Cell sensitivity, non-linearity and inverse effects

It has been claimed that the homeopathic principle of 'similarity' (or 'similia') and the use of individualized remedies in extremely low doses conflicts with scientific laws, but this opinion can be disputed on the basis of recent scientific advances. Several mechanisms to explain the responsiveness of cells to ultra-low doses and the similarity as inversion of drug effects, have again been suggested in the framework of hormesis and modern paradoxical pharmacology. Low doses or high dilutions of a drug interact only with the enhanced sensitivities of regulatory systems, functioning as minute harmful stimuli to trigger specific compensatory healing reactions. Here we review hypotheses about homeopathic drug action at cellular and molecular levels, and present a new conceptual model of the principle of similarity based on allosteric drug action. While many common drugs act through orthostatic chemical interactions aimed at blocking undesired activities of enzymes or receptors, allosteric interactions are associated with dynamic conformational changes and functional transitions in target proteins, which enhance or inhibit specific cellular actions in normal or disease states. The concept of allostery and the way it controls physiological activities can be broadened to include diluted/dynamized compounds, and may constitute a working hypothesis for the study of molecular mechanisms underlying the inversion of drug effects

Study of the action mechanisms of Arnica montana effects on macrophages

Objective: To test the effect of Arnica montana (Arnica m.) on human macrophages. Method: The human monocytic leukaemia cell line THP-1 was cultured and differentiated in mature macrophages with PMA and other differentiating agents. Macrophages were exposed to Arnica m. homeopathic dilutions (2c, 3c, 5c, 9c and 15c) or Control solvent. Total RNA was isolated and sequenced to perform quantitative evaluation. Results: Screening sorted out with a list of 20 genes that were significantly changed by Arnica m. 2c treatment: 7 up-regulated and 13 down-regulated. Most notably, a clearly up-regulated function concerned the proteinaceous extracellular matrix (ECM), including genes HSPG2, FBN2, FN1 (

Biological activity of interferon gamma and lipopolysaccharide on the nitric oxide production in C6 astroglioma cells and some unexpected effects of potentization

Background: A proinflammatory environment is a hallmark of several neurodegenerative diseases where astrocyte involvement is also well established. Astrocytes and microglia in central nervous system are mainly involved in the release of cytokines, oxygen free radicals and nitric oxide (NO). Several studies on C6 astroglioma cells, a widely used in vitro model for these events, demonstrated that co-stimulation of this cell line with bacterial lipopolysaccharide (LPS) and interferon gamma (IFN-) induces a synergistic nitric oxide synthase (iNOS) expression.1 In our laboratory we are using this versatile cell model in order to carefully investigate dose-response effects of various putative agonists or inhibitors and to assess the possible changes provoked in those agents by different procedures of dilution and succussion (agitation) (potentization according to the homeopathic terminology)

Investigations into Alzheimer's disease pathogenetic mechanisms using normal adult human astrocytes in culture

La malattia di Alzheimer \ue8 una delle principali patologie neurodegenerative legate alla terza et\ue0: i casi infatti che si sviluppano in et\ue0 precoce rappresentano meno dell\u20191% e sono dovuti ad alterazioni geniche specifiche. L\u2019eziologia di questa malattia resta ancora sconosciuta, sebbene la presenza di placche senili, composte principalmente di A\u3b21-40 e A\u3b21-42, o aggregati fibrillari intraneuronali della proteina tau, siano caratteristiche ormai note del suo stadio avanzato. Diversi processi patologici sono stati messi in relazione con il danno neuronale e il declino cognitivo tipici della malattia. Tra questi uno dei pi\uf9 studiati \ue8 il processo infiammatorio indotto da aggregati di amiloide negli spazi perineuronali e mediato principalmente da astrociti e cellule della microglia. Recentemente anche alterazioni dei meccanismi neurovascolari e della struttura della barriera emato-encefalica sono stati correlati con la progressione del processo patologico e con l\u2019induzione locale di angiogenesi: la presenza di VEGF, una delle principali citochine ad azione angiogenica, \ue8 stata dimostrata infatti in corrispondenza di cluster di astrociti reattivi in pazienti AD. Per chiarire il possibile legame tra la neoangiogenesi, la presenza di aggregati di amiloide e la secrezione di mediatori infiammatori, si \ue8 deciso di analizzare la modulazione dell\u2019espressione del complesso trascrizionale HIF-1\u3b1\u2022HIF-1\u3b2 in astrociti umani normali adulti (NAHA) a seguito del trattamento con la triade di citochine proinfiammatorie (CM-trio, IL-1\u3b2 + IFN-\u3b3 + TNF-\u3b1) e con A\u3b225-35, il frammento attivo dell\u2019A\u3b21-42. Il legame di HIF-1\u3b1\u2022HIF-1\u3b2 alla sequenza HRE presente nella regione promotoriale di geni correlati alla malattia, quali VEGF e BACE-1, \ue8 responsabile della loro induzione in risposta a stimoli quali stress ipossico. Entrambi i trattamenti, pur non alterando in nessun modo la trascrizione delle subunit\ue0 HIF-1\u3b1 e HIF-1\u3b2, inducono sia una maggiore stabilit\ue0 della frazione proteica di entrambi i fattori, sia un\u2019aumentata traslocazione del complesso a livello nucleare con conseguente legame alla sequenza HRE. Il trattamento invece con CM-trio determina una significativa riduzione dell\u2019espressione di un altro fattore implicato nell\u2019induzione della trascrizione del VEGF-A, PGC-1\u3b1, dimostrando che HIF-1\u3b1\u2022HIF-1\u3b2 \ue8 il solo complesso coinvolto nella modulazione del fattore angiogenico. La traslocazione nucleare dell\u2019eterodimero HIF-1\u3b1\u2022HIF-1\u3b2 e il suo legame alla sequenza promotoriale HRE corrisponde ad un aumento dell\u2019espressione di tre varianti di splicing del VEGF-A (121, 165 e 189), ma non all\u2019 accumulo della corrispondente frazione proteica a livello intracellulare. Analisi mediante ELISA hanno dimostrato un aumento del rilascio del VEGF-A nel medium di coltura di cellule trattate con A\u3b225-35, con la coppia IFN-\u3b3 + TNF-\u3b1, ma soprattutto con CM-trio. L\u2019insieme di A\u3b225-35 + CM-trio non \ue8 in grado di esercitare un\u2019azione sinergica sul rilascio, dimostrando che la triade di citochine proinfiammatorie \ue8 il principale induttore della secrezione. Il complesso HIF-1\u3b1\u2022HIF-1\u3b2 si \ue8 rivelato inoltre implicato nella regolazione della trascrizione di BACE-1, una delle secretasi coinvolte nella produzione di A\u3b2. Il trattamento con A\u3b225-35 stimola infatti la maggiore espressione dell\u2019mRNA e della proteina BACE-1, inducendo inoltre un aumento della sua attivit\ue0 e di quella della \u3b3-secretasi, altro enzima responsabile del processamento dell\u2019APP. La produzione di peptidi A\u3b2 in NAHA trattati con A\u3b225-35 \ue8 stata dimostrata tramite immunoblotting e immunofluorescenza; quest\u2019ultima tecnica, in particolare, ha permesso di evidenziare la presenza di aggregati di A\u3b225-35 nel citoplasma di astrociti trattati, delineando un loro possibile ruolo nella rimozione di aggregati di amiloide potenzialmente patologici.Alterations in neurovascular mechanisms and blood-brain barrier (BBB) disorders have been related to late-onset Alzheimer\u2019s disease (AD). An increased expression of vascular endhotelial growth factor (VEGF) has seen in cluster of reactive astrocytes in AD brains and into rat hippocampus A\u3b21-42 injected. We found that the A\u3b225-35, an amyloid \u3b21-42 active fragment, and proinflammatory cytokines (CM-trio, IL-1\u3b2 + IFN-\u3b3 + TNF-\u3b1) treatments has induced an increased protein steady state levels of the hypoxia-inducible factor transcriptional complex (HIF-1\u3b1\u2022HIF-1\u3b2) in normal adult human astrocytes (NAHAs). This transcription factor is the main regulator of the VEGF expression, but also the PGC-1\u3b1 cofactor seems to be related to its upregulation in AD. We analyzed the mRNA expression of both these factors, but only the complex HIF-1\u3b1\u2022HIF-1\u3b2 was implicated in the VEGF-A expression. Indeed, the enhanced HIF-1\u3b1\u2022HIF-1\u3b2 nuclear translocation and its binding at the hypoxia response element (HRE) sequence in the VEGF-A promoter, found after both treatments, were correlated with the increased VEGF-A mRNA splice variants (121, 165, and 189) expression. The CM-trio was the most powerful stimulus inducing the VEGF-A secretion in NAHAs culture medium, also induced, less than CM-trio, by the paired cytokines IFN-\u3b3 + TNF-\u3b1, A\u3b225-35 and CM-trio + A\u3b225-35 treatments. Hence the astrocytes in an AD brain can produce a raised amount of VEGF-A in response to A\u3b2 peptide and the proinflammatory cytokines promoting the neoangiogenic process. We found that the HIF-1\u3b1\u2022HIF-1\u3b2 complex was furthermore responsible for the \u3b2-secretase (BACE-1) mRNA and protein up-regulation in our experimental model treated with A\u3b225-35. After the same treatment we found moreover an enhancement of the \u3b2 and \u3b3-secretase activities, the two secretases involved in the amyloid precursor protein (APP) processing. The immunofluorescence and immunoblotting assays confirmed the A\u3b2 production in NAHAs, suggesting the existence of a positive feed-back loop for the A\u3b2 synthesis in astrocytes. This findings support the theory that astrocytes, as well as neurones, are involved in the AD decline of cognitive functions. Moreover, the immunofluorescence assay shows the A\u3b225-35 uptake, defining a possible role of astocytes as A\u3b2 peptides scavengers

Experimental neuropharmacology of Gelsemium sempervirens: Recent advances and debated issues

Gelsemium sempervirens L. (Gelsemium) is traditionally used for its anxiolytic-like properties and its action mechanism in laboratory models are under scrutiny. Evidence from rodent models was reported suggesting the existence of a high sensitivity of central nervous system to anxiolytic power of Gelsemium extracts and Homeopathic dilutions. In vitro investigation of extremely low doses of this plant extract showed a modulation of gene expression of human neurocytes. These studies were criticized in a few commentaries, generated a debate in literature and were followed by further experimental studies from various laboratories. Toxic doses of Gelsemium cause neurological signs characterized by marked weakness and convulsions, while ultra-low doses or high Homeopathic dilutions counteract seizures induced by lithium and pilocarpine, decrease anxiety after stress and increases the anti-stress allopregnanolone hormone, through glycine receptors. Low (non-Homeopathic) doses of this plant or its alkaloids decrease neuropathic pain and c-Fos expression in mice brain and oxidative stress. Due to the complexity of the matter, several aspects deserve interpretation and the main controversial topics, with a focus on the issues of high dilution pharmacology, are discussed and clarified. Keywords: Gelsemium sempervirens, Anxiety, Neurocytes, Animal models, Homeopathic medicine, Behavior, Gene expressio

Biological activity of interferon gamma and lipopolysaccharide on the nitric oxide production in C6 astroglioma cells and some unexpected effects of potentization

Background: The search for new therapeutic approaches with fewer side effects and better treatment efficacy to the Chagas Disease has been a major challenge. Aim: To evaluate the effects of Kalium causticum, Conium maculatum, and Lycopodium clavatum 13 cH in mice inoculated with the Y strain of Trypanosoma cruzi. Materials and methods: In a blind, controlled, randomized study, 102 male Swiss mice, eight weeks old, were inoculated with 1,400 trypomastigotes of the Y strain of T. cruzi and distributed into the following groups: CI (treated with 7% hydroalcoholic solution), Ca (treated with Kalium causticum 13cH), Co (treated with Conium maculatum 13cH), and Ly (treated with Lycopodium clavatum 13cH). The medicines were selected by three homeopaths using Lince Expert System Software (Albuquerque, NM, USA), considering the behavioral characteristics of the mice. The treatments were performed 48 hours before and 48, 96, and 144 hours after infection [1]. The following parameters were evaluated: infectivity, prepatent period, parasitemia peak, total parasitemia, tissue tropism, inflammatory infiltrate, and survival. Results: The prepatent period was greater in the Ly group than in the CI group (p = 0.02). The number of trypomastigotes on the 8th day after infection was lower in the Ca group than in the CI group (p < 0.05). Total parasitemia was significantly lower in the Ca, Co, and Ly groups than in the CI group. On the 12th day after infection, the Ca, Co, and Ly groups had fewer nests of amastigotes and amastigotes/nest in the heart than the CI group (p < 0.05) (Figure-I). A decrease in the number of nests and amastigotes in the intestine were observed in the Ly group compared with the CI group (p < 0.05). In the liver (day 12), Ly significantly prevented the formation of inflammatory foci compared with the other groups. In muscle, Co and Ly decreased the formation of inflammatory foci compared with CI (p < 0.05). Ly afforded greater animal survival compared with CI, Ca, and Co (p < 0.05). The animals in the Co group died prematurely compared with the CI group (p = 0.031). (Figure-II) Conclusion: All of the experimental homeopathic medications with 13cH dynamization studied herein reduced the parasite peak and total parasitemia. Ly had significantly more benefits in the treatment of mice infected with T. cruzi, reducing the number of blood parasites, amastigotes nests in tissue and the number of amastigotes per nest, resulting in the increasing animal survival. The data may contribute to changes in management strategies in individuals with Chagas disease

Arnica Montana effects on gene expression in a human macrophage cell line. Evaluation by quantitative Real-Time PCR

Background: Arnica montana is a popular traditional remedy widely used in complementary medicine, also for its wound healing properties. Despite its acknowledged action in clinical settings at various doses, the molecular aspects relating to how Arnica montana promotes wound healing remain to be elucidated. To fill this gap, we evaluated the whole plant extract, in a wide range of dilutions, in THP-1 human cells, differentiated into mature macrophages and into an alternative IL-4-activated phenotype involved in tissue remodelling and healing. Methods: Real-time quantitative Reverse Transcription Polymerase Chain Reaction (PCR) analysis was used To study the changes in the expression of a customized panel of key genes, mainly cytokines, receptors and transcription factors. Results: On macrophages differentiated towards the wound healing phenotype, Arnica montana affected the expression of several genes. In particular CXC chemokine ligand 1 (CXCL1), coding for a chief chemo- kine, exhibited the most consistent increase of expression, while also CXC chemokine ligand 2 (CXCL2), Interleukin8 (IL8) and bone morphogenetic protein (BMP2) were slightly up-regulated, suggesting a positive influence of Arnica montana on neutrophil recruitment and on angiogenesis. MMP1, coding for a metalloproteinase capable of cleaving extracellular matrix substrates, was down-regulated. Most results showed non-linearity of the dose-effect relationship. Conclusions: This exploratory study provides new insights into the cellular and molecular mechanisms of action of Arnica montana as a promoter of healing, since some of the genes it modifies are key regulators of tissue remodelling, inflammation and chemotaxis

Amyloid \u3b225-35, an amyloid \u3b21-42 surrogate, and proinflammatory cytokines stimulate VEGF-A secretion by cultured, early passage, normoxic adult human cerebral astrocytes

Cerebrovascular angiopathy affects late-onset Alzheimer\ufffds disease (LOAD) brains by possibly increasing vascular endothelial growth factor (VEGF). A expression, thereby stimulating endothelial cell proliferation and migration. Indeed, VEGFA gene upregulation, with increased VEGFA protein content of reactive astrocytes and microglia, occurs in LOAD brains, and neovascularization was observed one week after injecting amyloid \u3b2 (A\u3b2)1 1242 into rat hippocampus. We have now found, with cultured \ufffdnormoxic\ufffd normal adult human astrocytes (NAHAs), that fibrillar A\u3b225 1235 (an active A\u3b21 1242 fragment) or a cytokine mixture (the (CM)trio (interleukin [IL]1\u3b2+interferon [IFN] +tumor necrosis factor [TNF]-\u3b1), or pair (IFN-y +TNF-\u3b1) like those produced in LOAD brains) stimulates the nuclear translocation of stabilized hypoxiainducible factor (HIF)1\u3b1 protein and its binding to VEGFA hypoxiaresponse elements; the mRNA synthesis for three VEGFA splice variants (121, 165, 189); and the secretion of VEGFA165. The CMtrio was the most powerful stimulus, IFN-y +TNF-\u3b1 was less potent, and other cytokine pairs or single cytokines or A\u3b235 1225 were ineffective. While A\u3b225 1235 did not change HIF1\u3b2 protein levels, the CMtrio increased both HIF1\u3b1 and HIF1\u3b2 protein levels, thereby giving an earlier and stronger stimulus to VEGFA secretion by NAHAs. Thus, increased VEGFA secretion from astrocytes stimulated by A\u3b21 1242 and by microgliareleased cytokines might restore angiogenesis and A\u3b21 1242 vascular clearance.Peer reviewed: YesNRC publication: Ye

Angiogenic factor induction in and secretion by cultured phenotypically normal adult human astrocytes (NAHA) exposed to beta-amyloid peptides and proinflammatory cytokines

The induction and secretion of VEGF-A by cultured phenotypically normal adult human astrocytes (NAHAs) exposed to beta-amyloid peptides and proinflammatory cytokines is reported for the first time

Detection of nanostructures in solutions of Zincum metallicum and the vehicle lactose

Background: Nanoparticles (NP), because of their size (< 1μm) and high relative surface area, are highly reactive forms of their source material with biological, chemical, optical, electromagnetic, and thermal properties different from larger bulk forms of the same material. It has been speculated that NPs can occur in homeopathic products as a function of trituration and shaking into glass containers. Moreover, the presence of sugars additives like lactose and of silicates leaked from the glassware are reported to stabilize the nanoparticles.1 Up to now some authors observed nanoparticles in highly diluted samples2, 3, but further studies are needed to know the nature of the NPs. Actually, nanostructures in the solutions may derive from the source materials but also from stuff contaminations and also may be constituted by nano-bubbles.4, 5 Moreover, the mechanism by which the nanostructures can be formed and the effect of serial dilution/succussions of the NPs suspension should be studied.6 Many tools are available to analyze the nanostructures both in solutions or in dried samples and these give complementary information about the concentration, stability, structure and chemical nature of the NPs. In the present report we describe preliminary observations obtained by the nanoparticle tracking analysis (NTA) in Zincum metallicum (Zinc met.) solutions at low-grade dilution/dynamization and their lactose controls. This study is part of a formal Brazilian-Italian inter-university collaboration