Cadmium and myalgic encephalomyelitis/chronic fatigue syndrome; application of transcranial sonography to the study of cadmium-induced neuronal damage

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (CFS) is a neurological disease characterized by widespread inflammation and neuropathology. Aetiology and pathogenesis are unknown and it has been hypothesized that exposure to heavy metals is among the triggers of CFS. We recently hypothesized that cadmium, an occupational and environmental heavy metal pollutant, might be associated with some of the neurological findings typical of CFS. It is worth noticing that cadmium induces neuronal death in cortical neurons; cadmium-induced neuronal cell death and cadmium- induced low angiogenesis may be responsible for decrease in attention level and memory in exposed humans as well as to a diminished ability for training and learning in rats (symptoms patognomonic for CFS). In order to assess cadmiuminduced neuronal damage in vivo, we developed a modified procedure for transcranial sonography. This procedure allowed detailed visualization of the cortex of the temporal lobe, the meninges, the subarachnoidal space, and the meningeal and cortical vascularisation. The consequences of our observation could affect prevention, early diagnosis, and treatment of CFS with important repercussions in the definition of clinical cases and in forensic and insurance-related matters. CFS could then be considered as consequence of occupational or accidental exposure to cadmium. Implications in prevention and early diagnosis could entail the evaluation of symptoms typical of CFS in cadmium-exposed subjects as well as the search for signs of exposure to cadmium in subjects diagnosed with CFS. Also the management of the syndrome could be affected; nutritional supplementation of magnesium and zinc have been proposed in the prophylaxis and therapy of cadmium exposure

Treatment and Prevention of Cadmium-induced alterations on human neurons

The effects of Cadmium on the central nervous system are still relatively poorly understood and its role in neurodegenerative diseases has been debated (Matés et al., 2010). In this study we investigate the protective role of a Cadmium antagonist, Zinc, and of a molecule positively affecting neuronal viability such as GcMAF (Vitamin D-binding protein-derived macrophage activating factor) (Mohamad et al., 2002) in counteracting Cadmium-induced cell modifications. Cell line Sh-SY5Y is treated with Cadmium for 24 h with and without Zinc or GcMAF at different concentrations and exposure times. Cell viability, cell morphology and activation of apoptotic pathways are investigated. Results show that Zinc as well as GcMAF are able to partially or totally counteract the toxic effects of Cadmium on human neurons. This lead us to think at the real possibility of limiting, avoiding or reversing toxic effects of Cadmium on human neurons

Effects of vitamin D and paricalcitol on murine cardiomyocytes

Severe alterations of Cacium-Phospate metabolism are frequently associated with chronic kidney diseases. High levels of Phospate and low levels of vitamin D in subjects affected by chronic kidney disease are, in many cases, correlated with high risk of mortality. For this reason, administration of vitamin D represents the elective treatment. Nevertheless, vitamin D in itself induces a variety of side effects which in many cases can be avoided by the administration of vitamin D analogues. In this study we treated murine cardiomyocytes with different concentrations of vitamin D and paricalcitol (one of its analogues) for 48 hours. Cell morphology, cell proliferation, intracellular Calcium deposition, and cAMP level were investigated respectively by light microscopy, immunoenzymatic assay and Von Kossa staining. Results show that, in comparison to paricalcitol, vitamin D induces stronger side effects on cardiomyocytes (such as cell proliferation, morphological changes, activation of signal transduction pathways). From our data emerges that paricalcitol induces less stressful effects on murine cardiomyocytes in comparison to what observed with vitamin D treatment

Intra-tumoural nitric oxide release by macrophages activated by Gc-protein-derived Macrophage Activating Factor (GcMAF)

Over past decades, nitric oxide (NO) has emerged as a molecule of interest in cancer treatment because of its tumouricidal properties (Choudhari et al., 2013). Gcprotein- derived Macrophage Activating Factor (GcMAF) induces the synthesis and release of NO by activated macrophages. It was previously demonstrated that molecular complexes of oleic acid (OA) and GcMAF (OA-GcMAF) stimulate macrophage activation in cancer patients (Ward et al., 2014). Here we demonstrate that intratumoural injection of OA-GcMAF leads NO synthesis and release inside the tumour. Under ultrasound guidance, OA-GcMAF was injected into patients harbouring different types of solid tumours; a metastasis from a melanoma, and a metastasis from breast cancer. Intra-tumoural NO synthesis and release was monitored in real-time by power-doppler ultrasonography. One to two minutes after injection, we observed a significant increase in blood flow and in blood vessels diameter, a clear indication of vasodilation due to NO synthesis and release. These observations substantiate the dramatic clinical results previously observed by Ward et al. (2014), and open the way to further investigation in the role of GcMAF as a powerful anticancer agent

Variations in salivary testosterone, cortisol, and DHEA levels in professional rugby union players during the preseason training period

Excessive training and inadequate recovery can lead to states of overtraining and performance decrements. The anabolic-catabolic balance by testosterone, cortisol and DHEA-S resulted a useful endogenous indicator to evaluate and monitor the athlete’s training state and the effectiveness of the training program [1]. In this study sixteen professional rugby players provided saliva samples during the official preseason training period. Hormone saliva levels were determined by immunoenzymatic assay (Grifols, Italy). The results showed that both cortisol levels and DHEA-S to cortisol ratio levels significantly decreased during the initial reduced training sub-period according to the goal of the training program. Furthermore the testosterone to cortisol ratio levels significantly increased during the initial sub-period as weel as during the organic muscle conditioning period and after a 2-day recovery time following the conditioning period. Testosterone levels were positively correlated with the Gacon physical test and the maximum cardiac frequence during the initial reduced training sub-period. No correlation was observed between hormone levels and genotype. However ACE genotypes (I/D polymorphism) statistically correlated with the different morphotype related to the rugby player position. Our data shows that cortisol and testosterone to cortisol ratio levels can be considered useful tools to evaluate the athlete’s physical stress during the training program. According to data available in literature the morphotype related to the rugby player position is deeply affected by the ACE gene polymorphism

Effects of a physical activity program on functional fitness, oxidative stress and salivary cortisol levels in older adults

Quality of life into later life is influenced by multiple factors. The physical ability to perform common everyday activities represents a key factor to maintain a healthy and independent lifestyle. Moreover, aging is a process characterized by physiological alterations resulting in a progressive decline in biological functions, decreased resistance to stress, and increased susceptibility to diseases. Especially in elderly people, alterations such as imbalance between pro and antioxidant activity and/or hormonal dysregulation negatively affect the physical capacity, the emotional status and the overall general health and quality of life [1]. On the other hand, regular physical activity is considered an effective strategy for older adults to prevent and reduce the risk of developing those negative conditions arising from aging. A 24-week regular physical activity program (twice weekly, 1 hour per session) focused on functional fitness exercises was performed by 20 older adults (aged 55 years or more). A set of anthropometric (height, weight, BMI and body fat percentage) and physical measurements (grip strength, chair sit to stand, sit and reach and back scratch) assessing the functional fitness performance [2] were evaluated. Moreover, biochemical markers (d-ROMs and BAP tests as assessment of oxidative stress and antioxidant potential; salivary cortisol levels) were measured before and after the intervention program. The results confirm the benefits of a regular physical activity in older adults resulting in improved physical strength and flexibility in the functional fitness parameters, and in regulating pro and antioxidant activity and cortisol levels

A molecular model of the interaction between vitamin D binding protein-derived macrophage activating factor and vitamin D receptor

We previously demonstrated that the response of human monocytes to vitamin D binding protein-derived macrophage activating factor (DBP-MAF) is dependent on vitamin D receptor (VDR) polymorphisms [1]. Therefore, in order to verify the type of molecular interaction between DBP-MAF and VDR, we compared the amino acid sequences at their respective vitamin D binding sites. There are 23 hydrophobic amino acids (aa) near the amino terminus of DBP-MAF, and 23 aa near the carboxyl terminus of VDR. When these two sequence are aligned, it is possible to observe not only that in both proteins there is a long stretch (13-14) of hydrophobic aa, but that 4 hydrophobic aa are identical and 11 aa have similar functional valence. According to this model, the last 23 hydrophobic aa of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic aa of the DBP-MAF located at the external part of the plasma membrane, with vitamin D sandwiched in between the two vitamin D-binding proteins. Oleic acid, taken as an example of an unsaturated fatty acid bound to DBP-MAF, could stabilize the complex at the level of the plasma membrane. Therefore, it can be hypothesized that DBP-MAF and VDR have multiple sites of interaction at the level of the plasma membrane. Further studies will elucidate whether this interaction occurs only in the presence of vitamin D or whether the hydrophobic profile of the two proteins allows direct interaction without the need for vitamin D

Alpha-N-acetylgalactosaminidase levels in cancer patients are affected by Vitamin D binding protein-derived macrophage activating factor

It is well assessed that alpha-N-acetylgalactosaminidase (nagalase) accumulates in serum of cancer patients and is responsible for deglycosylation of vitamin D binding protein, which is the precursor of vitamin D binding protein-derived macrophage activating factor (GcMAF), eventually leading to immunodeficiency in advanced cancer patients. The increase in nagalase activity in cancer patients is due to the fact that cancer cells release nagalase, and nagalase activity reflects tumour burden, aggressiveness and progression of the disease up to the point that determination of nagalase activity is a non-invasive way of evaluation of cancer severity. Here we report the observation of a series of clinical cases describing the results obtained administering GcMAF to patients with diverse types of cancers. In all cases, GcMAF treatment was initiated at late stages of tumour progression. The response to GcMAF was robust. All patients (n= 20) presented with nagalase levels well above the threshold of normal values (2.84+0.26 nM/min/mg). All patients, but one, showed significant decrease of nagalase levels following GcMAF weekly injections (1.59+0.17. p<0.01). Nagalase decrease was associated with improved clinical conditions. No adverse side effects were reported. The observation reported here confirm and extend the results presented in [1] and open the way to further studies aimed at assessing the precise role and indications for GcMAF in the immunotherapy of cancer

Vitamin D binding protein-derived macrophage activating factor stimulates proliferation and signalling in a human neuronal cell line

Vitamin D (vitD), vitD binding protein-derived macrophage activating factor (DBP-MAF), and vitD receptor (VDR) are essential for adult neurogenesis [1], and this effect could be responsible for the recently reported effects of DBP-MAF on autism spectrum disorders (ASD) [2]. In order to test this hypothesis, we challenged a human neuronal cell line (SH-SY5Y, IZSLER) with DBP-MAF (Immuno Biotech), and we studied cAMP formation (cAMP EIA kit, Abnova), cell proliferation (MTT assay, Sigma Aldrich), apoptosis (human caspase 3 act, Invitrogen) and cell morphology. SH-SY5Y cells represent a validated in vitro model of human neurons in neurodegenerative diseases [3]. DBP-MAF induced rapid (15 min) formation of cAMP in a dose-dependent manner (0.4-40 ng/ml) as well as increase in cell proliferation at 24-48 and 72 h. Cell morphology was consistent with neurogenesis and an increase in the number of cells with high synthetic activity was observed. No apoptosis following DBP-MAF treatment was observed. Our results open the way to exploit these newly described effects to treat neurodegenerative disorders from Parkinson’s and Alzheimer’s diseases to Myalgic Encephalomyelitis and ASD

Gc-protein derived macrophage activating factor (GcMAF) counteracts the neuronal damage induced by oxaliplatin

Oxaliplatin-based regimens are effective in metastasised advanced cancers. However, a major limitation to their use is represented by neurotoxicity leading to peripheral neurophaty (Wolf et al., 2008). In this study we evaluate the effects of an immunotherapeutic agent (Gc protein-derived macrophage activating factor, GcMAF) in preventing oxaliplatin-induced neuronal damage and in restoring microglial activation. The effects of oxaliplatin was studied in human neurons (SH-SY5Y) and microglial cells (c13-nj). Cell density, morphology and viability as well as production of cAMP and expression of vascular endothelial growth factor (VEGF), markers of neuron regeneration and markers of microglia activation were determined. GcMAF reverted the damage inflicted by oxaliplatin on human neurons and preserved their viability; it also increased cAMP production, VEGF and neuromodulin expression. GcMAF did not revert the effects of oxaliplatin on microglial cell viability. However, it induced microglial activation resulting in an increased expression of a specific marker without any increase in cell number. Our results demonstrate that GcMAF may significantly contribute to neutralize the neurotoxicity induced by oxaliplatin, at the same time concurring to an integrated anti-cancer effect