Growth stage dependent effects of electromagnetic fields on DNA synthesis of Jurkat cells.

A 1.8 mT, bone healing, electromagnetic field (EMF) and power frequency EMFs of 0.1 and 0.4 mT significantly inhibit DNA synthesis in otherwise unstimulated Jurkat (E 6.1) cells. Inhibition is generally most prominent in cells from mid log phase growth. In complete medium the bone healing EMF inhibits [3H] thymidine uptake of the latter cells by almost 50% vs. 20-25% inhibition by 60 Hz fields. Cells in conditioned medium are even more sensitive to EMFs with inhibition up to ca. 60%. The effects of the 0.1 and 0.4 mT power frequency EMFs were very similar suggesting saturation at 0.1 mT or lower.

Comparative methodological investigations on the cytochemical localization of calcium in brain and inner ear of cichlid fish.

Four different methods for calcium precipitation are compared in the optic tectum and the inner ear of the cichid fish, Oreochromis mossambicus. Several parameters are investigated concerning their influences on the reaction product. Three procedures (bichromate, fluoride, and oxalate-pyroantimonate) produce fine-grained deposits, often flocculent in the latter method. The fourth method (potassium-pyroantimonate) generates predominantly coarse-grained reaction product. The calcium content of the deposits is always proven with energy-filtering transmission electron microscopy (EFTEM). In both tissues fine-grained reaction product is found in endoplasmic reticulum and synaptic vesicles, and in addition in some mitochondria and at the cytoskeleton. The coarse-grained deposits of the potassium-pyroantimonate method have a more unspecific distribution. This is the only method which produces extracellular deposits in the inner ear, whereas in the optic tectum extracellular precipitates are always present except with the oxalate-pyroantimonate procedure. Two factors have an influence on the reaction product: the duration of fixation and the type of resin. The prolongation of the fixation time up to 24 hours leads to an increase of the reaction product, which also becomes coarse-grained. These observations are corroborated by quantification with image analysis. Furthermore the use of an epoxy resin compared to acrylic resins decreases the amount of reaction product produced. We show that the application of several methods is meaningful in order to understand the calcium properties of the investigated tissue, but it is necessary to optimize a certain method for a given tissue.

Influence of altered gravity on the cytochemical localization of cytochrome oxidase activity in central and peripheral gravisensory systems in developing cichlid fish.

Cichlid fish larvae were reared from hatching to active free swimming under different gravity conditions: natural environment, increased acceleration in a centrifuge, simulated weightlessness in a clinostat and near weightlessness during space flight. Cytochrome oxidase activity was analyzed semiquantitatively on the ultrastructural level as a marker of regional neuronal activity in a primary, vestibular brainstem nucleus and in gravity receptive epithelia in the inner ear. Our results show, that gravity seems to be positively correlated with cytochrome oxidase activity in the magnocellular nucleus of developing fish brain. In the inner ear the energy metabolism is decreased under microgravity concerning utricle but not saccule. Hypergravity has no effect on cytochrome oxidase activity in sensory inner ear epithelia.

Ultrastructural aspects of otoliths and sensory epithelia of fish inner ear exposed to hypergravity.

The present electron microscopical investigations were directed to the question, whether alterations in the gravitational force might induce structural changes in the morphology of otoliths or/and inner ear sensory epithelia of developing and adult swordtail fish (Xiphophorus helleri) that had been kept either under long-term moderate hypergravity (8 days; 3g) or under short-time extreme hypergravity (10 minutes up to 9g). The otoliths of adult and neonate swordtail fish were investigated by means of scanning electron microscopy (SEM). Macular epithelia of adult fish were examined both by SEM and transmission electron microscopy (TEM). The saccular otoliths (sagittae) of normally hatched adult fish revealed an enormous inter- (and even intra-; i.e. left vs. right) individual diversity in shape and size, whereas the otoliths of utricles (lapilli) and lagenae (asterisci) seemed to be more constant regarding morphological parameters. The structural diversity of juvenile otoliths was found to be less prominent as compared to the adults, differing from the latter regarding their peculiar crystalline morphology. Qualitative differences in the fine structure (SEM) of otoliths taken from adult and larval animals kept under 3g in comparison to 1g controls could not be observed. The SEM and TEM investigations of sensory epithelia also did not reveal any effects due to 3g stimulation. Even extreme hypergravity (more than 7g) for 10 minutes did not result in distinct pathological changes.

Comparative electronmicroscopical investigations on the influences of altered gravity on cytochrome oxidase in the inner ear of fish: a spaceflight study.

The regional metabolic activity in the otolithic sensory epithelia of the inner ear of a cichlid fish (Oreochromis mossambicus) was investigated on light- and electronmicroscopical level using the cytochemical method for detection of cytochrome oxidase activity. In adult animals a characteristic distribution of mitochondria with high enzyme activity was found in sensory and non-sensory cells of otolithic sensory epithelia, which was correlated with regions with a high energy demand. These findings were the basis for studies on the influence of long-term altered gravity conditions in developing larvae: hypogravity (10(-4) g in spaceflight), normal gravity (1 g in a centrifuge in space and 1 g on earth) and hypergravity (3 g in a laboratory centrifuge). Cytochrome oxidase activity was quantified in different parts of the sensory hair cell synapse in the vestibular sensory epithelia utricle and saccule: apical and basal cytoplasm, postsynaptic area of the afferent synapse and presynaptic region of the efferent synapse. Our results show that the energy metabolism of utricle, but not of saccule is decreased after microgravity exposure during the 2nd German Spacelab Mission D-2. However, a general effect of the spaceflight is detectable in both sensory epithelia. Long-term exposure to increased acceleration (3 g) had no effects on cytochrome oxidase activity in inner ear sensory epithelia.

Models of the uniformity of electro-magnetic fields generated for biological experiments by Merritt coils.

Electromagnetic field (EMF) producing wire coils were described by Merritt et al, Rev. Sci. Instrum. 54 (7), 1983. Merritt coils produce large volume EMFs in which statistical numbers of biological experiments are performed. We build and use Merritt coils for cell/animal studies and are developing therapeutic EMF systems. Here we present models illustrating the EMFs produced by our coils and discuss the criteria that should be applied to the use of Merritt and other coils to achieve valid experimental results. In a companion paper at this meeting Nindl et al, describe biological experiments, using these Merritt coils, showing that EMFs may be useful in treating many inflammatory disease states. Although the large-volume EMFs produced by Merritt coils are convenient for biological experiments the EMFs are not perfectly uniform and the deviations can be a significant source of experimental error. The orientation and size of experimental objects are key contributors to these deviations. To evaluate our Merritt coils we solved the Biot-Savart law explicitly for ideal 3-coil and 4-coil Merritt systems and compared these theoretical EMFs with those of our systems. We present a detailed examination of deviations in magnetic field amplitude, as well as magnetic field direction, as a function of location within the coils. We find that spherically shaped experimental sets minimize these deviations. We developed simple formulae for accurately predicting deviations associated with Merritt coils.

Electromagnetic field effects: changes in protein phosphorylation in the Jurkat E6.1 cell line.

This study is aimed at expanding the role of electromagnetic field (EMF) therapy for treatment of inflammatory diseases and obtaining new information on the biophysical mechanism of action of weak EMFs. The mechanism of action of EMFs on biological systems is a question that has yet to be answered. Several models have been proposed to explain the coupling of low frequency fields to biological systems, although no consensus has been reached as to which most adequately portrays the true mechanism. Protein phosphorylation is a major cellular metabolic regulator. As such, it has the potential to be a valuable indicator of the impact of EMFs on cellular metabolism. Using a well-controlled EMF exposure system, we examined the regulatory role of EMFs on low molecular weight protein phosphorylation in Jurkat E6.1 cells, a transformed human leukemic T cell line. Jurkat cells were grown to mid-log phase, preloaded with 32P and exposed to EMF (0.1 mT, 60 Hz) or sham for 30 minutes. Cell proteins were separated by SDS-polyacrylamide gel electrophoresis and incorporated radioactivity of low molecular weight proteins (18-23 kDa) was quantified by AMBIS data analysis. Three of five experiments showed no difference in protein phosphorylation in EMF exposed samples compared to controls, while two experiments revealed an EMF effect. We identified stathmin, an important T cell signaling phosphoprotein, as one of the low molecular weight proteins present in our Jurkat cell system. Stathmin expression as well as its phosphorylation was decreased in samples that were exposed to EMFs compared to controls. These data indicate that phosphorylation of individual proteins might be masked by the presence of numerous other proteins in whole cell lysate experiments. Further studies testing other low molecular weight T cell signaling molecules may validate this hypothesis.

Effect of a wound healing electromagnetic field on inflammatory cytokine gene expression in rats.

In earlier studies, we have shown that pulsed electromagnetic fields (PEMFs) induce programmed cell death in cultured T cells and that rats exposed in vivo to PEMFs have decreased T-cell proliferative capacity. These data led us to hypothesize that PEMFs might be used to control proliferation of inflammatory lymphocytes and therefore beneficially affect inflammatory diseases. Tendinitis is characterized by painful inflammation of the tendon. Inflammation is characterized by massive infiltration of T lymphocytes, neutrophils and macrophages into the damaged tissue. These inflammatory cells produce a variety of cytokines, which are the cellular regulators of inflammation. The current study tests whether in vivo PEMF effects are mediated via systemic cytokine production in rat tendinitis. Inflammation was chemically induced in female Harlan Sprague Dawley rats Achilles' tendons and a wound healing PEMF (Electrobiology, Inc.) was applied for 4 hours immediately following injury. Spleens from control and experimental animals were harvested 24 hours later and total RNA was extracted from the tissues. Gene expression was analyzed by reverse transcription of mRNA, and polymerase chain reaction amplification (RT-PCR) using primers specific for the cytokines IFN-gamma, IL-1 beta, IL-6, TNF-alpha, and TGF-beta, as well as for the control beta-actin. RT-PCR products were separated on 1.5% agarose gels and band intensities were normalized to beta-actin gene expression of the same sample. TGF-beta was the only cytokine produced at high levels in rats with tendinitis in comparison to the other cytokines. PEMFs did not show an effect on any cytokine expression in the spleens, 24 hours after induction of tendinitis. Further studies need to test if cumulative exposures of PEMFs are able to regulate inflammatory cytokine expression either at the site of inflammation or at the local lymph nodes.

A system for simultaneous ultraviolet light and electromagnetic field exposure in in vitro experiments.

Ultraviolet light (UV) is a common treatment for skin diseases such as psoriasis, but bears the risk of carcinogenic side effects. We have biological evidence that electromagnetic fields (EMFs) can act additively with UV so that new therapeutic protocols combining UV and EMF might be developed to improve psoriasis phototherapy. In this study we report on a system that allows in vitro experiments testing this hypothesis. For simultaneous exposure of cell cultures to UVB and EMF, we built Merritt coils with an integrated UV exposure system. The coils can be operated in a sham or experimental mode (up to 1.5 mT and 20,000 Hz). Two UV bulbs were fitted inside the coils for UVB doses between 100-1000 J/m2/nm. In the exposure area the EMF is uniform within 0.0038%. For exposure, the cells are cultured in standard culture plates and placed in a specifically designed box. The box holds two plates in a top chamber covered with a Saran Wrap lid (91% UV transmission) so that cells are exposed to UVB and EMFs. The bottom chamber holds two plates, where cells are screened from UVB and only exposed to EMFs. Temperature control is maintained (+/- 1 degree C) by airflow vents on the side of the box and a fan placed 25 cm away from the cell culture box. To maintain sterility within the box the vents are covered with a bacterial filter. The box lid has additional ventilation through two air direction changes to create an additional bacterial barrier similar to that in culture plate lids.

Effect of ultraviolet B radiation and 100 Hz electromagnetic fields on proliferation and DNA synthesis of Jurkat cells.

The use of ultraviolet B light (UVB) has been proven to be highly effective for treatment of various inflammatory skin diseases, but UVB phototherapy is limited by its carcinogenic side effects. It is necessary to uncover effectors that augment UVB so that similar or improved efficacy can be obtained with lower UVB doses. We found that low frequency, low intensity electromagnetic fields (EMFs) can act as such an effector and synergistically inhibit T lymphocyte proliferation. We first characterized the effects of UVB on Jurkat cells, a model for cutaneous T lymphocytes, and determined UVB's dose dependent inhibition of cell proliferation and induction of apoptosis. Cells exposed to a sublethal UVB dose retained their sensitivity to UVB, but repetitive irradiation seemed to cause accumulation of delayed DNA damage. We then exposed cells to combinations of UVB plus EMFs and found that 100 Hz, 1 mT EMFs decrease DNA synthesis of UVB-activated Jurkat cells by 34 +/- 13% compared to UVB alone. The decrease is, however, most effective when relatively high UVB doses are employed. Since EMFs alone had only a very weak inhibitory effect (10 +/- 2%), the data suggest that EMFs augment the cell killing effects of UVB in a synergistic way. These findings could provide the basis for development of new and improved clinical phototherapy protocols.

Experiments showing that electromagnetic fields can be used to treat inflammatory diseases.

While it is well known that electromagnetic fields (EMFs) can induce repair of non-healing bone fractures, EMF therapy remains confined to orthopedic clinics mainly because the biological and physical mechanisms underlying the therapy are unknown. However, it is generally believed that non-invasive, EMF therapy might have a broad, albeit currently unrecognized clinical potential. In support of this view, we report that 0.1 mT, 60 Hz EMFs induce a 20% mean-increase in anti-CD3 binding to T cell receptors (TcRs) of Jurkat cells, a T lymphocyte cell line. Additionally, we show that 60 Hz sinusoidal EMFs and a commercial bone healing EMF modulate signal transduction pathways that regulate lymphocyte proliferation and that are normally triggered by activating the Jurkat TcR. Similar EMF effects are shown in human peripheral blood lymphocytes (hPBLs), exposed to EMFs in culture and in rat PBLs, when donor animals are exposed to a bone healing field (21 days, 4 hr/day). Although we do not yet satisfactorily understand the differences we obtain in cell and animal based experiments, our findings clearly demonstrate that EMFs can regulate lymphocyte proliferation in vitro and in vivo. Since T cells are key modulators of inflammation, the development of EMF based therapeutic devices to regulate their activity can be expected to provide important tools to treat numerous human inflammatory diseases such as psoriasis and arthritis.

Electromagnetic fields used clinically to improve bone healing also impact lymphocyte proliferation in vitro.

An important aspect of medical device development is the need to understand how a device produces a specific biological effect. The focus can then be on optimizing that effect by device modification and repeated testing. Several reports from this lab have targeted programmed cell death, or apoptosis, as a cellular pathway that is induced by exposure of transformed leukemic T-cells in culture to specific frequency and intensity electromagnetic fields (EMFs). An EMF delivery device capable of selectively inducing T-cell apoptosis in human tissues could be used to enhance healing by limiting the production of molecules that promote inflammatory disorders such as psoriasis and tendonitis. In the present study, we examined the normal T-cell response to EMF exposure in vitro. In the peripheral blood, 70-80% of the lymphocytes are T-cells, and thus is a rich source of normal cells that match the transformed T-cells used in other experiments (Jurkat cells). We isolated lymphocytes from the peripheral blood of humans and rats, cultured them in nutritive medium and exposed them to either a complex 1.8 mT pulsed EMF (Electrobiology, Inc.), a 0.1 mT, 60 Hz power frequency EMF or a 0.2 mT, 100 Hz sinusoidal EMF. Control lymphocytes were cultured similarly, without field exposure. Lymphocytes were then treated with T-cell mitogens and evaluated for proliferative capacity after an additional 72 hours culture. Results indicate that T-cell proliferation is modulated by in vitro exposure to defined EMFs. The potential use of an EMF delivery device capable of selectively inducing such T-cell effects is discussed.