and by quantification of total protein with Bradford assay on lysed cells, respectively. Repeated experiments were conducted in microplates, being careful to perform analogous manipulations to the drug dilutions and the corresponding controls. Statistical analysis was performed with paired T-test. Results and discussion: After stimulation with different doses of LPS, the responses of THP-1 cells can be monitored via the dosage of specific interleukins. The production of IL-1β (as proof) in response to different doses of LPS is shown in Figure 2. The cell model is very sensitive to variations in the amount of activator, exponentially increasing the amount of cytokine produced. The same was observed for the other cytokines investigated. The experiments with the immunoactive drugs were performed in intermediate condition of activation, that looked adequate to observe both increase and inhibition effects. Figure 2. Quantification of the cytokine IL-1β produced in THP-1 cell supernatants after activation with increasing concentrations of LPS, in presence or not of interferon-ï§ as co-activator. Histograms report mean values ± SD. Betamethasone, a potent glucocorticoid steroid with anti-inflammatory and immunosuppressive properties, was used as model immunomodulatory drug. Figure 3 describes the effects of different doses of the steroid on LPS-activated cells. Notably, we tested the effect of “physiological†doses of betamethasone in circulating blood and some of the doses that can be relevant in pharmacokinetics of the drugs (the therapeutic dosage in human correspond to the dose of 0.2 μM). As shown in Figure 3, the concentration of the proinflammatory cytokine TNF-α and IL-6 decreases linearly with the increase of the betamethasone dose, in particular when the cells were strongly stimulated with LPS (100 ng/ml). In contrast, if the LPS stimulus is weaker (LPS 10 ng/ml) the direction of effect depends on the dose of betamethasone: low doses (0.01 and 0.1 μM) seem to increase the concentration of TNF-α, while larger doses exert a inhibitory action, as expected (Figure 3A). This effect can be described as a typical hormetic behavior and it was observed also in the left part of Figure 3C, where the IL-10 production is increased with low doses of the immunosuppressive drug. Notably, in the case of IL-10, the steroid did not impair significantly the cytokine production in THP-1 cells activated with LPS 100 ng/ml. Actually, the activation pathway of the pro-inflammatory cytokine IL-10 is different from the TNF-α [3]. Figure 3. Effects of betamethasone on the production of the pro-inflammatory cytokines TNF-α and IL-6 and the anti-inflammatory cytokine IL-10. The dose response experiments were performed both in presence of LPS 10 or 100 ng/ml as cell activator. Bars report mean value ± SEM. The effects on THP-1 macrophages of Gelsemium sempervirens (2c, 3c, 4c, 5c, 9c and 30c) were compared with the corresponding potentized vehicles in cells activated with either high LPS dose (100 ng/ml) or low dose (10 ng/ml). Figure 4 shows the main results concerning three cytokines. This compound was tested both to extend our previous knowledge gathered with neuronal models [2] and because it is often used in influenza syndromes and in the Materia medica it is associated with symptoms of fever and inflammation. Figure 4. Percentage effect of Gelsemium s. treatment vs the corresponding succussed control at different dilutions on the THP-1 cytokine production. The variation of the cytokines concentration was measured in the supernatants of cells after 24h of treatment by ELISA assay. Values in graphs are mean % effect ± SEM, n=12, two replicated experiments. As shown in Figure 4A and 4B, the pro-inflammatory cytokines IL-6 and TNF-α were apparently modulated by Gelsemium s., although not in a linear fashion with increasing concentration. Analogous trends were obtained in the two LPS conditions. The highest doses (low dilutions) increased slightly the cytokine production (Gels 2c and 3c), while a maximum of inhibition was observed with 4c and 5c dilutions. Statistical inference was obtained in the data from the separate experiments for the TNF-α inhibition with Gels 4c (p

Abstract: Background: Gelsemium sempervirens (Gelsemium s.) is a highly toxic plant but is employed at low doses and/or high dilutions as an anxiolytic and antidepressant. Previous investigations in our laboratory [1,2] have shown a significant anxiolytic-like activity of Gelsemium s., using emotional response models in laboratory mice. Although there is some biochemical evidence of a possible role of neurosteroid metabolism [3] , the cellular and molecular mechanisms involved in the effects of Gelsemium s. at the level of nervous system are largely unknown. To help determine these pathways, we used human neurocytes (SH-SY5Y cell line) treated in vitro with different dilutions of Gelsemium s. and evaluated their vitality and gene expression changes. Methods: The drugs were produced by Boiron Laboratoires, Lyon (F), starting from a whole-plant-hydroalcoholic extract of Gelsemium s. Solutions 1C, 2C, 3C, 4C, 8C and 29C (C= centesimal dilution/dynamization prepared in 30% ethanol/distilled water) were provided in 30-ml glass bottles, wrapped in aluminium foil and were stored in the dark at room temperature in a metal cupboard. Control solutions (“placeboâ€ÂÂ) were serially diluted/dynamized 30% ethanol/distilled water. Before each experiment, 0.05 ml samples of Gelsemium s. and placebo were added to 5 ml of distilled sterile and apyrogenic water in a 15 ml Falcon polystyrene plastic tube, closed and shaken in mechanical shaker DinaA for 7.5 sec (150 strokes) to obtain the final 2C, 3C, 4C, 5C, 9C and 30C succussed dilutions, with ethanol concentration of 0.3% (v/v) (final 0.03% in the assay system). Human neuroblastoma cell line SHSY5Y was grown in DMEM-F12 medium (Lonza), with 10% fetal bovine serum (FBS), penicillin (100 units/ml) and streptomycin (100 mg/ml). To assess cell viability and metabolism, 20,000 cells per well were seeded in 96 microplate wells in 200 μl of medium. After overnight incubation, 22 μl of drug or placebo were added and the plate was incubated at 37°C with 5% CO2 in a humidified atmosphere for 72 hours. Then the viability test with reagent WST-1 (Roche) was performed for 3 hours and the absorbance was detected with multiplate reader. A total of 17 experiments, each done with six replicate microwells. To make a relative measurement of protein, the cells were lysed and a Bradford assay was done directly in the plate. The Student t-test and the sign rank test for paired data were utilized for data analysis. To obtain a profile of gene expression, cells were pre preconditioned with Gelsemium s./placebo dilutions for 24 h, then RNA was isolated and analysed by microarray and RT-PCR. SHSY5Y cells were plated onto Petri dishes (day 1) and the day after the medium was replaced with the medium with 2% FBS (day 2). After 24h, 10%v/v Gelsemium s. or placebo dilutions were added to the medium (day 3) and cells were incubated for a further 24h. On day 4, cells were then harvested and the RNA extracted using the Qiagen RNAeasy Mini Kit following the manufacturer’s instructions. Microarray analysis was performed on a custom 12 x 135 k human NimbleGen microarray containing 45033 genes with 3 probes per target gene. Four biological replicates were analysed for each condition. Analysis of differentially expressed genes was performed using linear modelling and empirical Bayes methods and p-values were adjusted for multiple testing with the Benjamini and Hochberg method. A Human Neurotransmitter Receptors and Regulators RT2 Profiler PCR array (Qiagen) was performed in profiling the expression of genes involved in modulating the biological processes of neurotransmitter biosynthesis, uptake, transport and signaling through neurotransmitter receptors. Results: In viability tests, cells treated with Gelsemium s. showed slightly higher metabolic activity (3-4 %) than those treated with placebo. Overall comparison of the data for the whole sample of placebo versus that of Gelsemium s. using the Student t-test showed a small but significant difference (p 〈 0.001). Furthermore, a non parametric approach comparing the two treatments at the same dilution yielded a significant difference under the sign test (p 〈 0.01) and Wilcoxon rank test for paired data (p 〈 0.05), so that the values of differences were also considered. The differences between groups having the same dilution (placebo 2C versus Gelsemium s. 2C etc.) were significant in four dilutions: 2 C, 3 C, 4C (p 〈 0.01), 9 C (p 〈 0.02), while 5 C and 30 C yielded non-significant values. No changes due to Gelsemium s. were detected using protein assay, suggesting that the viability test revealed effects on metabolic activity instead of on cell proliferation. In microarray analysis, transcripts expression was analyzed and genes differentially expressed by the Gelsemium s. dilutions were selected. A gene was considered to be differentially expressed if it showed an absolute value of log-ratio greater than or equal to 0.5, an index that translates to a fold-change of 1.4 in transcript quantity. Out of a total of 45,033 transcripts, exposure to Gelsemium s. 2C promoted the selective downexpression of 49 genes (p values adj

Abstract: Drosera rotundifolia has been traditionally used for the treatment of respiratory diseases in phytotherapy and homeopathy. The mechanisms of action recognized so far are linked to the known effects of specific components, such as flavonoids, but are not completely understood. In this study, the biological functions of D. rotundifolia were explored in vitro following the treatment of bronchial epithelial cells, which are the potential targets of the pharmacological effects of the herbal medicine. To do so, the whole plant ethanolic extract was 1000-fold diluted in water ( D. rotundifolia 3×) and added to a 16HBE human cell line culture for 3 h or 6 h. The effects on gene expression of the treatments and corresponding controls were then investigated by RNA sequencing. The differentially expressed genes were validated through RT-qPCR, and the enriched biological functions involved in the effects of treatment were investigated. D. rotundifolia 3× did not impair cell viability and was shown to be a stimulant of cell functions by regulating the expression of dozens of genes after 3 h, and the effects were amplified after 6 h of treatment. The main differentially expressed genes encoded ligands of epithelial growth factor receptor, proteins involved in xenobiotic detoxification and cytokines, suggesting that D. rotundifolia 3× could stimulate self-repair systems, which are impaired in airway diseases. Furthermore, D. rotundifolia 3× acts on a complex and multifaceted set of genes and may potentially affect different layers of the bronchial mucosa.