BackgroundIn the search for new therapies novel drugs and medications are being discovered, developed and tested in laboratories. Highly diluted substances are intended to enhance immune system responses resulting in reduced frequency of various diseases, and often present no risk of serious side-effects due to its low toxicity. Over the past years our research group has been investigating the action of highly diluted substances and tinctures on cells from the immune system.MethodsWe have developed and tested several highly diluted tinctures and here we describe the biological activity of M1, M2, and M8 both in vitro in immune cells from mice and human, and in vivo in mice. Cytotoxicity, cytokines released and NF-κB activation were determined after in vitro treatment. Cell viability, oxidative response, lipid peroxidation, bone marrow and lymph node cells immunophenotyping were accessed after mice in vivo treatment.ResultsNone of the highly diluted tinctures tested were cytotoxic to macrophages or K562. Lipopolysaccharide (LPS)-stimulated macrophages treated with all highly diluted tinctures decreased tumour necrosis factor alpha (TNF-α) release and M1, and M8 decreased IFN-γ production. M1 has decreased NF-κB activity on TNF-α stimulated reporter cell line. In vivo treatment lead to a decrease in reactive oxygen species (ROS), nitric oxide (NO) production was increased by M1, and M8, and lipid peroxidation was induced by M1, and M2. All compounds enhanced the innate immunity, but M1 also augmented acquired immunity and M2 diminished B lymphocytes, responsible to acquired immunity.ConclusionsBased on the results presented here, these highly diluted tinctures were shown to modulate immune responses. Even though further investigation is needed there is an indication that these highly diluted tinctures could be used as therapeutic interventions in disorders where the immune system is compromised.

The effects of mycotoxin citrinin on Ca2+ efflux and membrane permeabilization were studied in isolated rat liver mitochondria. The efflux rate observed when in presence of ruthenium red was higher when citrinin was added. Swelling experiments demonstrated Ca2+‐dependent membrane permeabilization by citrinin. Catalase, butylhydroxitoluene (BHT), and dithiothreitol (DTT) did not protect swelling caused by Ca2+ plus citrinin. The protection conferred by ATP–Mg2+ and cyclosporin A in the latter experiments are strong indications of pore formation. These results suggest that citrinin can induce permeability transition by a mechanism that does not involve oxidative damage. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 291–297, 1998

The ability of the mycotoxin citrinin to act as an inhibitor of iron-induced lipoperoxidation of biological membranes prompted us to determine whether it could act as an iron chelating agent, interfering with iron redox reactions or acting as a free radical scavenger. The addition of Fe3+ to citrinin rapidly produced a chromogen, indicating the formation of citrinin-Fe3+ complexes. An EPR study confirms that citrinin acts as a ligand of Fe3+, the complexation depending on the [Fe3+]:[citrinin] ratios. Effects of citrinin on the iron redox cycle were evaluated by oxygen consumption or the o-phenanthroline test. No effect on EDTA-Fe2+-->EDTA-Fe3+ oxidation was observed in the presence of citrinin, but the mycotoxin inhibited, in a dose-dependent manner, the oxidation of Fe2+ to Fe3+ by hydrogen peroxide. Reducing agents such as ascorbic acid and DTT reduced the Fe3+-citrinin complex, but DTT did not cause reduction of Fe3+-EDTA, indicating that the redox potentials of Fe3+-citrinin and Fe3+-EDTA are not the same. The Fe2+ formed from the reduction of Fe3+-citrinin by reducing agents was not rapidly reoxidized to Fe3+ by atmospheric oxygen. Citrinin has no radical scavenger ability as demonstrated by the absence of DPPH reduction. However, a reaction between citrinin and hydrogen peroxide was observed by UV spectrum changes of citrinin after incubation with hydrogen peroxide. It was also observed that citrinin did not induce direct or reductive mobilization of iron from ferritin. These results indicate that the protective effect on iron-induced lipid peroxidation by citrinin occurs due to the formation of a redox inactive Fe3+-citrinin complex, as well as from the reaction of citrinin and hydrogen peroxide.

Background Any dairy herd that continually has a somatic cell count (SCC) above 200,000 cells/ml has an indication of mammary gland inflammation (mastitis). Routine use of antibiotics to prevent mastitis is prohibited by organic farming regulations. This limitation has lead researchers to focus on cows natural defense mechanisms [1]. Calcarea carbonica derivative complex (M8) is a complex high diluted medication comprised of comprised of Calcarea carbonica 16x, Aconitum napellus 20x, Arsenicum album 18x, Asa foetida 20x, Conium maculatum 17x, Ipecacuanha 13x, Phosphorus 20x, Rhus toxicodendron 17x, Silicea 20x, Sulphur 24x, and Thuya occidentalis 19x. Dilution procedures have followed standard methodology described at the Brazilian Homeopathic Pharmacopoeia. This medication has enhanced immune system responses both in vitro and in vivo in a murine model [2]. Aims In the present study, we investigate the response of dairy cows after M8 treatment. Methodology The study was performed as a randomized, observer double-blinded and placebo-controlled trial, with a stratified design, using lactation number and SCC as stratification factors. The study sample consisted of 42 lactating dairy cows (Holstein) in one high producing dairy herd with 52 cows in milk in southern Brazil, divided into two experimental groups (n=21). Exclusion criteria were cows with clinical mastitis or receiving any other medical treatment. Pre- and post-milking teat disinfection was practiced in the herd. All cows were clinically examined, with udder and milk samples being appraised according to Rosenberger (1990) [3]. During 3 months one group received daily M8 treatment, the other placebo. Oral administration of 5 ml/day/cow was performed using an automatic dosage dispenser. Monthly, milk production, SCC, fat and total protein content were carefully recorded for each animal by an official milk recording program. SCC were log transformed for analysis. ANOVA and Tukey test were used to compare the averages. The Bartlett´s test was used for homogeneity of variance evaluation. Results There were no significant differences (P=0.435) among the groups in the initial evaluation (Values of SCC x103 : Placebo 67,37±80,48; Treatment 359,39±677,02). After 3 months, the M8 treated group showed a decrease (134,00±178,76 P= 0.047) in SCC when compared with control group (391,71±686,60). Fat and protein did not differ between groups and time analysed. Milk production decreased in the placebo group during time (Before: 34,97±6,69kg; After:28,69±4,33kg), whereas the treatment group did not change total amount (Before: 28,7±6,54kg; After: 26,39±5,73kg; P > 0.05). Conclusions These results indicate that the M8 influenced positively SCC and suggest that it may be considered as a possible tool to promote bovine mastitis prophylaxis. Keywords: Calcarea carbonica complex, mastitis, somatic cell count [1] McDougall S, Parker KI, Heuer C, Compton CWR. A review of prevention and control of heifer mastitis via non-antibiotic strategies. Vet Microbiol. 2009; 154:177–85. [2] Oliveira CC, Abud APR, Oliveira SM, Guimarães FSF, Andrade LF, Di Bernardi RP, Coletto ELO, Kuczera D, Da Lozzo EJ, Gonçalves JP, Trindade ES, Buchi DF. Developments on drug discovery and on new therapeutics: highly diluted tinctures act as biological response modifiers. BMC Complementary and Alternative Medicine. 2011; 11(101): 2-11. [3]Rosenberger, G. Die klinische Untersuchung des Rindes, 3. Neubearbeitete Auflage, Paul Parey Verlag, Berlin, 1990.

Paracelsus once wrote: "All things are poison and nothing is without poison, only the dose permits something not to be poisonous." Latter Hahnemann formulated the law of similars, preparations which cause certain symptoms in healthy individuals if given in diluted form to patients exhibiting similar symptoms will cure it. Highly diluted natural complexes prepared according to Hahnemann’s ancient techniques may represent a new form of immunomodulatory therapy. The lack of scientific research with highly diluted products led us to investigate the in vivo and in vitro actions of commonly used medications. Here we describe the results of experimental studies aimed at verifying the effects of Mercurius solubilis, Atropa Belladonna, Lachesis muta and Bryonia alba. All medications were at 200cH dilution. Animals were maintained for 7 days and were allowed to drink the medications, which were prepared in a way that the final dilution and agitation (200cH) was performed in drinking water. The medication bottle was changed and sucussed every afternoon. Co-culture of non treated mice bone marrow cells and in vitro treated peritoneal macrophages were also performed. After animal treatment the bone marrow cells were immunophenotyped with hematopoietic lineage markers on a flow cytometer. We have determined CD11b levels on bone marrow cells after culture and co-culture with treated macrophages and these macrophages were processed to scanning electron microscopy. We have observed by morphological changes that macrophages were activated after all treatments. Mercurius solubilis treated mice showed an increase in CD3 expression and in CD11b on nonadherent bone marrow cells after co-culture with in vitro treatment. Atropa Belladonna increased CD45R and decreased Ly-6G expression on bone marrow cells after animal treatment. Lachesis muta increased CD3, CD45R and, CD11c expression and decreased CD11b ex vivo and in nonadherent cells from co-culture. Bryonia alba increased Ly-6G, CD11c and CD11b expression ex vivo and when in co-culture CD11b was increased in adherent cells as well as decreased in nonadherent cells. With these results we have demonstrated that highly diluted medications act on immune cells activating macrophages, and changing the expression profile of hematopoietic lineage markers. Highly diluted medications are less toxic and cheaper than other commonly used medications and based on our observations, it is therefore conceivable that this medications which are able to act on bone marrow and immune cells may have a potential therapeutic use in clinical applications in diseases were the immune system is affected and also as regenerative medicine as it may allow proliferation and differentiation of progenitor cells.