RESUMO
BACKGROUND: The homeopathic medicines Silicea terra (Sil) and Zincum metallicum (Zinc) modulate macrophage activity and were assessed in an experimental study in-vitro for their effects on macrophage-BCG (Bacillus Calmette-Guérin) interaction. METHODS: RAW 264.7 macrophages were infected with BCG, treated with different potencies of Sil and Zinc (6cH, 30cH and 200cH) or vehicle, and assessed 24 and 48 h later for bacilli internalization, hydrogen peroxide (H2O2) and cytokine production, and lysosomal activity. RESULTS: Treatment with vehicle was associated with non-specific inhibition of H2O2 production to the levels exhibited by uninfected macrophages. Sil 200cH induced significant reduction of H2O2 production (p < 0.001) compared with the vehicle and all other treatments, as well as higher lysosomal activity (p ≤ 0.001) and increased IL-10 production (p ≤ 0.05). Such effects were considered specific for this remedy and potency. The number of internalized bacilli was inversely proportional to Zinc potencies, with statistically significant interaction between dilution and treatment (p = 0.003). Such linear-like behavior was not observed for Sil dilutions: peak internalization occurred with the 30cH dilution, accompanied by cellular degeneration, and IL-6 and IL-10 increased (p ≤ 0.05) only in the cells treated with Sil 6cH. CONCLUSION: Sil and Zinc presented different patterns of potency-dependent effect on macrophage activity. Bacterial digestion and a balanced IL-6/IL-10 production were related to Sil 6cH, though reduced oxidative stress with increased lysosomal activity was related to Sil 200cH. Degenerative effects were exclusively related to Sil 30cH, and potency-dependent phagocytosis was related only to Zinc.
Assuntos
Bacillus/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Materia Medica/farmacologia , Zinco/farmacologia , Brasil , Humanos , Mycobacterium bovis/efeitos dos fármacosRESUMO
BACKGROUND: The mechanism by which highly diluted and agitated solutions have their effect is still unknown, but the development in recent years of new methods identifying changes in water and solute dipole moments is providing insights into potential modes of action. OBJECTIVE: The objective of the current study was to compare the biological effects of Antimonium crudum (AC) previously obtained by our group and already described in the literature with now measurable physico-chemical effects on solvatochromic dyes. METHODS: Different dilutions of AC and succussed water have been characterized with respect to their effect on the visible spectra of the solvatochromic dyes methylene violet (MV), a pyridinium phenolate (ET33), and a dimethylamino naphthalenone (BDN) compared with in-vitro action against Leishmania amazonensis-infected macrophages. RESULTS: Dye responses varied according to the dye used and the level of AC dilution and results were found to corroborate previously published in-vivo and in-vitro effects of AC. In addition, a very significant enhancement in the absorbance increase of MV was seen using the supernatant from AC 200cH-treated cells (15%; p < 0.0001) over that seen with AC 200cH itself (4%; p = 0.034), suggesting the amplification of ultra-high dilution effects by biological systems. Furthermore, supernatants from AC-treated cells increased the range of dilutions of AC that were capable of producing effects on the spectra of MV. The effect of AC dilutions on dye ET33 was eliminated by a weak electric current passed through potency solutions. CONCLUSION: The data confirm a correspondence between the biological effects of dilutions of AC in-vitro and physico-chemical effects on solvatochromic dyes as measured by changes in their visible spectra. Results also indicate high dilutions of AC are sensitive to exposure to electric currents and biological systems.
Assuntos
Antimônio/química , Antimônio/farmacologia , Corantes/química , Homeopatia , Solventes/química , Corantes/farmacologia , Leishmania mexicana/efeitos dos fármacos , Macrófagos , Solventes/farmacologia , Espectrofotometria UltravioletaRESUMO
INTRODUCTION: Encephalitozoon cuniculi (E. cuniculi), a fungus that acts as an intracellular pathogen, causes a marked neurological syndrome in many host species and is a zoonotic concern. Although no well-established treatment for this syndrome is known, previous successful clinical experience using homeopathic phosphorus has been described in which symptom remission with no mortality occurred in 40/42 animals by means of unknown immunological mechanisms. The latter observation was the main motivation for this study. OBJECTIVE: To verify, in an in-vitro model, if macrophages infected with E. cuniculi can change in function after treatment with different potencies of phosphorus. MATERIALS AND METHODS: RAW 264.7 macrophages were infected with E. cuniculi in-vitro and treated with various homeopathic potencies of phosphorus. The vehicle was used as a control solution (0.06% succussed ethanol). After 1 and 24 hours, the following parameters were analyzed: parasite internalization (by the Calcofluor staining method), lysosome activity (by the acridine orange method), cytokine/chemokine production (by the MAGPIX system), and cell ultrastructure. Automatic image analysis was used when applicable, and the experiments were performed in triplicate. RESULTS: Treatment with vehicle alone increased interleukin (IL)-6, tumor necrosis factor alpha and monocyte chemotactic protein -1 production (p ≤ 0.05) and reduced the number of internalized parasites (p ≤ 0.001). A progressive and time-dependent increase in RANTES (regulated on activation, normal T-cell expressed and secreted) and lysosome activity (p ≤ 0.002) was observed only after treatment with the highest potency of phosphorus (Phos 200cH), together with decreased apoptosis rate, intense parasite digestion, and the presence of non-internalized spores. CONCLUSIONS: Phos 200 cH has a modulatory action on the activity of infected macrophages, especially a specific increase in RANTES, a key element in the prognosis of E. cuniculi-infected and of immunosuppressed patients bearing infections.
Assuntos
Encephalitozoon cuniculi/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Fósforo/uso terapêutico , Animais , Encephalitozoon cuniculi/patogenicidade , Encefalitozoonose/tratamento farmacológico , Homeopatia/métodos , Homeopatia/normas , Macrófagos/microbiologia , Fosfatos/uso terapêutico , CoelhosRESUMO
INTRODUCTION: According to the "silica hypothesis" formulated to explain homeopathy, the information of starting materials would be transferred to cells by silica nanoparticles detached from the glassware walls by serial dilution and agitation through epitaxy. We compared the biological activity, electrical current and silicon microparticle content (by means of scanning electron microscopy/energy-dispersive X-ray spectroscopy) of high dilutions (HDs) of arsenic prepared in plastic and glass vials to investigate the role of silica in their biological effects in vitro. MATERIALS AND METHODS: Co-cultures of macrophages and yeast (Saccharomyces cerevisiae) were treated with different HDs of arsenic prepared in plastic and glass vials. Macrophage morphology, phagocytosis index, nitric oxide (NO), and cytokine production were evaluated. RESULTS: Measurable amounts of silicon microparticles were detected only in the HDs prepared in glass vials, but ultra-centrifugation eliminated them. Specific and non-specific results were observed. Non-specific pro-inflammatory effects were seen in all dilutions prepared in plastic vials, including elevation of pro-inflammatory cytokines, NO and macrophage phagocytic index. Only the 200th centesimal dilution of arsenic produced specific decrease in interleukin-6 production in macrophages, and it was independent of the vial type or the presence of microparticles of silica in the medicine samples. The nature of the vials had an impact on the electric flow in the respective fluids. CONCLUSION: The non-specific, pro-inflammatory effects might be attributed to organic residuals detached from the vials' plastic walls during manipulation. Instead, specific silica-independent effects of the homeopathic medicine can be attributed to the decrease of interleukin-6 after treatment with the 200th centesimal dilution of arsenic.
Assuntos
Arsenicais/isolamento & purificação , Condutividade Elétrica , Silício/isolamento & purificação , Citocinas/isolamento & purificação , Homeopatia/métodos , Humanos , Microscopia Eletrônica de Varredura/métodosRESUMO
Existem poucos testes validados para avaliar prejuízos cognitivos em usuários de cocaína/crack, sendo estes restritos a poucos domínios. O Montreal Cognitive Assessment (MoCA) é um teste que avalia múltiplos prejuízos cognitivos, validado, por exemplo, para o diagnóstico de demência, e doença de Alzheimer, mas não para usuários de cocaína/crack. Nós comparamos o desempenho de usuários crônicos de cocaína/crack com indivíduos saudáveis no teste MoCA. Nós também avaliamos o desempenho destes indivíduos no teste Trail Making Test (TMT) para comparar os resultados. Sujeitos controles e usuários eram ambos do sexo masculino e adultos, com pelo menos 10 anos de escolaridade (para evitar falsos erros cognitivos). No teste MoCA os usuários de cocaína/crack apresentaram escores inferiores aos controles. No TMT (A, B e B-A) também. Esses resultados revelaram prejuízos cognitivos, como, por exemplo, na linguagem e memória dos usuários. Porém, a correlação de escores entre os testes MoCA e TMT foi evidenciada somente no grupo controle, sugerindo não só a diferença, mas outros importantes resultados obtidos com a realização de ambos os testes, de modo a garantir um rastreamento mais completo e múltiplo de prejuízos cognitivos. Portanto, nós sugerimos o uso do MoCA como um teste complementar ao TMT para avaliar prejuízos cognitivos em usuários crônicos de cocaína/crack.
There are few validated tests to measure cognitive impairments in cocaine/crack users; these tests are restricted to single/few domains. The Montreal Cognitive Assessment (MoCA) test evaluates multiple cognitive impairments and is valid for dementia, and Alzheimer's disease diagnosis, for example, but not for cocaine/crack users. We compared the performance of chronic cocaine/crack users and healthy individuals in the MoCA test. We also performed the Trail Making Test (TMT) to compare the results. Controls and cocaine/crack users groups were both formed by adult males, with at least 10 years of schooling (to eliminate false cognitive errors). In the MoCA test, cocaine/crack users had lower scores than control subjects. In TMT (A, B, and B-A), they also performed worse than controls. These results revealed cognitive impairments, such as on language, memory, and flexibility in cocaine/crack users. However, a correlation between MoCA and TMT scores was only evidenced in control group; not for cocaine/crack users. This suggests that different and important results could be obtained from both tests, for a more complete and multiple screening on cognitive impairments. Therefore, we suggest the use of MoCA as a complementary test of TMT to evaluate cognitive impairments in chronic cocaine/crack users.
Assuntos
Humanos , Cocaína , Cocaína Crack , Testes Neuropsicológicos , Transtornos Relacionados ao Uso de Substâncias , Usuários de Drogas , Reabilitação PsiquiátricaRESUMO
Existem poucos testes validados para avaliar prejuízos cognitivos em usuários de cocaína/crack, sendo estes restritos a poucos domínios. O Montreal Cognitive Assessment (MoCA) é um teste que avalia múltiplos prejuízos cognitivos, validado, por exemplo, para o diagnóstico de demência, e doença de Alzheimer, mas não para usuários de cocaína/crack. Nós comparamos o desempenho de usuários crônicos de cocaína/crack com indivíduos saudáveis no teste MoCA. Nós também avaliamos o desempenho destes indivíduos no teste Trail Making Test (TMT) para comparar os resultados. Sujeitos controles e usuários eram ambos do sexo masculino e adultos, com pelo menos 10 anos de escolaridade (para evitar falsos erros cognitivos). No teste MoCA os usuários de cocaína/crack apresentaram escores inferiores aos controles. No TMT (A, B e B-A) também. Esses resultados revelaram prejuízos cognitivos, como, por exemplo, na linguagem e memória dos usuários. Porém, a correlação de escores entre os testes MoCA e TMT foi evidenciada somente no grupo controle, sugerindo não só a diferença, mas outros importantes resultados obtidos com a realização de ambos os testes, de modo a garantir um rastreamento mais completo e múltiplo de prejuízos cognitivos. Portanto, nós sugerimos o uso do MoCA como um teste complementar ao TMT para avaliar prejuízos cognitivos em usuários crônicos de cocaína/crack.(AU)
There are few validated tests to measure cognitive impairments in cocaine/crack users; these tests are restricted to single/few domains. The Montreal Cognitive Assessment (MoCA) test evaluates multiple cognitive impairments and is valid for dementia, and Alzheimer's disease diagnosis, for example, but not for cocaine/crack users. We compared the performance of chronic cocaine/crack users and healthy individuals in the MoCA test. We also performed the Trail Making Test (TMT) to compare the results. Controls and cocaine/crack users groups were both formed by adult males, with at least 10 years of schooling (to eliminate false cognitive errors). In the MoCA test, cocaine/crack users had lower scores than control subjects. In TMT (A, B, and B-A), they also performed worse than controls. These results revealed cognitive impairments, such as on language, memory, and flexibility in cocaine/crack users. However, a correlation between MoCA and TMT scores was only evidenced in control group; not for cocaine/crack users. This suggests that different and important results could be obtained from both tests, for a more complete and multiple screening on cognitive impairments. Therefore, we suggest the use of MoCA as a complementary test of TMT to evaluate cognitive impairments in chronic cocaine/crack users.(AU)
Assuntos
Humanos , Cocaína Crack , Cocaína , Transtornos Relacionados ao Uso de Substâncias , Testes Neuropsicológicos , Usuários de Drogas , Reabilitação PsiquiátricaRESUMO
We have previously reported decreased expression and activities of lysosomal cathepsins B and L in diabetic kidney. Relevant morphological changes were observed in proximal tubules, suggesting that these cells are implicated in the early stages of the disease. The aim of the present study was to investigate the mechanisms that lead to these changes. The effects of high glucose (HG) and advanced glycation end products (AGEs) on cell viability, lysosomal enzymes and other effectors of cell signaling of cultured kidney cells were studied. HG increased viable mesangial cells (ihMC) in 48 h, while epithelial tubular cells were not affected (LLC-PK1 and MDCK). In contrast, the number of viable cells was markedly decreased, for all cell lines, by AGE-BSA. Concerning lysosomal enzymes, the main cysteine-protease expressed by these cells was cathepsin B, and its concentration was much higher in epithelial than in mesangial cells. Exposure to HG had no effect on the cathepsin B activity, but AGE-BSA caused a marked decrease in LLC-PK1, and increased the enzyme activities in the other cell lines. The levels of nitric oxide (NO) was increased by AGE-BSA in all cell lines, suggesting oxidative stress, and Western blotting has shown that, among the investigated proteins, cathepsin B, mTOR and transcription factor EB (TFEB) were the most significantly affected by exposure to AGE-BSA. As mTOR induces anabolism and inhibits autophagy, and TFEB is a master transcription factor for lysosomal enzymes, it is possible that this pathway plays a role in the inhibition of lysosomal enzymes in proximal tubule cells.
Assuntos
Glucose/farmacologia , Produtos Finais de Glicação Avançada/farmacologia , Rim/citologia , Animais , Bovinos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cães , Humanos , Lisossomos/enzimologia , Soroalbumina Bovina/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: In previous results mice treated with high dilutions of antimony presented reduction of monocyte migration to the site of infection with increase in B lymphocytes population in the local lymph node. AIMS: To know the mechanisms involved, a series of in vitro studies was done, using co-cultures of macrophages (RAW 264.7) and Leishmania (L.) amazonensis treated with different dilutions of antimony (Antimonium crudum or AC), in different times. METHODOLOGY: Spreading, phagocytosis, the oxidative activity of macrophages, the viability of free promastigotes and the cytokines/chemokines concentration in the supernatant were evaluated. The assays were performed in quadruplicate. RESULTS: Cells treated with AC 30cH (10-58M) and AC 200cH (10-398M) presented a temporary reduction of the spreading after 02h of incubation, followed by increase after 48h, being the most significant increase observed after the AC 200cH treatment. However, the percentage of internalized parasites at 48, 96 and 120h of incubation was also higher in cells treated with AC 200cH. It is suggested that the AC 200cH improves the ability of phagocytes to internalize the parasites, but not to digest them. The cytokines-chemokines panel corroborated these results. Both dilutions potentiated the parasite-induced reduction of cytokines production, especially IL-6, IL 12 p40 and γ-IFN, after 48h of incubation. In addition, the production of MIP-1 beta (CCL4), a chemokine involved in chronic inflammation, was also reduced after 120h. A specific effect of AC 30cH was seen by the inhibition of two peaks of CCL2 (MCP-1) observed in infected macrophages, at 24 and 120h. Since this cytokine is an important chemokine for monocytes, it explains the results obtained formerly in vivo. The morphology of macrophages after acridine orange staining revealed that the treatment with AC 30cH reduced substantially the acid vacuoles in the cytoplasm, indicating a certain inability of these cells to digest the parasites. On the other hand, a large peak of VEGF-A, associated with increase of internalized parasites was observed after 120h of treatment with AC 200cH, which could be associated to the regulation of the chronic inflammation events by M1-M2 polarization. There was no statistical difference among groups regarding the production of TNF, NO and H2O2, showing that the drugs do not alter macrophage cytotoxic activity. A clear quantitative and qualitative variation of the modulatory effects of AC 30cH and 200cH was seen, in function of time. CONCLUSIONS: Both dilutions were able to potentiate the decrease of most of cytokines and chemokines induced by the parasite infection in vitro, which explains the clinical improvement seen previously in vivo, however, the mechanisms involved and the epidemiological significance of these findings are still under discussion.
Assuntos
Antimônio/farmacologia , Leishmania/imunologia , Leishmaniose/imunologia , Macrófagos/imunologia , Monocinas/imunologia , Animais , Leishmaniose/patologia , Macrófagos/parasitologia , Camundongos , Células RAW 264.7RESUMO
BACKGROUND: Anoikis resistance is one of the abilities acquired along tumor progression. This characteristic is associated with metastasis development, since tumorigenic cells must survive independently of cell-matrix interactions in this process. In our laboratory, it was developed a murine melanocyte malignant transformation model associated with a sustained stressful condition. After subjecting melan-a melanocytes to 1, 2, 3 and 4 cycles of anchorage impediment, anoikis resistant cells were established and named 1C, 2C, 3C and 4C, respectively. These cells showed altered morphology and PMA independent cell growth, but were not tumorigenic, corresponding to pre-malignant cells. After limiting dilution of 4C pre-malignant cells, melanoma cell lines with different characteristics were obtained. Previous data from our group showed that increased Timp1 expression correlated with anoikis-resistant phenotype. Timp1 was shown to confer anchorage-independent growth capability to melan-a melanocytes and render melanoma cells more aggressive when injected into mice. However, the mechanisms involved in anoikis regulation by Timp1 in tumorigenic cells are not clear yet. METHODS: The ß1-integrin and Timp1 expression were evaluated by Western blotting and CD63 protein expression by flow cytometry using specific antibodies. To analyze the interaction among Timp1, CD63 and ß1-integrin, immunoprecipitation assays were performed, anoikis resistance capability was evaluated in the presence or not of the PI3-K inhibitors, Wortmannin and LY294002. Relative expression of TIMP1 and CD63 in human metastatic melanoma cells was analyzed by real time PCR. RESULTS: Differential association among Timp1, CD63 and ß1-integrins was observed in melan-a melanocytes, 4C pre-malignant melanocytes and 4C11- and 4C11+ melanoma cells. Timp1 present in conditioned medium of melanoma cells rendered melan-a melanocytes anoikis-resistant through PI3-K signaling pathway independently of Akt activation. In human melanoma cell lines, in which TIMP1 and beta-1 integrin were also found to be interacting, TIMP1 and CD63 levels together was shown to correlate significantly with colony formation capacity. CONCLUSIONS: Our results show that Timp1 is assembled in a supramolecular complex containing CD63 and ß1-integrins along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway, independently of Akt phosphorylation. In addition, our data point TIMP1, mainly together with CD63, as a potential biomarker of melanoma.