Macrophage activity regulation by high dilution of aspirin and its possible mechanism

Under LPS-stimulus, platelets can activate macrophagesby a cell-to-cell contact or through cytokine degranulation. Rebound effects of anti-thrombotic agents, such as prostanoids and COX inhibitors can lead to thrombosis, infarct, and stroke. Aspirin has been prescribed for decades due to its powerful antiplatelet action, but it is also related to paradoxical effects such as withdrawal syndrome peaks, resistance, and thrombogenesis. Ultra-diluted aspirin can also produce the same effect in one hour, regardless of Cox-2, by still unknown pathways. Antithrombotic effects of aspirin are also reversed by its high dilutions.Aims: This study aims to characterize the effects of aspirin 15cH on macrophages challenged with LPS, a Cox-2 activator.Methodology: RAW 264.7 macrophages were sown in 24 wells plates using R10medium, boosted with 1µg/ml LPS,and treated with aspirin 15 cH and controls. The activity was evaluated after 24 hours. Supernatants were evaluated for cytokines, nitric oxide, and dielectric oscillations, through solvatochromic dyes (Cartwright's method).Results and discussion: macrophage spreading was increased by aspirin 15 cH, anLPS-like effect. Paradoxically, a significant reduction of this effect was noted when both, LPS and aspirin 15 cH, were added. Succussed water reversed the effect of LPS, leading to TNF (p<0.05) production close to baseline levels. Also, the single treatment with succussed water inhibited IL-10 production (p<0.05), but aspirin 200 µg/mL (positive control) highly increased it (p<0.0001), showing the validity of the model. Nitric oxide production was strengthened by LPS presence (p<0.0001), as expected, but partially downregulated after treatment with aspirin 200 µg/mL, water and succussed water. A pilot study with solvatochromic dyes showed no significant difference among treatments.Conclusion: The main data suggest that aspirin 15 cH increases macrophage activity but presents a paradoxal effect when mixed with LPS. On the other hand, succussed water itself has modulatory effects on macrophages.

Characterization of physicochemical markers for homeopathic medicines and biological supernatant samples

Solvatochromic dyes are probes to detect variations on the dipole moment of solvents after the insertion of homeopathic potencies. Recent studies have shown they can be useful tools in laboratory and field studies to detect the activity of homeopathic remedies.Objective: Determine whether solvatochromic dyes can be a diagnostic tool for cells infected by different agents and/or markers to identify the activity of homeopathic medicines. Methods: Ethilicum1cH, Siliceaterra6, 30, 200cH; Zincummetallicum6, 30, 200cH and Phosphorus6, 30 and 200cH were analyzed by pouring the samples (in a 1:60 rate) into a series of seven dyes (rhodamine, ET 33, ET 30, coumarin 7, NN DMIA, Nile red, methylene violet) diluted in absolute ethanol using pre-established working concentrations. Oscillations of dye absorbance were observed at visible light spectrophotometry according to the remedy and potency. Water and succussed water were used as controls. In a second moment, the absorbance profile of the remedies will be compared with those of biological samples (supernatants) and checked with the biological effect previously obtained from each treatment.Supernatants of RAW 264.7 macrophages stimulated by Calmette-Guérin bacilli (BCG) or infected with Encephalitozoon cuniculiwill be analyzed. Results: Preliminary results have shown that Siliceaterra6cH, Phosphorus30 and 200cH and Zincummetallicum6, 30 and 200cH reduced the absorbance of methylene violet (p=0.01). Repetitions and analysis of supernatants are expected to be performed in the next steps of the study. Future perspectives: Establish a pattern of reactivity of the studied medicines with different dyes and the putative relation with the corresponding supernatants, as an attempt to obtain a "physicochemical signature" for each kind of infection and/or treatment.

Immobilisation of solvatochromic dyes on transparent cellulose films; an improved methodology for the study of homeopathic potencies

Highly diluted and succussed solutions (homeopathic potencies) have been shown to interact with a wide range of solvatochromic dyes based on changes in their UV-visible spectra. Studies so far have involved free dyes in solution, but there is a pressing need to find ways to investigate the potency-dye interaction using isolated dye molecules in order to ask more searching physico-chemical questions regarding the fundamental nature of potencies. Aims and Methods:The aims of the present study have been tolook for ways to covalently immobilizesolvatochromic dyes onto transparent cellulose films and hence be in a position to investigate dye-potency interactions without the complication of dye-dye interactions, including dye aggregation, which can occur with free dyes in solution. Results: to date a total of nine different dyes have been immobilised on cellulose films using epoxide activation of hydroxyl groups on the cellulose surface. Using this methodology studies have begun looking at the time course of potency action on one of these immobilised dyes, Brooker's merocyanine. Results show that the interaction ofArsenicum10M with this dye consists of three phases ­an initial growth phase, a sustained plateau of interaction and a final decline phase lasting several days.Conclusions: amethodology has been developed that successfully immobilises solvatochromic dyes onto transparent cellulose film. These films can then be used in a spectrophotometer to study at a much more detailed level how potencies interact with dyes compared with using free dyes in solution. Results indicate that the information gained in this way provides new insights regarding the fundamental nature of potencies. Specifically, studies using immobilised Brooker's merocyanine with Arsenicum10M reveal that the lifetime of the potency is much longer than expected and that its action consists of three distinct phases, suggesting a resonant interaction with the dye.