Dorly de Freitas Buchi

Effect of an acidic heteropolysaccharide (ARAGAL) from the gum of Anadenanthera colubrina (Angico branco) on peritoneal macrophage functions

Brazilian flora are a source of interesting polysaccharides which, either in their native state or when submitted to structural modifications, might have potential applications as biological response modifiers (BRM). A complex acidic heteropolysaccharide, containing mainly galactose and arabinose (ARAGAL), isolated from the gum of the native leguminous tree Anadenanthera colubrina (Angico branco), was studied for its immunological properties on peritoneal exudate cells, namely their superoxide anion production, phagocytic activity, morphological alterations and percentage content of activated macrophages. Activation of macrophages showing increased cytoplasm, bright and large nuclei, various cytoplasmatic projections and spreading ability, was detected following in vitro cell exposure to ARAGAL or in cells obtained from treated animals. In vitro exposure to ARAGAL increased the occurrence of activated macrophages in a time- and a dose-dependent pattern, since approximately 82% of the cells were activated in the presence of 300 microg/ml of ARAGAL after 24 h of incubation and approximately 91% after 48 h. The occurrence of activated macrophages was also evident in cell preparations from ARAGAL-treated mice, their percentage showing a dose-dependent pattern. There were approximately 60, 75 and 75% following treatment with 100, 250 and 500 mg/kg of ARAGAL, respectively. A phagocytic assay showed that 25 microg/ml ARAGAL was sufficient to impose a maximum phagocytic ability, although this effect was dose-dependent. O(2)(-) production by macrophages from ARAGAL-treated mice was 70% higher than that of cells from untreated mice. Moreover, cells from treated mice responded to PMA, the effect being 25% higher than that of the control using untreated mice. These results thus suggest a possible role of ARAGAL from A. colubrina as a BRM.

Galactofuranose-rich heteropolysaccharide from Trebouxia sp., photobiont of the lichen Ramalina gracilis and its effect on macrophage activation.

A structural study of the carbohydrates from Trebouxia sp., the algal symbiont of the lichen Ramalina gracilis demonstrated a galactofuranan-rich heteropolysaccharide, which was predominated by (1-->5)-linked galactofuranosyl units with side-chains in position 6 on approximately 11.0% of the units. The side-chains have very complex branched structures. This polysaccharide showed cell eliciting activity on peritoneal macrophages in vitro at all concentrations tested (1-150 microg/mL), and at 150 microg/mL an increase of 60% of macrophage activation in comparison to the control group was observed. A potential role of these carbohydrates in lichen recognition process is also discussed.

Activation of bone marrow cells treated with Canova in vitro

Canova is a Brazilian complex homeopathic medication produced from Aconitum, Thuya, Bryonia, Lachesis and Arsenicum. Previous studies demonstrated that Canova induces up‐regulation in numbers of leukocytes. The bone marrow microenvironment is composed of growth factors, stromal cells, extracellular matrix, and progenitor cells that differentiate into mature blood cells. As it is the major site of blood cell formation, we studied in vitro Canova effects on bone marrow cells of mice. Swiss mouse femurs were dissected, cleaned, and the marrow was flushed. The cells were plated, treated or not, incubated for different times and processed for light, scanning electron, and confocal microscopy, and also flow cytometry. The treatment did not modify the expression of the analyzed surface markers or cytokine production. All microscopy techniques showed that a monocytic lineage (CD11b+) and stromal cells (adherent cells) were activated by treatment. Canova also increased cell clusters over adherent cells, suggesting proliferation areas.

Effectiveness of Vernonia scorpioides ethanolic extract against skin inflammatory processes.

ETHNOPHARMACOLOGICAL RELEVANCE Vernonia scorpioides (Asteraceae) is a native Brazilian medicinal plant that is commonly used to treat skin disorders. Considering the traditional use of Vernonia scorpioides and the lack of information about its pharmacological properties, we investigated the topical anti-inflammatory effect of the ethanolic extract of Vernonia scorpioides (EEVS) on acute and chronic cutaneous inflammation models in mouse. MATERIALS AND METHODS The topical anti-inflammatory effect of EEVS was evaluated against acute models (12-O-tetradecanoylphorbol acetate (TPA)- and arachidonic acid (AA)-induced mouse ear oedema) and chronic models (multiple applications of croton oil). RESULTS The EEVS caused a dose-related inhibition of oedema in both the TPA- and AA-induced acute models (DI(50)=0.24 and 0.68 mg/ear with an inhibition of 80 ± 5% and 65 ± 5%, respectively, for 1mg/ear). In addition, the TPA-induced increase in myeloperoxidase activity (MPO) in the ear was reduced (77 ± 8%) by the topical application of EEVS. In the chronic model, the EEVS reduced all parameters evaluated: oedema formation (31 ± 2%), epidermal hyperproliferation (histology) and MPO (25 ± 10%). However, the topical treatment of EEVS had no effect on N-acetyl-β-d-glucosaminidase activity. The EEVS effectively interfered in the ear oedema on the delayed-type hypersensitivity reaction induced by oxazolone. The topical treatment with EEVS performed on both phases or only on the elicitation phase caused the inhibition of the ear oedema-induced by oxazolone in 42.9% and 63.4%, respectively, when compared to control animals (sensitized and challenged). CONCLUSIONS The results suggest that EEVS is effective as a topical anti-inflammatory agent in acute and chronic inflammatory processes and that its action is markedly influenced by the inhibition of neutrophil migration into inflamed tissue as well as by epidermal hyperproliferation.

Lymphocyte proliferation stimulated by activated human macrophages treated with Canova

INTRODUCTION Canova (CA) is a homeopathic medication with immunomodulatory properties, recommended for patients with a depressed immune system. CA has been reported to increase in leukocyte numbers, cellular differentiation and reduction in tumor size. AIM AND METHOD Since CA may stimulate lymphocyte differentiation, proliferation, and/or survival, the aim of the present study was to compare the mitotic index (MI) of phytohemagglutinin-stimulated human lymphocytes cultured in a medium supplemented with human macrophages activated by CA, with lymphocytes cultured in a medium without CA-treated macrophages. RESULTS In this study, the MI of lymphocyte cultured received the medium containing CA-stimulated macrophages showed a higher proliferation index (p<0.01) than the lymphocytes cultured in a medium without CA-treated macrophages. Our results suggest that CA treatment, in addition to activating macrophages, indirectly induces lymphocyte proliferation and has potential as a new adjuvant therapeutic approach.

Stimulation of lymphocyte anti-melanoma activity by co-cultured macrophages activated by complex homeopathic medication

BackgroundMelanoma is the most aggressive form of skin cancer, and the most rapidly expanding cancer in terms of worldwide incidence. Chemotherapeutic approaches to treat melanoma have been uniformly disappointing. A Brazilian complex homeopathic medication (CHM), used as an immune modulator, has been recommended for patients with depressed immune systems. Previous studies in mice have demonstrated that the CHM activates macrophages, induces an increase in the number of leukocytes and improves the murine response against Sarcoma-180.MethodsHere we studied the interaction of mouse lymph node lymphocytes, co-cultured in vitro with macrophages in the presence or absence of the CHM, with B16F10 melanoma cells.ResultsLymphocytes co-cultured with macrophages in the presence of the CHM had greater anti-melanoma activity, reducing melanoma cell density and increasing the number of lysed tumor cells. There was also a higher proportion of activated (CD25+) lymphocytes with increased viability. Overall, lymphocytes activated by treatment destroyed growing cancer cells more effectively than control lymphocytes.ConclusionCo-culture of macrophages with lymphocytes in the presence of the CHM enhanced the anti-cancer performance of lymphocytes against a very aggressive lineage of melanoma cells. These results suggest that non-toxic therapies using CHMs are a promising alternative approach to the treatment of melanomas. In addition, they are attractive combination-therapy candidates, which may enhance the efficacy of conventional medicines by improving the immune response against tumor cells.

In vitro and in vivo anticancer properties of a Calcarea carbonica derivative complex (M8) treatment in a murine melanoma model

BackgroundMelanoma is the most aggressive form of skin cancer and the most rapidly expanding cancer in terms of worldwide incidence. Chemotherapeutic approaches to treat melanoma have had only marginal success. Previous studies in mice demonstrated that a high diluted complex derived from Calcarea carbonica (M8) stimulated the tumoricidal response of activated lymphocytes against B16F10 melanoma cells in vitro.MethodsHere we describe the in vitro inhibition of invasion and the in vivo anti-metastatic potential after M8 treatment by inhalation in the B16F10 lung metastasis model.ResultsWe found that M8 has at least two functions, acting as both an inhibitor of cancer cell adhesion and invasion and as a perlecan expression antagonist, which are strongly correlated with several metastatic, angiogenic and invasive factors in melanoma tumors.ConclusionThe findings suggest that this medication is a promising non-toxic therapy candidate by improving the immune response against tumor cells or even induce direct dormancy in malignancies.

Mercurius solubilis: actions on macrophages

BACKGROUND Macrophages play central roles in homeostasis as well as host defence in innate and acquired immunity, auto-immunity and immunopathology. Our research group has demonstrated the effects of highly diluted toxic substances in macrophages. AIM To investigate if highly diluted Mercurius solubilis (Merc sol), can activate or modulate macrophage functions. METHODS We evaluated the effects of Merc sol in the 6, 12, 30 and 200 centesimal high dilutions (CH) potencies on mice peritoneal macrophages (in vitro and in vivo). Merc sol was added to mices drinking water for 7 days (in vivo treatment) and animals were euthanised and cells were collected. In vitro treatment was performed on macrophages and bone-marrow cell cultures. RESULTS Macrophages showed activated morphology, both when Merc sol was added directly to the cell culture and to drinking water. The in vitro experiments showed enhanced morphological activation, increased interferon (IFN)γ release in the supernatant at lower dilutions and interleukin (IL)-4 production at higher dilutions. Increase in nitric oxide and decrease in superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)) were also observed. In vivo treatment caused a decrease in O(2)(-) and increase in H(2)O(2) production by macrophages. DISCUSSION Taken together, the results allow us to conclude that highly diluted Merc sol modulates reactive oxygen species (ROS), reactive nitrogen species (RNS) and cytokine secretion, which are central mediators of the immune system, wound healing and body homeostasis.

B1 and B2 kinin receptor participation in hyperproliferative and inflammatory skin processes in mice

BACKGROUND Kinins are released during dermal injury and inflammation and seem to contribute to the pathogenesis of cutaneous diseases. OBJECTIVE Participation of kinins in skin inflammatory process was evaluated using knockout mice and non-peptide kinin receptor antagonists. METHODS Chronic skin inflammation was induced by multiple applications of TPA in mice ear. RESULTS The B(2) knockout mice (B(2)(-/-)) showed a significant increase of ear weight (23 ± 10%) and epidermal cellular hyperproliferation and acanthosis formation upon histological analysis when compared with wildtype mice. Also, evaluation of PCNA levels by Western blot and immunohistochemistry confirmed the increase in the epidermis hyperproliferation in the ear skin of B(2)(-/-) mice. In contrast, no modification in these parameters was detected in B(1) knockout mice (B(1)(-/-)). However, mice lacking both kinin receptors (B(1)B(2)(-/-)) presented a considerable reduction of epidermis thickness and in PCNA levels. Following the establishment of skin inflammation (5th day of TPA application) treatment with the non-peptide antagonists SSR 240612 (B(1) receptor antagonist), FR 173657 (B(2) receptor antagonist), or SSR 240612 plus FR 173657 topically applied, caused a significant inhibition of ear weight (20 ± 5%, 34 ± 4% and 32 ± 6%, respectively). In the histological analysis, the antagonists produced a reduction in epidermal hyperplasia and acanthosis formation; but the treatment with a combination of the two antagonists did not increase efficacy. CONCLUSION Kinin receptors seem to be involved in the control of the keratinocyte hyperproliferative process, and non-peptide kinin receptor antagonists may be useful tools in the treatment of hyperproliferative skin disorders.

Developments on drug discovery and on new therapeutics: highly diluted tinctures act as biological response modifiers

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.