Gabriele Morucci

Cadmium modulates proliferation and differentiation of human neuroblasts

Cadmium is an environmental pollutant inducing numerous pathological effects, including neurological disorders and brain diseases. However, little is known about the molecular mechanisms of cadmium in affecting neurons and in inducing neurotoxicity in the development of the human brain. We have recently established, cloned, and propagated in vitro a primary long‐term cell culture (FNC‐B4) obtained from the human fetal olfactory neuroepithelium. In the present study, we show that different concentrations of cadmium chloride (CdCl2) induced dose‐dependent biological effects in FNC‐B4 cells. A low concentration (10 μM) of CdCl2 stimulated neuroblast growth, whereas a high concentration (100 μM) inhibited the growth and the viability of neuroblasts inducing morphological and cytoskeletal alterations as well as apoptotic cell death. We also observed that CdCl2 affected, in a dose‐dependent manner, the differentiation of FNC‐B4 neuroblasts, with increased mRNA and protein levels of differentiation markers and decreased expression levels of neuronal stem markers. Furthermore, differentiated cells co‐expressed glial and neuronal markers. We suggest that CdCl2 in FNC‐B4 neuroblasts might represent a selective cue by which, in a heterogeneous primary culture, the more differentiated mature cells die, whereas the undifferentiated cells, at the same time glial and neuronal progenitors, are forced to access a state of differentiation.

A Novel Role for a Major Component of the Vitamin D Axis: Vitamin D Binding Protein-Derived Macrophage Activating Factor Induces Human Breast Cancer Cell Apoptosis through Stimulation of Macrophages

The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH)(2)D3), its two binding proteins that are the vitamin D receptor (VDR) and the vitamin D-binding protein-derived macrophage activating factor (GcMAF). In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This al1ows 1,25(OH)(2)D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects.

C-reactive protein levels and vitamin d receptor polymorphisms as markers in predicting cachectic syndrome in cancer patients.

AbstractBackground and Objective: In patients with advanced cancer, cachexia correlates with low performance status and poor quality of life. In addition, cachexia may be associated with reduced response to chemo-radiotherapy and a poor prognosis in cancer patients. Nearly all forms of cachexia are closely associated with chronic inflammation and elevated levels of inflammatory and pro-inflammatory circulating factors, including C-reactive protein (CRP), which is considered a valid laboratory and clinical marker. Among the different pathways involved in the production of inflammatory cytokines and chemokines, the vitamin D-vitamin D receptor (VDR) axis plays a fundamental role.In this study, we explore the possible association between CRP and key factors pertaining to the vitamin D axis — in particular, VDR gene polymorphisms — in cancer patients with cachexia. Although certain tumor types are more commonly associated with cachexia, even within the same tumor type there are significant differences in the extent and duration of cachexia. Such variations may be due to polymorphisms of the VDR gene that could lead to cachexia-prone genotypes or to cachexia-resistant genotypes. Identification of such genotypes could be very helpful in the management of cancer patients. Methods: Forty-three cancer patients were recruited by the Nutritional Unit of the Prato Hospital. Data on age, gender, type of cancer, stage of cancer, and nutritional assessment, as well as transferrin, ferritin, albumin, and CRP levels, were collected. Genomic DNA was extracted from peripheral blood leukocytes and amplified by polymerase chain reaction. BsmI, ApaI, TaqI, and FokI polymorphisms of the VDR gene were investigated using the respective restriction enzymes. For the different VDR polymorphisms, the absence or presence of the restriction sites were designated with capital or small letters, respectively. For example, for the BsmI polymorphism, the presence of the undigested fragment identified the B allele, whereas the presence of the digested fragment identified the b allele. Results: Cancer patients with cachexia have higher CRP levels compared with non-cachectic cancer patients, independently from the genotype. In cachectic patients, the presence of specific VDR BsmI and TaqI alleles was associated with higher CRP levels. In particular, the VDR b and T alleles were more frequent in cachectic cancer patients with elevated CRP levels than in cachectic patients with normal CRP levels. Conclusion: From these results, we hypothesize that there is an association between BsmI and TaqI VDR gene polymorphisms and the cachectic syndrome. In particular, we propose that in cancer patients, the concomitance of b and T alleles with elevated CRP levels may represent an early clinical predictor for the development of a more aggressive form of cachexia.

Angiotensin-converting enzyme/vitamin D receptor gene polymorphisms and bioelectrical impedance analysis in predicting athletic performances of Italian young soccer players.

Micheli, ML, Gulisano, M, Morucci, G, Punzi, T, Ruggiero, M, Ceroti, M, Marella, M, Castellini, E, and Pacini, S. Angiotensin converting enzyme/vitamin D receptor gene polymorphisms and bioelectrical impedance analysis in predicting athletic performances of Italian young soccer players. J Strength Cond Res 25(8): 2084-2091, 2011—We evaluated the association between 2 genetic polymorphisms known to be involved in fitness and performance, and anthropometric features, body composition, and athletic performances in young male soccer players with the goal of identifying genetic profiles that can be used to achieve maximal results from training. One hundred twenty-five medium-high-level male soccer players were genotyped for angiotensin-converting enzyme (ACE) I/D, and vitamin D receptor (VDR) FokI gene polymorphisms and scored for anthropometric measurements, body composition, and athletic performance. Body mass index, fat mass, fat-free mass, resistance, reactance, impedance, phase angle (PA), and body cell mass were measured. Athletic performance was evaluated by squat jump, countermovement jump (CMJ), 2-kg medicine ball throw, 10- and 20-m sprint time. We observed that the homozygous ff genotype of the VDR gene was significantly more represented in young soccer players than in a matched sedentary population. Values of reactance and PA were differently distributed in ACE and VDR genotypes with high mean values in subjects with DD (ACE) and FF (VDR) genotypes. No correlation was observed between ACE or VDR genotypes and 2-kg medicine ball throw, 10- and 20-m sprint times. The ID genotype of ACE was associated with the best performances in squat jump and CMJ. Our results suggest that determination of ACE and VDR genotypes might help select those young athletes harboring the most favorable genetic potential to succeed in soccer.

GC protein-derived macrophage-activating factor decreases α-N-acetylgalactosaminidase levels in advanced cancer patients

α-N-acetylgalactosaminidase (nagalase) accumulates in the serum of cancer patients and its activity correlates with tumor burden, aggressiveness and clinical disease progression. The administration of GC protein-derived macrophage-activating factor (GcMAF) to cancer patients with elevated levels of nagalase has been associated with a decrease of serum nagalase activity and with significant clinical benefits. Here, we report the results of the administration of GcMAF to a heterogeneous cohort of patients with histologically diverse, advanced neoplasms, generally considered as “incurable” diseases. In most cases, GcMAF therapy was initiated at late stages of tumor progression. As this is an open-label, non-controlled, retrospective analysis, caution must be employed when establishing cause-effect relationships between the administration GcMAF and disease outcome. However, the response to GcMAF was generally robust and some trends emerged. All patients (n = 20) presented with elevated serum nagalase activity, well above normal values. All patients but one showed a significant decrease of serum nagalase activity upon weekly GcMAF injections. Decreased nagalase activity was associated with improved clinical conditions and no adverse side effects were reported. The observations reported here confirm and extend previous results and pave the way to further studies aimed at assessing the precise role and indications for GcMAF-based anticancer immunotherapy.

New Frontiers in Sport Training: Genetics and Artistic Gymnastics

Abstract Morucci, G, Punzi, T, Innocenti, G, Gulisano, M, Ceroti, M, and Pacini, S. New frontiers in sport training: Genetics and artistic gymnastics. J Strength Cond Res 28(2): 459–466, 2014—The increasing understanding of the genetic influences in sport has prompted an association study between the athletic performances and the polymorphisms of the angiotensin-converting enzyme (ACE), the &agr;-actinin-3 (ACTN3), and the vitamin D receptor genes. The details of these gene polymorphisms can provide useful information to improve and plan new modern training programs for elite athletes. Eighty Italian male high level gymnasts were trained and tested for gymnastic-specific exercises and tested in all the mens artistic gymnastic apparatus (floor, pommel horse, rings, vault, parallel bars, and horizontal bar), and then genotyped. The training parameters of volume, intensity, and density of each gymnast were periodically measured during the season in each apparatus from the tests performed, and the seasonal average values were calculated. Gene polymorphisms were determined by polymerase chain reaction restriction fragment length polymorphism assay and studied in association with the performance results. The performances of ACE II gymnasts were significantly lower than that of the ACE ID/DD gymnasts in the apparatus expressing power features, confirming the predisposition of these athletes toward power-oriented sport. Gymnasts with ACTN3 RR/RX genotypes did not show a predisposition to the power-oriented apparatus, having worse performances compared with that of the ACTN3 XX gymnasts. Similarly, gymnasts with ACE II + ACTN3 RR/RX combined genotypes showed lower performances in comparison with that of the other gymnasts. Vitamin D receptor polymorphisms showed no significant association with the athletic performances. Because ACE insertion/deletion (I/D) and ACTN3 R577X polymorphisms heavily affect the physical performance of elite male gymnasts, the Italian Gymnastic Federation trainers have started to customize the current high-level training programs.

Effects of Vitamin D3 and Paricalcitol on Immature Cardiomyocytes: A Novel Role for Vitamin D Analogs in the Prevention of Cardiovascular Diseases

Cardiovascular diseases are more prevalent in patients with chronic kidney disease than in the general population and they are considered the leading cause of death in patients with end-stage renal disease. The discovery that vitamin D3 plays a considerable role in cardiovascular protection has led, in recent years, to an increase in the administration of therapies based on the use of this molecule; nevertheless, several studies warned that an excess of vitamin D3 may increase the risk of hypercalcemia and vascular calcifications. In this study we evaluated the effects of vitamin D3, and of its selective analog paricalcitol, on immature cardiomyocytes. Results show that vitamin D3 induces cAMP-mediated cell proliferation and significant intracellular calcification. Paricalcitol, however, induces cell differentiation, morphological modifications in cell shape and size, and no intracellular calcification. Furthermore, vitamin D3 and paricalcitol differently affect cardiomyoblasts responses to acetylcholine treatment. In conclusion, our results demonstrate that the effects of vitamin D3 and paricalcitol on cardiomyoblasts are different and, if these in vitro observations could be extrapolated in vivo, they suggest that paricalcitol has the potential for cardiovascular protection without the risk of inducing intracellular calcification.

Effects of oxaliplatin and oleic acid Gc-protein-derived macrophage-activating factor on murine and human microglia

The biological properties and characteristics of microglia in rodents have been widely described, but little is known about these features in human microglia. Several murine microglial cell lines are used to investigate neurodegenerative and neuroinflammatory conditions; however, the extrapolation of the results to human conditions is frequently met with criticism because of the possibility of species‐specific differences. This study compares the effects of oxaliplatin and of oleic acid Gc‐protein‐derived macrophage‐activating factor (OA‐GcMAF) on two microglial cell lines, murine BV‐2 cells and human C13NJ cells. Cell viability, cAMP levels, microglial activation, and vascular endothelial growth factor (VEGF) expression were evaluated. Our data demonstrate that oxaliplatin induced a significant decrease in cell viability in BV‐2 and in C13NJ cells and that this effect was not reversed with OA‐GcMAF treatment. The signal transduction pathway involving cAMP/VEGF was activated after treatment with oxaliplatin and/or OA‐GcMAF in both cell lines. OA‐GcMAF induced a significant increase in microglia activation, as evidenced by the expression of the B7‐2 protein, in BV‐2 as well as in C13NJ cells that was not associated with a concomitant increase in cell number. Furthermore, the effects of oxaliplatin and OA‐GcMAF on coculture morphology and apoptosis were evaluated. Oxaliplatin‐induced cell damage and apoptosis were nearly completely reversed by OA‐GcMAF treatment in both BV‐2/SH‐SY5Y and C13NJ/SH‐SY5Y cocultures. Our data show that murine and human microglia share common signal transduction pathways and activation mechanisms, suggesting that the murine BV‐2 cell line may represent an excellent model for studying human microglia.

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

Oxaliplatin-based regimens are effective in metastasized advanced cancers. However, a major limitation to their widespread use is represented by neurotoxicity that leads to peripheral neuropathy. In this study we evaluated the roles of a proven immunotherapeutic agent [Gc-protein-derived macrophage activating factor (GcMAF)] in preventing or decreasing oxaliplatin-induced neuronal damage and in modulating microglia activation following oxaliplatin-induced damage. The effects of oxaliplatin and of a commercially available formula of GcMAF [oleic acid-GcMAF (OA-GcMAF)] were studied in human neurons (SH-SY5Y cells) and in human microglial cells (C13NJ). Cell density, morphology and viability, as well as production of cAMP and expression of vascular endothelial growth factor (VEGF), markers of neuron regeneration [neuromodulin or growth associated protein-43 (Gap-43)] and markers of microglia activation [ionized calcium binding adaptor molecule 1 (Iba1) and B7-2], were determined. OA-GcMAF reverted the damage inflicted by oxaliplatin on human neurons and preserved their viability. The neuroprotective effect was accompanied by increased intracellular cAMP production, as well as by increased expression of VEGF and neuromodulin. OA-GcMAF did not revert the effects of oxaliplatin on microglial cell viability. However, it increased microglial activation following oxaliplatin-induced damage, resulting in an increased expression of the markers Iba1 and B7-2 without any concomitant increase in cell number. When neurons and microglial cells were co-cultured, the presence of OA-GcMAF significantly counteracted the toxic effects of oxaliplatin. Our results demonstrate that OA-GcMAF, already used in the immunotherapy of advanced cancers, may significantly contribute to neutralizing the neurotoxicity induced by oxaliplatin, at the same time possibly concurring to an integrated anticancer effect. The association between these two powerful anticancer molecules would probably produce the dual effect of reduction of oxaliplatin-induced neurotoxicity, together with possible synergism in the overall anticancer effect.

Macrophages of the mucosa-associated lymphoid tissue (MALT) as key elements of the immune response to vitamin D binding protein-macrophage activating factor

Macrophages are key elements of the immune response and vitamin D binding protein-macrophage activating factor (DBP-MAF, also known as GcMAF) has been successfully used in treatment of immunodeficiency (J Med Virol 81:16-26, 2009). Here we report the effects of DBP-MAF on the immune system of HIV/AIDS patients as well as the effects of an original probiotic preparation, putatively containing DBP-MAF. Eight HIV/AIDS patients were treated with 100 ng/week DBP-MAF (from www.gcmaf.eu) i.v. for 15 weeks. During treatment, patients did not assume antiretroviral drugs. Blood monocyte count rose in six patients, indicating a response to DBP-MAF consistent with the effects of DBP-MAF described in Immunol Cell Biol 76:237-44, 1998. Individual response appeared to be associated with vitamin D receptor (VDR) gene polymorphisms ( BsmI and FokI ). Within the time frame of administration, however, no significant increase in CD4 cell count or decrease in viral load was observed. Therefore, searching for an alternative approach, we tested an original milk-derivative (MAF 3 14®) that contains microorganisms introduced in order to maximize natural DBP-MAF production. We hypothesized that natural DBP-MAF, once ingested, activated the Mucosa-Associated Lymphoid Tissue (MALT) widely diffused in the walls of the entire gastrointestinal tract. In fact, enzymes of certain strains of microorganisms contained in yogurt and kefir are able to convert milk Gc-protein into active DBP-MAF and it is known that kefir modulates the immune response in mice, increasing the phagocytic activity (i.e. activating) of peritoneal and pulmonary macrophages (Immunobiology 211:149-56, 2006). It is also known that probiotic yogurt consumption is associated with an increase of CD4 count among people living with HIV/AIDS (J Clin Gastroenterol 44:e201-5, 2010). Thus, members of the research team consumed 125 ml/day of MAF 3 14® for three weeks. Participants did not assume any drug or supplement and did not modify their usual diet and lifestyle. Blood analyses were performed two weeks before beginning consumption, and after three week consumption. After three week consumption, CD4 count dramatically increased in those of us who started with low CD4 count (subject # 1, before consumption CD4: 372; CD8: 206. After consumption: CD4: 609; CD8: 448), or abnormal CD4/CD8 ratio (subject # 2, before consumption: CD4: 857; CD8: 794. After consumption: CD4: 1279; CD8: 640). Also these effects appeared to be associated with VDR gene polymorphisms.