Effects of vitamin D-binding protein-derived macrophage-activating factor on human breast cancer cells

BACKGROUND: Searching for additional therapeutic tools to fight breast cancer, we investigated the effects of vitamin D-binding protein-derived macrophage activating factor (DBP-MAF, also known as GcMAF) on a human breast cancer cell line (MCF-7). MATERIALS AND METHODS: The effects of DBP-MAF on proliferation, morphology, vimentin expression and angiogenesis were studied by cell proliferation assay, phase-contrast microscopy, immunohistochemistry and western blotting, and chorioallantoic membrane (CAM) assay. RESULTS: DBP-MAF inhibited human breast cancer cell proliferation and cancer cell-stimulated angiogenesis. MCF-7 cells treated with DBP-MAF predominantly grew in monolayer and appeared to be well adherent to each other and to the well surface. Exposure to DBP-MAF significantly reduced vimentin expression, indicating a reversal of the epithelial/mesenchymal transition, a hallmark of human breast cancer progression. CONCLUSION: These results are consistent with the hypothesis that the known anticancer efficacy of DBP-MAF can be ascribed to different biological properties of the molecule that include inhibition of tumour-induced angiogenesis and direct inhibition of cancer cell proliferation, migration and metastatic potential

Effects of Cadmium and vitamin D binding protein-derived macrophage activating factor (DBP-MAF) in human breast cancer cells

We previously demonstrated that chronic exposure of the human breast cancer cell line MCF-7 to non-cytotoxic concentrations of Cadmium reduced viability and angiogenic potential of this cell line. In order to better understand these effects, cells, after Cadmium exposure, were treated with vitamin D binding protein-derived macrophage activating factor (DBP-MAF). DBP-MAF is a potent macrophage-activating factor derived from vitamin D binding protein, a polymorphic serum glycoprotein with multiple functions also known as a group specific component or Gc protein. Besides stimulating macrophages, DBP-MAF has anti-tumour properties. Our data demonstrate that the decrease of MCF-7 cell viability following Cadmium treatment was completely reversed when DBP-MAF was present in the cell medium. Following this observation, we further investigated the role of DBP-MAF in modulating angiogenesis, morphology and cytoskeleton structure of MCF-7 cell line. As shown by chorioallantoic membrane assay, DBP-MAF inhibited MCF-7 cancer cell-stimulated angiogenesis. Concerning cell morphology (studied by contrast phase light microscopy and after Papanicolaou staining), following DBP-MAF treatment, cell shape and growth pattern were significantly modified. Vimentin expression (studied by immunohistochemistry and Western blot analysis), considered a hallmark of human breast cancer progression, after DBP-MAF treatment, significantly varied. Intermediate filament status changes, consisting in a shift from a keratin-rich to a vimentin-rich network (epithelial-mesenchymal transition), were observed. In conclusion, we demonstrate that the anti-cancer effects of DBP-MAF can be attributed to multiple actions independent of macrophage stimulation such as reversal of Cadmium effects on cell viability, reversal of morphological malignant phenotype and inhibition of cancer cell-stimulated angiogenesis. For these reasons, DBP-MAF might represent an useful tool to control progression and differentiation of human breast cancer

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

Role of angiotensin-converting enzyme and vitamin D receptor gene polymorphisms in cancer anorexia-cachexia syndrome

The ubiquitin-proteasome pathway is a crucial connection between aberrant immune system activation, systemic inflammation and Cancer Anorexia-Cachexia Syndrome (CACS), a syndrome that culminates in hyper-activation of the ubiquitin-proteasome pathway. Angiotensin directly up-regulates this pathway, while vitamin D down-regulates it indirectly through the insulin-like growth factor-1 pathway. We investigated the genetic predisposition towards CACS in a cancer population, examining Insertion/Deletion (I/D) polymorphism of angiotensin-converting enzyme gene and FokI and BsmI polymorphisms of vitamin D receptor gene. Sixty-two cancer patients were recruited and divided into three groups: primary cachectic (C1, n = 14; dysmetabolic body weight loss ≥5% in 6 months); secondary cachectic (C2, n = 34; similar weight loss, mechanic or iatrogenic origin); and non-cachectic (NC, n = 16). C2+NC were merged in the control group. The three groups showed significant differences in average prognostic inflammatory nutritional index (C1: 26.4±23.4; C2: 5.4±5.6; NC: 0.37±0.5), C-reactive protein serum levels (C1: 6.6±2.1; C2: 2.4±2.2; NC: 1.0±2.0 mg/dL), albumin serum levels (C1: 3.1±0.6; C2: 3.5±0.4; NC 3.7±0.6 g/dL), weight loss (C1: 22±8; C2: 15±6.7; NC 5±6%) and life expectancy (C1: 6.4±3.3; C2: 25±28; NC: 45±25 months). However, none of the chosen polymorphisms showed any statistically significant correlation with CACS. The complexity of the changes of the immune system in the chronic inflammation state associated with CACS is far greater than expected and further studies are required to identify genetic independent markers of progression toward CACS.

Protective effects of selenium on cadmium neurotoxicity

Prolonged exposure to Cadmium may cause serious toxic effects due to its accu- mulation on both central and peripheral nervous systems. Cadmium could be uptak- en from the nasal mucosa via the olfactory pathways and gain direct access into the nervous system circumventing the blood-brain barrier. However, mechanisms under- lying the cadmium uptake and neurotoxicity remain not completely understood. Oxidative damage, interference with calcium-, copper- and zinc-dependent processes, dysregulation of cell repair mechanisms, estrogen-like effects, and epigenetic modifi- cations may be considered mechanisms for cadmium-induced neurotoxicity [1]. Our previous data demonstrated that zinc chloride counteracts the toxic effects of cadmium chloride (CdCl2) on human neurons in vitro [2]. Thus, in this study we evaluate the efficacy of Sodium Selenite (Na2SeO3) in preventing and/or counteracting the damages induced by exposure to cadmium chloride on a human neuronal cell line. In this study we treated SH-SY5Y human neurons with different sub-toxic concentrations of CdCl2 for 24 h with and without a 24 h pre-treatment with Na2SeO3. Cell viability, morphological modifications, and protein expression of specific neuronal plasticity and apoptosis (Gap43 and caspase 3) markers were evaluated. Our results suggest that toxic effects of CdCl2 can be prevented and reverted by Na2SeO3 suggesting a role for selenium compounds in protecting neuronal cells and rebuilding the complex network connections

The effect of Gcmaf complexed with oleic acid on multiple myeloma cultures

Abstract: Deglycosylated vitamin D-binding protein-derived macrophage-activating factor (GcMAF) is known to be a strong immune stimulatory natural molecule. Data in literature demonstrate that GcMAF has a direct role in decreasing cell proliferation of different cancer cell lines. In this study we evaluate the direct effect of GcMAF complexed with oleic acid (OA-GcMAF) on human multiple myeloma cells (KMS-12- BM), as well as the effect on the same cell line of human macrophages (CRL9853) previously activated by OA-GcMAF. Cell viability and living cell number were evaluated respectively by tetrazolium dye cell viability assay and by Trypan blue staining. Interactions between activated macrophages and myeloma cells were studied by time lapse photography. Our results show that OA-GcMAF decreases the cell viability of KMS-12-BM with a dose-dependent pathway. Furthermore OA-GcMAF activates human macrophages, which in turn phagocytise myeloma cancer cells. OA-GcMAF confirms its double effect on cancer cells: a direct inhibition of their viability and, at the same time, an efficient macrophage activation leading to a significant depletion of cancer cell population. Introduction: In recent years the interest of vitamin D-binding protein-derived macrophage-activating factor (GcMAF) as a potent immunotherapeutic agent has increased. The GcMAF has been shown to be effective in stimulating murine macrophages in vitro to phagocytose human breast carcinoma cultures (1, 2), as well as inhibiting the growth of prostate cancer cells (3). It has also been the agent that has been referenced as reducing tumour burden in several clinical approaches (4, 5). In previous studies GcMAF has been used to stimulate Raw 264.7 cells (murine macrophage cell line) that were observed in vitro to phagacytose MCF-7 cells (human breast carcinoma). In this study we demonstrate the effect of GcMAF stabilized with oleic acid (OA-GcMAF) directly on KMS-12-BM multiple myeloma cells and on co-culture of stimulated human macrophages (CRL9853) and KMS-12-BM. Materials and Methods: Cell lines: KMS-12-BM: human multiple myeloma cell line was purchased from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and cultured in RPMI 1640 supplemented with 10% FBS and 2mM L-Glutamine (Life Technologies, Paisley, UK). Cultures were passaged every 3-4 days. CRL9853: human spleen macrophage was purchased from ATCC (American Type Culture Collection, Teddington, UK) and routinely cultured in IMDM supplemented with 10% FBS and 2mM L-Glutamine (Life Technologies). Cultures were passaged every 3-4 days. Prior to assay, CRL9853 cells were activated culturing them for 72h in the presence of OAGcMAF at a concentration 100ng/ml in complete medium. Stimuli: OA-GcMAF, commercially available, was prepared at Immuno Biotech Ltd. (Guernsey, Channel Island) with a proprietary procedure previously described (6). Cell viability assay: Cell viability was evaluated by the reduction of a tetrazolium salt (WST-8) as an index of cell dehydrogenases’ activity. KMS-12-BM cells were seeded into a 96-well plate at a density of 3x104 cells/well in their appropriate starvation medium (without FBS). After incubation for 24h the cell line was treated for 24h with the following different concentrations of OA-GcMAF ([8-80-800 pM). At the end of the treatment, the medium was replaced with 100μl of fresh starvation medium plus 10μl of WST-8. The 96-well plate was incubated for 3h at 37°C and the optical density (O.D.) was directly measured at A450nm by Multiscan FC photometer (ThermoScientific, Milano, Italy). Cell counting – Trypan blue assay: To corroborate the results obtained by cell viability assay, a viable cell count was performed. Briefly, KMS-12-BM cells were plated into a 6-well plate at a density of 2x105 cells/well in starvation medium. After 24h incubation, human multiple myeloma cells were treated with OA-GcMAF at the increasing concentrations (8-80-800 pM) for 24h. At the end of the treatment, a volume of cell suspension was collected and the viable cell number was counted by Trypan Blue staining. Video-time lapse photography: KMS-12-BM cells were seeded into a 24-well plate at a density of 1x106 cells/well with a 1ml volume. The cells were allowed to settle for a minimum of 2h prior to the addition of the OA-GcMAF-activated CRL9853 macrophages. The staging mat was set at a temperature of 37°C and allowed to equilibrate prior to placement of the 24-well plate. The OA-GcMAF-activated CRL9853 macrophages were added to a final concentration of 5x105 cells per well in 1ml. HEPES (Fisher Scientific, Loughborough, UK) was added to each well to provide a final concentration of 25mM to stabilize the culture pH. Once activated macrophages and the HEPES were added, the 24-well plate was observed microscopically and an image selected. An initial frame was taken and a timelapse film initiated. A frame was taken every 3 minutes until filming was stopped. Results: Cell viability assay: Cell viability (Figure 1), evaluated both by tetrazolium dye cell assay (A) and by Trypan blue staining (B), decreased when KMS-12-BM cells were treated with increasing concentrations of OAGcMAF. In particular, when cells were treated with OAGcMAF (800 pM) a significant reduction (p<0.01) in cell viability was observed in comparison to the untreated control cells. Discussion: It has been shown that Gc-MAF activates Raw 264.7 murine macrophages to phagocytose and destroy MCF-7 human breast carcinoma cells (1). An identical effect has been recorded for the first time with the stimulation of human macrophages CRL9853 on KMS-12-BM cell line . The OA-GcMAF-stimulated human macrophages seek out surround and phagocytose the myeloma cell lines destroying them. This demonstrates that the CRL9853 behave as postulated against KMS-12- BM cell line. In addition the effect of increased cell death in the presence of OA-GcMAF alone as indicated by direct evaluation of viable cell counts and viability assay also provides supports to the previous data generated (1). This study provides some inferred evidence to support the in vivo clinical data that has recently been published (6) providing some insight into the method of potential tumour removal by stimulated macrophages. In conclusion OA-GcMAF has demonstrated two major effects: a direct decrease of KMS-12- BM cell viability and an efficient activation of human macrophages, which become able to phagocytose and destroy the myeloma cells. The studies will be expanded further to encompass additional cancer cell lines

Transcranial sonography in the diagnosis, follow-up and treatment of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

We used a modified transcranial sonography technique to study the cortex of the temporal lobe, a brain region involved in the processing of functions that are often compromised in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) patients. We studied the meninges, the subarachnoidal space and the cortex. The spatial resolution and the ability to visualize structures of 200-300 µm size, led us to hypothesize that the linear structures parallel to the subarachnoidal space could be referred to the neuronal layers of the cortex. In real-time mode, we could observe pulsation of the meninges and the cortex synchronous with the heart beat and independent of blood flow. This pulsation was more evident at the level of the meninges, but it was also appreciable at the level of the layers of the cortex and it was not accompanied by any type of flow. In addition to these findings, we observed that the subject undergoing the procedure experienced a series of changes that might prove potentially useful in the treatment of ME/CFS. In particular, we observed a decrease of tachycardia accompanied by an increase in systolic blood pressure and by a significant increase in muscle strength measured by the degree of muscle fibre shortening at the level of the biceps brachii. These findings, together with the low cost and simplicity of the procedure, suggest that modified transcranial sonography has a significant potential in the study and treatment of ME/CFS

Aortic arch branching pattern variation: its incidence on a 20.030 cases review.

Aim of the study: the main objective of this work was to study the frequency of variation of the aortic arch branching pattern in a wide and varied population. Introduction: Variations in the branching pattern of the aortic arch are clinically relevant because of the direct influence that their presence can have on the success of cardio-vascular procedures, neck or thorax surgery, in trauma management or in intensive care unit patient’s. In most cases these anatomical variations are asymptomatic and considered clinically benign, but some particular aortic branching patterns had been associated with surgical complications or with vascular diseases in medical non surgical patients. Methods: this paper analyzes the aortic arch branching patterns of 20.030 cases reported by 40 anatomical or radiological studies. Results: 84,52% of the studied population has a three branches pattern and 14,65% has a two branches pattern. The four primary arteries arising directly from the aortic arch were seen in 0,81% of the cases and only 0,02% had them all arising from a common trunk