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 mice's 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.

Calcarea carbonica e associações (M8) como tratamento adjuvante de carcinoma inflamatório mamário em cão / Calcarea carbonica derivative complex (M8) as adjuvant treatment of inflammatory mammary carcinoma in a dog

Background: Inflammatory mammary carcinoma (IMC) is locally aggressive, fast growing, highly malignant tumor that affects humans and dogs. Affected dogs usually are presented with generalized edema, pain, erythema, and skin ulceration in mammary glands. Surgery is not recommended and an effective treatment has not been established [1]. Calcarea carbonica derivative complex (M8) has demonstrated anticancer properties in a murine model, by improving innate immune response against tumor cells [2,3]. M8 is a complex high diluted medication comprised of a 10%-20% concentration of Calcarea carbonica, Aconitum napellus, Arsenicum album, Asa foetida, Conium maculatum, Ipecacuanha, Phosphorus, Rhus tox, Silicea, Sulphur, and Thuya occidentalis, all in decimal dilutions of Hahnemann in distilled water and submitted to vigorous shaking. Aim: Describe an association of M8 and piroxicam (Non-steroidal anti-inflammatory drug) to treat a dog with IMC. Discussion: A 7 years old, mixed breed intact female dog was presented to the Federal University of Parana - Veterinary Hospital, Curitiba (HV-UFPR) for mammary glands examination. The owners related inflammation of mammary glands with clinical course of approximately 10 days, which was treated for mastitis (cephalexin and metergoline) without clinical improvement. Clinical examination revealed erythema, increased skin warmth, pain on palpation, and plaque involving the 4th and 5th right mammary glands. Abdominal ultrasound and serum biochemistry were unremarkable. Thoracic radiographs showed suspicious images of pulmonary metastasis. Fine needle biopsy was taken for cytologic examination. Cytological interpretation was a malignant epithelial neoplasm, probably a mammary carcinoma. Diagnosis of IMC was based on clinical signs and cytopathology. Dog was treated with oral (0.5 mL) and topical M8 twice a day for 15 days, and pyroxican, 0.3mg/kg, PO, q24h. Clinical improvement was observed 7 days after starting treatment. Until present date (70 treatment days with M8), dog has no clinical signs of IMC, and does not show signs of disease progression. Conclusion: The present report suggests that M8 associated with piroxicam contributes to improvement of IMC dog?s quality of life and survival rate. However, further clinical studies are needed to evaluate response to treatment in patients diagnosed with IMC.(AU)

Introdução: O carcinoma inflamatório mamário (CIM) é um tumor altamente maligno que acomete cães e pessoas, apresentando-se localmente invasivo e com crescimento rápido. Em cães, os sinais clínicos incluem edema e eritema generalizado das mamas acometidas, dor local e ulceração. A intervenção cirúrgica é contra-indicada e não há consenso sobre tratamento clínico eficaz [1]. Estudos em modelo murino demonstraram que Calcarea carbonica e associações (M8) possuem propriedades anticancerígenas através de estímulo da resposta imune inata [2,3]. O M8 é altamente diluído, composto de 10 a 20% de Calcarea carbonica, Aconitum napellus, Arsenicum album, Asa foetida, Conium maculatum, Ipecacuanha, Phosphorus, Rhus tox, Silicea, Sulphur, e Thuya occidentalis, todos na diluição decimal de Hahnemann em água destilada e submetido à agitação vigorosa. Objetivo: Descrever a associação de M8 e piroxicam (antiinflamatório não esteroidal) no tratamento de cão com CIM. Discussão: Uma cadela não castrada, sem raça definida, de 7 anos de idade foi trazida ao Hospital Veterinário da Universidade Federal do Paraná - Curitiba (HV-UFPR) com histórico de inflamação mamária com evolução de 10 dias e não responsiva ao tratamento para mastite (com cefalexina e metergolina). Ao exame físico, as mamas abdominais caudais e inguinais direita apresentavam-se em placa, com aumento de temperatura, edema e eritema localizados e presença de sensibilidade dolorosa ao toque. A ultrassonografia abdominal e bioquímica sérica não apresentaram alterações significativas, enquanto que a radiografia torácica evidenciou imagem sugestiva de metástase pulmonar. Realizou-se biópsia aspirativa por agulha fina para análise citológica, a qual foi compatível com neoplasia epitelial maligna, provavelmente carcinoma mamário. O diagnóstico de CIM baseou-se nos sinais clínicos e resultados citopatológicos. Instituiu-se tratamento com M8 oral (0,5mL a cada 12 horas) e tópico (nas mamas envolvidas), em associação com piroxicam (0,3mg/kg, PO, a cada 24 horas). Observou-se melhora clínica significativa após 7 dias de tratamento e até a presente data (70 dias de tratamento com M8) a paciente não apresenta sinais clínicos de CIM e de progressão da doença.Conclusão: O presente caso sugere que a associação de M8 e piroxicam contribui para melhora da qualidade de vida e aumento da taxa de sobrevida em cães com CIM. No entanto, mais estudos são necessários para avaliar a resposta clínica de pacientes com CIM tratados com M8.(AU)

Avaliação da produção de citocinas pelos macrófagos J774. G8 após o tratamento com bioterápicos / Evaluation of cytokine production by J774. G8 macrophages after treatment with biotherapics

Strains of macrophages, such as murine J774.G8 macrophages, are susceptible to influenza A infection [1]. One of the responses to viral infection involves the production of various types of immunostimulatory cytokines by infected cells [2].In all cases, there were no significant differences compared to control groups. However, the production of TNF-? detected in macrophages treated by intact and inactivated biotherapics presented a tendency to increase after infection. In fact, similar results were previously detected in other experiments conducted only with the intact biotherapic [3]. The release of the cytokine MCP1 in all experimental situations presented a tendency to decrease after the viral infection when compared to untreated macrophages. No statistically significant difference was detected in the production of IL 12 and IL 10. These experiments will be repeated to confirm the data obtained.(AU)

Tratamento in vivo com M8, uma solução complexa altamente diluída, reduziu a malignidade de um modelo de melanoma de camundongo / In vivo treatment with M8, a highly diluted tinctures complex, reduced the malignancy of a mouse melanoma model

Background: Cancer is a class of disease responsible for 13% of death cause worldwide. Among all types of cancers, one of the most aggressive and with the highest death rate is melanoma. It is highly metastatic and current treatments with chemotherapeutic drugs do not yield satisfactory results. Therefore, the interest on new therapeutics for cancer treatment has been increasing on research. Highly diluted tinctures (HDT) 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. Previous results have demonstrated in vitro inhibition of invasion ability and in vivo anti-metastatic potential of B16F10 lung metastasis model after mice treatment with M8 inhalation.Conclusion: Even though further investigation are necessary to elucidate the mechanisms of action of M8 treatment there is an indication that these highly diluted tinctures could be a promising therapy to treat metastatic melanoma.(AU)

Introdução: O Câncer é uma classe de doenças responsáveis por 13% das causas de mortes no mundo todo. Entre todos os tipos de cânceres, um dos mais agressivos e com maior índice de mortalidade é o melanoma. Ele é altamente metastático, e os tratamentos atuais com drogas quimioterapeuticas não geram resultados satisfatórios. Portanto, o interesse em novos agentes terapeuticos para o tratamento do câncer tem aumentado na pesquisa. Soluções altamente diluídas (CAD) são destinadas à aumentar a resposta do sistema imunológico resultando em menores frequencias de várias doenças, e também não apresentam riscos de graves efeitos colaterais, devido à sua toxicidade reduzida. Resultados anteriores demostraram a inibição in vitro da habilidade de invasão e potencial antimetastático in vivo do modelo de metástase pulmonar da B16F10, após o tratamento dos camundongos pela inalação do M8.Conclusão: Mais pesquisas são necessárias para esclarecer os mecanismos de ação do tratamento com M8. Entretanto, há um indicativo de que soluções altamente diluídas podem ser terapias coadjuvantes para o tratamento do melanoma metastático.(AU)

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.

A shorter fixation protocol for transmission electron microscopy: an alternative to spend less time.

The performance of a moderately shorter fixation protocol for transmission electron microscopy (TEM) was evaluated by analyzing the cell structure quality after the processing. The relevance of this experimental technique is mainly based on reducting time of the steps of conventional protocols: fixation, washes, dehydration, and epoxy resin infiltration. Two sources of murine cells were used, the peritoneal and mesenteric lymph node cells. A fixation and material processing faster than usual methods can save time and improve results. Samples analysis indicated good preservation of different cell structures and organelles after this protocol.

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.

Gene expression profiling of macrophages following mice treatment with an immunomodulator medication.

Canova (CA) is a complex homeopathic medication used in diseases where the immune system is depressed. Previous studies demonstrated that it is neither toxic nor mutagenic and activates macrophages. We now evaluate CA effects on cytokine production and gene expression from mice macrophages. The global view of changes in expression of genes with known functions can provide a vivid picture of the way in which cell adapts to a changing environment or a challenge. We found a decrease in IL-2 and IL-4 production and a differential expression in 147 genes from CA group. These genes are mainly involved in transcription/translation, cell structure and dynamics, immune response, cytoprotection, enzymatic process, and receptors/ligands. With gene expression analysis we state that this medication provokes a reaction that involves alterations in gene expression profile mainly in the ones involved with macrophages activation, corroborating the laboratorial research and the clinical data.

Activation of mononuclear bone marrow cells treated in vitro with a complex homeopathic medication.

Canova is a Brazilian homeopathic medication with immunomodulatory properties, recommended for patients where the immune system is depressed. 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. We now report the effect of in vitro administration of the medication on the mononuclear differentiation of the bone marrow cell. Swiss mice femurs were dissected cleaned and the cells of the marrow were flushed. The cells were plated, treated or not, incubated for different times and processed for light, transmission and scanning electron, and confocal microscopy analysis. Bone marrow cells showed an enhanced proliferation in vitro in response to Canova medication and Canova plus M-CSF and an increase was also observed in the numbers of the cell niches and ring-shaped nuclei cells. Confocal and transmission and scanning electron microscopy showed the stages of monocyte maturation, with resting and activated cells. With Canova treatment there was a marked increase in cell size, which is mainly attributable to the augmented cytoplasm, an increase in the number of mitochondria, expansion of the RER and an enlarged Golgi. The response to Canova treatment indicates that it influences mononuclear differentiation and activation of bone marrow progenitor and stromal cells.

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.

Histological and immunohistochemical evaluation of sarcoma 180 in mice after treatment with and alfa-D-glucan from the lichen Ramalia celastri

The antitumoral activity of an alfa-D-glucan from the lichen Ramalia celastri was investigated using the tumor Sarcoma 180 (S-180). Mice were inoculated with the tumor and 24 h later received a single dose of alfa-D-glucan (200 mg/kg). Thirty-five days after inoculation the mice were sacrificed and the tumors were examined histopathologically. Morphological analyses showed that the tumor was invasive and that it produced typical and atypical mitoses, neovascularization and an infiltration of inflammatory cells. In treated mice, inflammatory cells were more frequent and the presence of nuclear fragments suggested tumor cell death by apoptosis. The tumors of control and alfa-D-glucan treated mice were negative for laminin but expressed fibronectin, the intensity and distribution of which varied in the connective tissue surrounding the tumor mass, in treated mice than in control mice, but in tumor cells, the expression was greater in control mice. The results indicate that alfa-D-glucan can inhibit tumor growth and affect host defense cell responses. The differences in fibronectin distribution between the control and alfa-D-glucan treated mice, suggest that this protein may play an important role in limiting the invasiveness of malignant cells.

Acid phosphatase activity in ungerminated conidia from Colletotrichum graminicola as determined by spectrophotometric and cytochemical methods

The acid phosphatase in ungerminated conidia from Colletotrichum graminicola, a corn pathogen, was investigated using spectrophotometric and cytochemical methods. Acid phosphatase activity was studied in a homogenate obtained by fragmentation of ungerminated conidia. With p-nitrophenylphosphate as substrate, the apparent Vmax and Km were 1.000 nmol p-nitrophenol/mg of protein/min and 0.631 mM, respectively. The pH and temperature optima were 5.5 and 60 graus Celsius, respectively. A cytochemical ultrastructural assay showed deposition of the reaction product inside vacuoles but not extracellularly on the cell surface. The permeabilization of conidia with Triton X-100 increased acid phosphatase activity eight fold. Compared to other procedures, our method was fast, easy to perform and gave consistent results.