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

Gc protein-derived macrophage-activating factor decreases alpha-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

Therapeutic effects of highly purified de-glycosylated GcMAF in the immunotherapy of patients with chronic diseases.

The de-Glycosylated vitamin D binding protein is a powerful Macrophage Activating Factor (GcMAF) that shows multiple biological effects that could be exploited in the immunotherapy of tumours, viral infections and autism. Here we report the observation of a series of clinical cases describing the results obtained administering highly purified GcMAF to patients with diverse types of chronic diseases. These are heterogeneous and refer to patients with different types of diseases at different stages. In some cases, patients underwent other complementary treatments such as stem cell infusion or administration of supplements. In patients harbouring tumours, GcMAF treatment was initiated at late stages of tumour progression. Therefore, since this is an open-label, non-controlled, retrospective analysis, caution must be employed when ascribing cause and effect to any treatment outcome. However, the response to GcMAF was robust and certain trends emerge evident. In all cases (n = 7), GcMAF subcutaneous injections were associated with improvement of clinical conditions. No adverse side effects were reported. The observation reported here confirm and extend the results previously presented by several Authors and open the way to further trials aimed at assessing the precise role and indications for GcMAF in the immunotherapy of chronic diseases

Three Dimensional Electrical Impedance Tomography

The electrical resistivity of mammalian tissues varies widely and is correlated with physiological function. Electrical impedance tomography (EIT) can be used to probe such variations in vivo, and offers a non-invasive means of imaging the internal conductivity distribution of the human body. But the computational complexity of EIT has severe practical limitations, and previous work has been restricted to considering image reconstruction as an essentially two-dimensional problem. This simplification can limit significantly the imaging capabilities of EIT, as the electric currents used to determine the conductivity variations will not in general be confined to a two-dimensional plane. A few studies have attempted three-dimensional EIT image reconstruction, but have not yet succeeded in generating images of a quality suitable for clinical applications. Here we report the development of a three-dimensional EIT system with greatly improved imaging capabilities, which combines our 64-electrode data-collection apparatus with customized matrix inversion techniques. Our results demonstrate the practical potential of EIT for clinical applications, such as lung or brain imaging and diagnostic screening

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

The prediction of floods in Venice: methods, models and uncertainty (review article)

This paper reviews the state of the art in storm surge forecasting and its particular application in the northern Adriatic Sea. The city of Venice already depends on operational storm surge forecasting systems to warn the population and economy of imminent flood threats, as well as help to protect the extensive cultural heritage. This will be more important in the future, with the new mobile barriers called MOSE (MOdulo Sperimentale Elettromeccanico, Experimental Electromechanical Module) that will be completed by 2021. The barriers will depend on accurate storm surge forecasting to control their operation. In this paper, the physics behind the flooding of Venice is discussed, and the state of the art of storm surge forecasting in Europe is reviewed. The challenges for the surge forecasting systems are analyzed, especially in view of uncertainty. This includes consideration of selected historic extreme events that were particularly difficult to forecast. Four potential improvements are identified: (1) improve meteorological forecasts, (2) develop ensemble forecasting, (3) assimilation of water level measurements and (4) develop a multimodel approach

Heart Preservation during 24 Hours for Transplantation

(J. Extra-Corpor. Technol. 19[3] p. 305-311 Fall 1987, 11 ref.) To promote the development of heart transplantation by developing a complete technique of prolonged preservation of the graft in an independent, miniaturized and computerized container, a surgical technique is described, which allows continuous protection of the myocardium during removal, preservation and grafting. The use of cardioplegia allows diastole arrest, an essential point for good preservation. The preservation system consists of a refrigerated and thermostatic container. The heart is perfused by a micropump and the perfusate is oxygenated and bubblefree due to a special filter. Captors are placed in the perfusion, thermic catheter, K ++, pH, lactate, electrodes and the results are analyzed by a microcomputer and transmitted to a feedback control system which adjusts the parameters. This device can be portable. With this type of device, good results were obtained as evidenced by: • ckmb release was low • the histological lesions are rare and reversible • after transport, the heart maintains electrical and mechanical activity