Enrico D'Emilia’s research while affiliated with Istituto Nazionale per l'Assicurazione Contro gli Infortuni sul Lavoro and other places

Cell therapy is an innovative strategy for tissue repair, since adult stem cells could have limited regenerative ability as in the case of myocardial damage. This leads to a local contractile dysfunction due to scar formation. For these reasons, refining strategy approaches for “in vitro” stem cell commitment, preparatory to the “in vivo” stem cell differentiation, is imperative. In this work, we isolated and characterized at molecular and cellular level, human Amniotic Mesenchymal Stromal Cells (hAMSCs) and exposed them to a physical Extremely Low Frequency Electromagnetic Field (ELF-EMF) stimulus and to a chemical Nitric Oxide treatment. Physically exposed cells showed a decrease of cell proliferation and no change in metabolic activity, cell vitality and apoptotic rate. An increase in the mRNA expression of cardiac and angiogenic differentiation markers, confirmed at the translational level, was also highlighted in exposed cells. Our data, for the first time, provide evidence that physical ELF-EMF stimulus (7 Hz, 2.5 �T), similarly to the chemical treatment, is able to trigger hAMSC cardiac commitment. More importantly, we also observed that only the physical stimulus is able to induce both types of commitments contemporarily (cardiac and angiogenic), suggesting its potential use to obtain a better regenerative response in cell-therapy protocols.

Heretofore only observed in living systems, we report that weak-field ion cyclotron resonance (ICR) also occurs in inanimate matter. Weak magnetic field (50 nT) hydronium ICR at the field combination (7.84 Hz, 7.5 µT) markedly changes water structure, as evidenced by finding an altered index of refraction exactly at this combined field. This observation utilizes a novel technique which measures the scattering of a He-Ne laser beam as the sample is exposed to a ramped magnetic field frequency. In addition to the hydronium resonance, we find evidence of ICR coupling to a more massive structure, possibly a tetrahedral combination of three waters and a single hydronium ion. To check our observations, we extended this technique to D2O, successfully predicting the specific ICR charge-to-mass ratio for D3O(+) that alters the index of refraction.

We have performed a series of experiments applying high voltage between two electrodes, immersed in two beakers containing bidistilled water in a way similar to experiments conducted by Fuchs and collaborators, which showed that a water bridge can be formed between the two containers. We also observed the formation of water bridge. Moreover, choosing different pairs of electrodes depending on the material they are made up of, we observed that copper ions flow can pass along the bridge if the negative electrode is made up of copper. We show that the direction of the flux not only depends on the applied electrostatic field but on the relative electronegativity of the electrodes too. These results open new perspectives in understanding the properties of water. We suggest a possible explanation of the obtained results.

Several years ago just before Christmas, in a small meeting room at the Institute of Pharmacology at the University of Rome, we had the opportunity to attend a meeting on "The role of QED in medicine" by Emilio Del Giudice and Giuliano Preparata. Before that meeting, we were more oriented towards a mechanistic view of Biochemistry and Medicine, believing that chemical reactions could only take place when a random collision between molecules with a gain in energy takes place. We envisioned water as just a solvent in which was possible to dissolve a solute. After we listened to Giuliano's and Emilio's speech on the "New physics of water", and on "The possible origin of coherence in cell, tissues and the interaction of very weak and low frequency magnetic fields with the ions, systems of the cell", we realized that living organisms are complex electrochemical systems which evolved in a relatively narrow range of well-defined environmental parameters. Environmental natural electro-magnetic fields are an ubiquitous factor in nature. If nature gave certain organisms the ability to receive information about the environment via invisible electromagnetic signals, then there must also the capability to discriminate between significant and meaningless ones. Bearing in mind that electromagnetic fields can be perceived by living organisms by means a resonance effect, we should not be amazed if they can be able to induce different biological effects. The work that we will present in memory of Emilio is based on the hypotheses that an aqueous system a chemical differentiation agent such as retinoic acid (RA) were electronically captured and transferred to the culture medium of Neuroblastoma Cell Line (LAN-5) and the proliferation rate was assessed to assess cell responses to the electromagnetic information transfer through the aqueous system. Like those enfolded in living organisms could play a synergic role in modulating biological functions, generating dissipative structures under appropriate patterns of electromagnetic signals providing basis for storing and retrieving biological activities. An external electro-magnetic stimulus from a source molecule can be stored, translated, and transferred by the aqueous systems to the biological target, selectively driving their endogenous activity and mimicking the effect of a source molecule.

SUMMARY Aims: To evaluate the in vitro effects of extremely low-frequency magnetic field (ELF-MF) on growth and biofilm formation by Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia strains from cystic fibrosis patients. The motion of selected ions (Fe, Ca, Cu, Zn, Mg, K, Na) was stimulated by the ion resonance effect, then influence on growth and biofilm formation/viability was assessed by spectrophotometry or viability count. Generally, exposure to ELF-MF significantly increased bacterial growth and affected both biofilm formation and viability, although with differences with regard to ions and species considered. Exposure to ELF-MF represents a possible new approach for treatment of biofilm-associated cystic fibrosis lung infections.

"In vivo" control of osteoblast differentiation is an important process needed to maintain the continuous supply of mature osteoblast cells for growth, repair and remodelling of bones. The regulation of this process has also an important and significant impact on clinical strategies and future applications of cell therapy. In this paper we studied the effect of non pulsed sinusoidal Electro Magnetic Field (EMF) radiation tuned at calcium-ion cyclotron frequency of 50 Hz exposure treatment for bone differentiation of human mesenchymal stem cells (hMSC) alone or in synergy with dexamethasone, their canonical chemical differentiation agent. Five days of continuous exposure to calcium-ion cyclotron resonance (Ca2+-ICR) affect hMSC proliferation, morphology and cytoskeletal actin reorganization. By quantitative Real-Time PCR, we also observed an increase of osteoblast differentiation markers' expression such as Runx2, Alkaline Phosphatase, (ALP), Osteocalcin (OC), and Osteopontin (OPN) together with the Osteoprotegerin (OPG) mRNA modulation. Moreover, in these cells, the increase of the protein expression of Osteopontin and Alkaline Phosphatase was also demonstrated. These results demonstrate bone commitment of hMSCs through a non invasive and biocompatible differentiating physical agent treatment and highlights its possible applications in new regenerative medicine protocols.

Abstract There is an ongoing question regarding the structure forming capabilities of water at ambient temperatures. To probe for different structures, we studied effects in pure water following magnetic field exposures corresponding to the ion cyclotron resonance of H3O(+). Included were measurements of conductivity and pH. We find that under ion cyclotron resonance (ICR) stimulation, water undergoes a transition to a form that is hydroxonium-like, with the subsequent emission of a transient 48.5 Hz magnetic signal, in the absence of any other measurable field. Our results indicate that hydronium resonance stimulation alters the structure of water, enhancing the concentration of EZ-water. These results are not only consistent with Del Giudice's model of electromagnetically coherent domains, but they can also be interpreted to show that these domains exist in quantized spin states.

In regenerative medicine finding a new method for cell differentiation without pharmacological treatment or gene modification and minimal cell manipulation is a challenging goal. In this work we reported a neuronal induced differentiation and consequent reduction of tumorigenicity in NT2 human pluripotent embryonal carcinoma cells exposed to an extremely low frequency electromagnetic field (ELF-EMF), matching the cyclotron frequency corresponding to the charge/mass ratio of calcium ion (Ca(2+)-ICR). These cells, capable of differentiating into post-mitotic neurons following treatment with Retinoic Acid (RA), were placed in a solenoid and exposed for 5 weeks to Ca(2+)-ICR. The solenoid was installed in a μ-metal shielded room to avoid the effect of the geomagnetic field and obtained totally controlled and reproducible conditions. Contrast microscopy analysis reveled, in the NT2 exposed cells, an important change in shape and morphology with the outgrowth of neuritic-like structures together with a lower proliferation rate and metabolic activity alike those found in the RA treated cells. A significant up-regulation of early and late neuronal differentiation markers and a significant down-regulation of the transforming growth factor-α (TGF-α) and the fibroblast growth factor-4 (FGF-4) were also observed in the exposed cells. The decreased protein expression of the transforming gene Cripto-1 and the reduced capability of the exposed NT2 cells to form colonies in soft agar supported these last results. In conclusion, our findings demonstrate that the Ca(2+)-ICR frequency is able to induce differentiation and reduction of tumorigenicity in NT2 exposed cells suggesting a new potential therapeutic use in regenerative medicine.

Controlling cell differentiation and proliferation with minimal manipulation is one of the most important goals for cell therapy in clinical applications. In this work, we evaluated the hypothesis that the exposure of myoblast cells (C2C12) to nonionizing radiation (tuned at an extremely low-frequency electromagnetic field at calcium-ion cyclotron frequency of 13.75 Hz) may drive their differentiation toward a myogenic phenotype. C2C12 cells exposed to calcium-ion cyclotron resonance (Ca(2+)-ICR) showed a decrease in cellular growth and an increase in the G(0)/G(1) phase. Severe modifications in the shape and morphology and a change in the actin distribution were revealed by the phalloidin fluorescence analysis. A significant upregulation at transcriptional and translational levels of muscle differentiation markers such as myogenin (MYOG), muscle creatine kinase (MCK), and alpha skeletal muscle actin (ASMA) was observed in exposed C2C12 cells. Moreover, the pretreatment with nifedipine (an L-type voltage-gated Ca(2+) channel blocker) led to a reduction of the Ca(2+)-ICR effect. Consequently, it induced a downregulation of the MYOG, MCK, and ASMA mRNA expression affecting adversely the differentiation process. Therefore, our data suggest that Ca(2+)-ICR exposure can upregulate C2C12 differentiation. Although further studies are needed, these results may have important implications in myodegenerative pathology therapies.

Background: Recently the authors reported the experimental evidence of the developing concept of Electro Magnetic Information Transfer (EMIT) of specific molecular signals directly and continuously on target cell picking up the molecular signals from the source chemical effector. This was in agreement with the pioneering work of Jaques Benveniste suggesting that the electronic transmission of the 4-phorbol-12-myristate-13-acetate (PMA) signals could be transferred to target neutrophils by an oscillator when coupled to two electromagnetic coils demonstrating the same biologic activity and so mimicking the biologic function of the original chemical active molecule. The present work is the further development of recent research designed to verify the hypotheses that water could record and replay the EMIT from biologic active chemical molecules. Methods: Retinoic acid, a well-known chemical differentiating agent, was placed at room temperature in the input coil connected to an oscillator (VEGA select 719), while culture medium for human neuroblastoma cell (LAN-5) and NT2/D1 stem teratocarcinoma human cells was placed into the output coil and exposed to signals for 1 hour. At the end the oscillator was switched off and LAN-5 neuroblastoma and NT2/D1 stem teratocarcinoma cells were seeded, respectively, into the medium conditioned as reported into an incubator under controlled conditions. After 5 days of incubations, cells were examined by different strategies such as morphological and biochemical parameters. Results: It was demonstrated that the electromagnetic signals coming from the retinoic acid molecule could be recorded and stored by the aqueous system of the cell culture medium. Cells seeded in the electronically conditioned medium received physical information generating a statistically significant decrease in metabolic activity and changes in phenotypical structure with protrusion typical of differentiated neuronal cells. Conclusions: These experimental results provide some evidence that water could be tuned in a resonant manner by the EMIT procedure appropriately carried through a carrier frequency provided by the oscillator in a manner that seems related to the chemical structure of the source molecule as, in this case, retinoic acid.