Clinical reports show improvement of the quality of life of cancer patients following treatment with homeopathic medicine Carbo animalis, although the literature on this subject is still rare and the mechanisms of action unknown. Previous results obtained by our group indicated that treatment of mice with Carbo animalis 6cH increased the migration of macrophages and B1 cells (phagocyte progenitors) to Ehrlich tumor primary site. In the present study we analyzed the interaction of macrophages with murine breast cancer cells (4T1) after treatment with Carbo animalis in different homeopathic dilutions: 6cH, 30cH and combination 30cH + 6cH (MIX). All dilutions and vehicles were physically and chemically analyzed for conductance and composition of microparticles in suspension by means of energy-dispersive x-ray spectroscopy (EDX). Exposure of macrophages co-cultured with 4T1 cells to treatments over a period of 48 hours led to the following significant changes: 1) reduction of TNF-alpha production in co-cultures compared to simple macrophage culture, indicating an inhibitory effect of the tumor on macrophage activity; b) higher expression of pseudopodia and IL-6 production after exposure to all treatments; c) increase of the cell number per microscopic field, increase in the interaction (cell-cell contact) between macrophages and 4T1 cells and increase in production of GM-CSF, IL-12p40 and MCP-1 after treatment with Carbo animalis 6cH; d) increase of VEGF production in cells treated with Carbo animalis 30cH. There was no change in nitric oxide production with any treatment. Physical-chemical analysis of the drugs revealed significant increase in the conductivity of Carbo animalis 30cH and MIX samples compared to the vehicle (30% alcohol). The microparticles exhibited an extremely diverse composition, including Na, Si, Cl, F, Pb, S, Ca, Mg, Al, K, Nb, B and Be . Only Carbo animalis 6cH exhibited boron and beryllium with frequency above 50% per particle, and this drug induced the most significant changes on the macrophage-tumor interaction. However, the role of these elements in the immunomodulatory activity observed both in vivo and in vitro still deserves further investigation.

In previous studies [1,2] we showed that treatment of mice with Antimonium crudum (Ant-c) 30cH was able to significantly reduce monocyte migration to the infection site after injection of Leishmania (L) amazonensis into the subcutaneous tissue, resulting in clinical improvement. Follow up was performed with an in vitro model, which showed that treatment of co-cultures of RAW 264.7 macrophages and parasites with Ant-c 30cH inhibited two parasite-induced CCL2 peaks 48 and 120 hours after infection together with early inhibition of lysosome activity. These findings explained the results previously obtained in vivo. In turn, treatment with Ant-c 200cH resulted in an early and transitory peak of cell spreading at 48 hours. The coherence between the in vivo and in vitro results indicates that this is a good model to study more thoroughly the mechanisms of action of homeopathic medicines, being the first step to establish correlations between the biological effects and the physical and chemical features of Ant-c 30cH and 200cH. In the present study, the same experimental model was replicated, through comparison of vehicle (30% cereal alcohol), Ant-c 200cH, Zincum metallicum (Zinc) 200cH and Arsenicum album (Ars) 200cH, to confirm the specificity of Ant-c effects. In addition, Ant-c 200cH was ultra-centrifuged, and only the superficial phase was applied to the culture medium. This procedure intended to separate the heavier particles from the lighter ones suspended in the homeopathic medicine. The physical-chemical profile of the medicines was assessed. Solid contaminants (microparticles) in the suspension were analyzed. Conductivity was assessed through measurement of the electron current induced by a micro-amperimeter (Ryodoraku®) connected to 2 clean electrodes immersed in the samples, prepared immediately before the analysis, diluted in pure water (MilliQ, Millipore®) and filtered in 22-µ filter (Millipore®). Pure water was used as control. The device was calibrated immediately before measurements. The microparticle profile was assessed with a scanning electronic microscope - SEM (JEOL JSM 6510®) coupled to an energy dispersive spectroscopy (EDS) system to identify the nature of the elements present in each particle. The size and the number of particles were analyzed from the images generated by electronic microscopy with an automatic image analysis system (Metamorph®). For this purpose, all materials used was cleansed through immersion in pure acetone and subjected to 30-minute sonication before insertion into the microscope to avoid secondary contamination. The samples of medicines were subjected to ultra-centrifugation (10000rpm for 60 minutes) to induce particle sedimentation in the bottom of microtubes. 10 microliters of each sample were collected from the bottom of tubes and placed on a copper stub and kept in a closed recipient until the material was fully dry. The samples were directly analyzed with the microscope. Metallization was not necessary, because the analyzed particles had metallic nature. The biological effects of Ant-c 200cH reproduced the previous ones: spreading and phagocytosis index were significantly higher in the co-cultures treated with Ant-c 200cH compared to vehicle and other, non-specific treatments (Ars 200cH and Zinc 200cH) (p=0.05). However, these results were not exhibited by centrifuged Ant-c 200cH. Analysis of the supernatant after 48-hour incubation revealed increase of the GM-CSF content only in cultures treated with Ant-c 200cH and centrifuged-Ant-c 200cH. No change was observed in the cytokine profile in the cultures treated with Ars 200cH or Zinc 200cH. Morphological analysis of Ant-c samples on SEM showed that the microparticles in Ant-c 30cH were smaller compared to Ant-c 6cH, most of them having half-moon shape. Curiously, agglomerates of particles were detected in Ant-c 200cH. Contaminant particles suspended in pure water contained Pb, Zn, Ca, Na, Au, Hg, Nb and Si, therefore, not related to any specific biological effect of Ant-c. P was identified only in Ant-c 30cH (6.51%) and Ant-c 200cH (13.56%). This wide-range profile of different microparticles did not change after centrifugation, which indicates that the weight of these particles is not conditioned by the nature of their component elements. Conductivity was lower in the vehicle (30% alcohol) compared to Ant-c 6, 30 and 200cH (p=0.0001); the conductivity of Ant-c 200cH was the highest (p=0.008). Also Ars 200cH exhibited higher conductivity (p=0.001) compared to the vehicle. Taken together, these data suggest that the biological effect of Ant-c 200cH on macrophage spreading and phagocytosis might be partially related to the size of the microparticles found in suspension. However, specific effects relative to cytokine production did not depend on microparticle size or content. The changes in conductivity changes exhibited correlation with presence of some elements, such as P, but not with any biological effect. To summarize, the results point to the relevance of eventual false-positive effects relative to phagocytosis in macrophages treated with homeopathic medicines in vitro, due to the interference of larger sized microparticles. They also points to the specificity of GM-CSF expression after 48-hours of co-culture exposure to Ant-c 200cH, centrifuged or not, which suggests it was independent from microparticle content and conductivity. The physical-chemical features of homeopathic medicines related to their specific biological effects are still unknown. Additional studies are needed in this regard.

Silica (silicon dioxide) is found in nature, being composed of 2 chemical elements abundant on the Earth's crust, oxygen and silicon. As homeopathic medicine, silica is used for treatment of chronic ulcers due to its ability to modulate the macrophage activity. Zinc is a cofactor of several immune mediators, especially thymulin, which is also capable of modulating the macrophage activity and recruitment of B1 cells in mice. In the present study, we assessed the homeopathic medicines Silicea terra and Zincum in an in vitro experimental model to determine their effects on the interaction between RAW 264.7 macrophages and BCG (Bacillus Calmette-Guérin). In this step of the study, we assessed oxidative activity through measurement of hydrogen peroxide and nitrite/nitrate in the cell culture supernatant after 24 hours of treatment. Several homeopathic potencies were used for both drugs (6cH, 30cH, 200cH). The tests were performed in duplicate, and the data were analyzed by means one-way ANOVA. The results show no effect on nitric oxide (NO) production, but reduction of hydrogen peroxide production (p≤ 0.001) after treatment with the vehicle (0.03% alcohol). Such reduction was reversed with treatments Silicea terra30cH and Zincum metallicum 30cH (p≤ 0.001). Treatment with Silicea terra 200cH induced significant reduction of hydrogen peroxide production, even when compared to the vehicle (p≤ 0.005). Taken together, the results indicate that only treatment of BCG-challenged macrophages with Silicea terra 200cH was able to significantly reduce the oxidative activity of these cells after 24 hours of incubation. Since NO production in vitro usually occurs after 96 to 120 hours of incubation, one might infer that the negative result obtained in the present study might be associated with the time-point of assessment. Other aspects of the macrophage-BCG interaction are still under evaluation.

Mice bearing Leishmania (L) amazonensis infection and treated with Antimonium crudum (AC) 30cH presented significant reduction of the monocyte migration to the site of infection with clinical improvement. In vitro, the treatment of infected macrophages with AC 30cH produced inhibition of the parasite-induced peaks of CCL2 (a chemokine for monocytes migration) and inhibition of lysosome activity, explaining the results obtained previously in vivo. In the following studies, physical-chemical parameters of the remedy and respective controls were evaluated, to search for a correlation with the former described biological effects. The study of polarity changes in different water-based dilutions of AC using Cartwright´s method, revealed dilution-dependent variations in the absorbance of three solvatochromic dyes ET 33, BDN and methylene Violet (MV), used as “probes” to evaluate the dipole features of the medicine. The electrical activity of the homeopathic preparations appears to be dilution-dependent and related to their biological effects. Further experiments were performed using samples of the supernatant of infected macrophages after 96 hours of incubation with AC in different dilutions. These samples were processed using the same procedures as used for the original medicines and were analyzed by MV method. All tested potencies presented a sharp increase of absorbance at 580 nm, in relation to all controls (supernatant from untreated cells and cells treated with succussed water), as performed by one-way ANOVA, being F = 176.208; p = 0.001 and ?2 = 0.988. This results strongly suggest that biological systems could amplify the electric signal and the following changes in the medium polarity.

Background: There are few published researches about the exclusive use of Carsinosinum in several potencies to treat cancer. The name Carcinosinum refers to any homeopathic preparation of epithelial cancerous tissues and is especially indicated when there are any hereditary and familial antecedents of cancer, tuberculosis, diabetes, pernicious anemia or a combination of two or more of these diseases. Homeopathic complexes which include Conium Maculatum, Sabal Serrulata, Thuja Occidentalis and Carcinosinum can reduce in 23% the incidence of prostate cancer in vivo and in 38% the tumor volume, compared to untreated groups. Another in vivo study revealed reduction of symptoms and increase of survival time in mice bearing Ehrlich ascitic carcinoma, after treatment with Carcinosinum 200cH. In vitro, Carcinosinum 200cH can increase the expression of the pro-apoptotic gene p53. However, mice treated with Carcinosinum 6cH had the highest percentage and diversity of symptoms compared to other treatments, which demonstrate the importance of homeopathic potency in pro or anti-carcinogenic action. Considering that the literature on this subject is still rare and focused on genotypic and clinical effects, the present study was proposed, with the aim of identifying the possible phenotypic changes, including viability, HER-2 expression and metastatic skills, using 4T1 cells in vitro as a model, after treatment with Carcinosinum in different homeopathic working dilutions (12cH; 30cH; 200cH), prepared mechanically (Denise Machine, Autic®) in our laboratory using sterile pure water, from a commercial matrix (HN Cristiano, São Paulo, Brazil) stocked in 70% hydro-alcoholic solution. The final dilutions were inserted in the culture medium in a volume equal to 10%, at the time of cell seeding. The same succussioned vehicle used to prepare the medicines (70% hydro-alcoholic solution), from the same batch and diluted 1:100 in sterile pure water, was used as control. All treated cells were cultivated in bottles of 25ml with cell density of 5 x 105 cells / ml and, after 24 hours of treatment, they were analyzed for the apoptosis index using the Annexin V kit and measured by the Countess® system. The morphology of the 4T1 cells was monitored by staining fixed cell smears with hematoxylin-eosin method. The samples were evaluated in quadruplicate and the data were analyzed by one-way ANOVA. The results obtained up to now show that the treatment with Carcinosinum 12cH produced a different pattern of cell death compared to the other treatments, with significant reduction in apoptosis index (one-way ANOVA, p=0.01) and clear hydropic degeneration phenotypic pattern. The analysis of HER-2 expression and metastatic skill will be the next step of this research.