Clara Bonafini

The calcium-sensing receptor: a novel Alzheimer's disease crucial target?

Alzheimers disease (AD) is the most common human neurodegenerative ailment, the most prevalent (>95%) late-onset type of which has a still uncertain etiology. The progressive decline of cognitive functions, dementia, and physical disabilities of AD is caused by synaptic losses that progressively disconnect key neuronal networks in crucial brain areas, like the hippocampus and temporoparietal cortex, and critically impair language, sensory processing, memory, and conscious thought. ADs two main hallmarks are fibrillar amyloid-β (fAβ) plaques in extracellular spaces and intracellular accumulation of fAβ peptides and neurofibrillary tangles (NFTs). It is still undecided whether either or both these AD hallmarks cause or result from the disease. Recently, the dysregulation of calcium homeostasis has been advanced as a novel cause of AD. In this case, a suitable candidate of AD driver would be the Aβ peptides-binding/activated calcium-sensing receptor (CaSR), whose intracellular signalling is triggered by Aβ peptides. In this review, we briefly discuss CaSRs roles in normal adult human astrocytes (NAHAs) and their possible impacts on AD.

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 A. 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, A. montana affected the expression of several genes. In particular CXC chemokine ligand 1 (CXCL1), coding for an chief chemokine, 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 A. 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 A. montana as a promoter of healing, since some of the genes it modifies are key regulators of tissue remodelling, inflammation and chemotaxis.

Arnica montana Stimulates Extracellular Matrix Gene Expression in a Macrophage Cell Line Differentiated to Wound-Healing Phenotype

Arnica montana (Arnica m.) is used for its purported anti-inflammatory and tissue healing actions after trauma, bruises, or tissue injuries, but its cellular and molecular mechanisms are largely unknown. This work tested Arnica m. effects on gene expression using an in vitro model of macrophages polarized towards a “wound-healing” phenotype. The monocyte-macrophage human THP-1 cell line was cultured and differentiated with phorbol-myristate acetate and Interleukin-4, then exposed for 24h to Arnica m. centesimal (c) dilutions 2c, 3c, 5c, 9c, 15c or Control. Total RNA was isolated and cDNA libraries were sequenced with a NextSeq500 sequencer. Genes with significantly positive (up-regulated) or negative (down-regulated) fold changes were defined as differentially expressed genes (DEGs). A total of 20 DEGs were identified in Arnica m. 2c treated cells. Of these, 7 genes were up-regulated and 13 were down-regulated. The most significantly up-regulated function concerned 4 genes with a conserved site of epidermal growth factor-like region (p<0.001) and three genes of proteinaceous extracellular matrix, including heparin sulphate proteoglycan 2 (HSPG2), fibrillin 2 (FBN2), and fibronectin (FN1) (p<0.01). Protein assay confirmed a statistically significant increase of fibronectin production (p<0.05). The down-regulated transcripts derived from mitochondrial genes coding for some components of electron transport chain. The same groups of genes were also regulated by increasing dilutions of Arnica m. (3c, 5c, 9c, 15c), although with a lower effect size. We further tested the healing potential of Arnica m. 2c in a scratch model of wound closure based on the motility of bone marrow-derived macrophages and found evidence of an accelerating effect on cell migration in this system. The results of this work, taken together, provide new insights into the action of Arnica m. in tissue healing and repair, and identify extracellular matrix regulation by macrophages as a therapeutic target.

Response to Commentary: Arnica montana Effects on Gene Expression in a Human Macrophage Cell Line. Evaluation by Quantitative Real-Time PCR

We recently investigated the effects of Arnica montana (Arnica) on the THP-1 myelomonocytic cell line, differentiated by phorbol-myristate acetate and IL-4 in the wound healing phenotype (M2) (1). Our study was the object of a commentary by Chirumbolo and Bjorklund (2), based on some recalculations and on their extrapolations from the values of SEM. Even though we had correctly reported the dosage of sesquiterpenes – reference substances for Arnica pharmacopeia – the authors inferred that this information was insufficient and they recalculated the doses in terms of helenalin: “in the starting 30% alcoholic preparation of Arnica (1c), a percentage of 0.036% sesquiterpene lactones, should correspond to 0.72 μg, i.e., 72 ng in the dilution 2c.” This calculation is flawed because our dose was referred to the mother tincture and not to the 1c dilution. Moreover, helenalin is not the only active ingredient in Arnica, which contains many other substances (3, 4). Finally, 0.72 μg in 1c are not 72 ng in 2c but 7.2 ng.

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.

Critical comments and methodological variations in Arnica montana's research studies

We read the article entitled “Bias and misleading concepts in an Arnica research study” [1], that criticizes our recent paper published in PlosONE [2]. The commentary claims the existence of “biases and misleading concepts” in the experimental line of Arnica montana (Arnica) studies. However, these arguments are based on incorrect interpretations of the data we reported. In their section 1, the authors criticize the molar estimation of the sesquiterpene lactones in the Arnica dilutions. However, the content of sesquiterpene lactones in Ref. [2] was determined by liquid chromatography in conformity with the European Pharmacopoeia Guidelines, which expresses the active constituents of Arnica as dihydrohelenalin tiglate. This is considered the most abundant type, even if a variety of chemical ester variants could be present depending on the cultivar, the part of the plant, the time and methods of harvesting. The correctness of calculation of standard sesquiterpene lactones was already illustrated [3] in response to a previous letter of the same authors [4]. The authors calculate that we used 51.1 mM ethanol in the assay system [2], a dose that they consider toxic for the cells. However, the final ethanol in cell culture was 5.1 mM, not 51.1 mM. Consequently, all the considerations about the purported confounding effects ascribed to ethanol are wrong, since they refer to ethanol doses ten times higher. Moreover, the same small dose of ethanol was used in control samples, and there was no toxicity at all. Note that a refutation of the toxicity of ethanol in homeopathic medicines was already published [5]. The cited paper [1] casts doubt as to the UVevis spectrum of the Arnica solution, suggesting that this may be due to ethanol. Actually, as clearly indicated in themethods, the spectrumwas obtained using a double-beam spectrophotometer procedure, using as reference-blank sample the same solvent control, so it is impossible that the published profile [2] corresponds to ethanol. Concerning the preparation of samples and the problem of filtering that is raised, all the procedural handlings were conducted in parallel,

Astrocytes may drive a Beta-Amyloid contagion in human Alzheimer's disease brains

mean MMSE1⁄427.2 62.2) we developed automated unimodal and multimodal classifiers based on support vector machines. The pool of features provided to the classifiers included MMSE, Trails B, AVLT delayed recall, plasma levels of ApoE, BDNF, TNF alpha, IL13, IL6 and clusterin, and hippocampal volume. All classifiers included age and sex. Three separate classifiers were built to predict mean cortical, lateral parietal and precuneal SUVR as the outcome variable. Results: All classifiers consistently ranked ApoE genotype as the most powerful predictor of brain amyloidosis. ApoE genotype alone predicted brain amyloidosis with 75-77% accuracy (AUC1⁄40.67-0.75). When cognitive, imaging and peripheral blood protein variables were added, classifier performance improved. Greatest classifier accuracy in predicting PIB+ vs. PIBsubjects was seen for the lateral parietal (accuracy1⁄485%, AUC1⁄40.87) and precuneal regions (accuracy1⁄485%, AUC1⁄40.89). The variables selected by these classifiers included ApoE genotype, MMSE, Trails B, AVLT delayed recall, clusterin, TNF alpha and BDNF. The precuneal classifier also included IL13 and ApoE protein. The average cortical classifier used ApoE genotype, MMSE, Trails B, hippocampal volume, clusterin, BDNF and IL6 and achieved 80% accuracy (AUC1⁄40.81). Conclusions: Automated classifiers based on ApoE4 genotype, cognitive and peripheral blood protein variables can identify the presence of brain amyloidosis with high accuracy. Such methods could have implications for clinical trial design and enrollment in the near future.