Modulation of cytokine production by interferential current in differentiated HL-60 cells

The influence of interferential current (IFC) on the release of four cytokines was investigated. IFC is an amplitude-modulated 4 kHz current used in therapeutic applications. Human promyelocytes (HL-60) were differentiated to monocytes/macrophages by treatment with calcitriol. Release of tumor necrosis factor alpha (TNFalpha) and interleukines 1beta, 6, and 8 (IL-1beta, IL-6, and IL-8) into the supernatant was measured after exposure to IFC at different modulation frequencies. TNFalpha release was stimulated about twofold by 4 kHz sine waves alone. The influences of exposure time (5-30 min) and current density (2.5-2500 microA/c m(2)) were tested. A maximum field effect was found at an exposure time of 15 min and a current density of 250 microA/cm(2). With these exposure conditions (15 min and 250 microA/cm(2) ), cells were treated at different modulation frequencies and reacted for TNFalpha, IL-1beta, and IL-8 release in a complex manner. Within the frequencies studied (0-125 Hz), we found stimulation as well as depression of the release. In a second run the cells were activated by pretreatment with 10 microg/ml lipopolysaccharide (LPS) and exposed in the same way as the nonactivated cells. Again the modulation frequency influenced, in a complex way, the induction of TNFalpha, IL-1beta, and IL-8, resulting in a pattern of stimulation and depression of release different from that found in nonactivated cells. For IL-6 production no significant changes were detected in activated or non-activated cells.     

Effect of Frequency and Waveform on Inactivation of Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Salsa by Ohmic Heating

The effect of frequency of alternating current during ohmic heating on electrode corrosion, heating rate, inactivation of food-borne pathogens, and quality of salsa was investigated. The impact of waveform on heating rate was also investigated. Salsa was treated with various frequencies (60 Hz to 20 kHz) and waveforms (sine, square, and sawtooth) at a constant electric field strength of 12.5 V/cm. Electrode corrosion did not occur when the frequency exceeded 1 kHz. The heating rate of the sample was dependent on frequency up to 500 Hz, but there was no significant difference (P > 0.05) in the heating rate when the frequency was increased above 1 kHz. The electrical conductivity of the sample increased with a rise in the frequency. At a frequency of 60 Hz, the square wave produced a lower heating rate than that of sine and sawtooth waves. The heating rate between waveforms was not significantly (P > 0.05) different when the frequency was >500 Hz. As the frequency increased, the treatment time required to reduce Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium to below the detection limit (1 log CFU/g) decreased without affecting product quality. These results suggest that ohmic heating can be effectively used to pasteurize salsa and that the effect of inactivation is dependent on frequency and electrical conductivity rather than waveform.     

Frequency change-induced alternative potential waveform dependence of membrane damage to cells cultured on an electrode surface

In the present study, alternative potential stimulation with rectangular pulse, sine and triangular waveforms at 10 and 100Hz was applied to cells cultured on an ITO electrode. As a result, we found that the alternating potential waveform dependence induced by the frequency on membrane damage of cells cultured on an electrode surface. The cell membrane damage was promoted by a rectangular pulse wave in comparison with sine and triangular waves, when alternating electrical potentials of 0 to +1.0V at 100Hz were loaded. In contrast, this waveform dependence was not observed when the frequency was 10Hz. Furthermore, it was found that cell membrane damage was induced at positive potentials more than +0.8V under the present experimental conditions.     

INFLUENCE OF LOW-FREQUENCY ELECTRIC FIELDS ON PROLIFERATION AND IL-8 RELEASE OF HL-60 CELLS

The influence of capacitively coupled extremely low-frequency (ELF) electric fields on proliferation and on interleukin (IL)-8 release of exponentially growing HL-60 cells was examined. The cell suspensions were treated with the field component of interferential current (IFC) using different exposure protocols. Modulation frequencies of 10 and 100 Hz were applied with field strengths between 0.075 and 11.54 V_pp/cm for 48 hr using a 5-min exposure time at every 3 hr. At a field strength of 1 V_pp/cm, the influence of the time between two exposure sessions was examined for different modulation frequencies. All exposure protocols applied have no effect on cell proliferation (p>0.05), but statistical significant reduction (p<0.05) of the IL-8 release at selected modulation frequencies and interval times could be observed.     

A preliminary study of oscillating electromagnetic field effects on human spermatozoon motility

Some effects of extremely low frequency electromagnetic fields (ELF-EMFs) on human spermatozoa are reported. Significant increases in the values of the motility and of the other kinematic parameters have been observed when spermatozoa were exposed to an ELF-EMF with a square waveform of 5 mT amplitude and frequency of 50 Hz. By contrast, a 5 mT sine wave (50 Hz) and a 2.5 mT square wave (50 Hz) exposure did not produce any significant effect on sperm motility. The effects induced by ELF-EMF (50 Hz; 5 mT) during the first 3 h of exposure persisted for 21 h after the end of the treatment. These results indicate that ELF-EMF exposure can improve spermatozoa motility and that this effect depends on the field characteristics.     

Effect of moderate electric field frequency on growth kinetics and metabolic activity of Lactobacillus acidophilus

Moderate electric fields (MEF) have been previously shown to alter the metabolic activity of microbial cells; thus, the effect of frequency and electric field would be of considerable interest. We investigated herein the effects of MEF frequency on microbial growth kinetics and bacteriocin (Lacidin A) production of Lactobacillus acidophilus OSU 133 during fermentation. The following fermentation treatments were compared: conventional (for 40 h), MEF (1 V cm(-1), for 40 h), combination of MEF (1 V cm(-1), for the first 5 h) and conventional (for 35 h) at various frequency levels (45, 60, and 90 Hz) all at 30 degrees C, and control (conventional) fermentation at 37 degrees C. MEF treatments with purely sinusoidal waveforms at all frequencies at 30 degrees C produced a shorter lag phase than conventional fermentation. However, no lag phase reduction was found for a 60 Hz waveform that contained high-frequency harmonics. There was, however, a significant increase in the bacteriocin production under early MEF treatment at 60 Hz with high-frequency harmonics. On the basis of these observations, the fermentation process is accelerated by applying pure sinusoidal MEF at the early stage of growth while a significant increase in the bacteriocin production occurs when sinusoidal field at 60 Hz with harmonics is applied at the early stage of the growth.     

ELF magnetic fields increase amino acid uptake into Vicia faba L. roots and alter ion movement across the plasma membrane

Vicia faba seedlings, subjected to a 10 microT 50 Hz square wave magnetic field for 40 min together with a radioactive pulse, showed a marked increase in amino acid uptake into intact roots. A more modest increase was observed with a 100 microT 50 Hz square wave. An increase in media conductivity at low field intensities from 10 microT 50 Hz square wave, 100 microT 50 Hz sine wave, and 100 microT 60 Hz square wave fields, indicated an alteration in the movement of ions across the plasma membrane, most likely due to an increase in net outflow of ions from the root cells. Similarly, marked elevation in media pH, indicating increased alkalinity, was observed at 10 and 100 microT for both square and sine waves at both 50 and 60 Hz. Our data would indicate that low magnetic field intensities of 10 and 100 microT at 50 or 60 Hz can alter membrane transport processes in root tips.     

Changes in IL-8 release and intracellular content in DMSO-differentiated HL-60 cells after treatment with 4-hydroxynonenal

4-Hydroxynonenal (HNE), a chemotactic aldehyde produced by lipid peroxidation, has been shown to trigger exocytosis in HL-60 cells induced to differentiate toward the granulocytic cell line by DMSO. In this work we studied HNE effects on the intracellular content of IL-8 and its release in DMSO-differentiated HL-60 cells. Cell incubation at 37 degrees C in the presence of 0.1 microM HNE induced a significant increase of IL-8 release after 30 min; the degree of HNE-induced IL-8 secretion became quite strong after 1 h, whereas the intracellular content showed no statistically significant changes. By contrast, 1 microM HNE induced a low decrease of the chemokine release; however, the used HNE concentrations failed to increase the release of lactate dehydrogenase (LDH), a test used to assay cell viability. The addition of 0.1 microM IL-8 to DMSO-differentiated HL-60 cells induced a strong increase of exocytosis, measured by beta-glucuronidase secretion. Exocytosis stimulation by IL-8 was much higher than that given by the aldehyde; the addition of various HNE concentrations to cells incubated in the presence of IL-8 decreased the secretion given by the cytokine alone. However, HNE-induced exocytosis was likely to be a direct action of the aldehyde and was not mediated through the stimulation of IL-8 release since HNE was unable to modify IL-8 secretion during the short time of 10 min used in the exocytosis assay.     

IL-5 up-regulates cysteinyl leukotriene 1 receptor expression in HL-60 cells differentiated into eosinophils

The cysteinyl leukotrienes, leukotriene (LT) C(4), LTD(4), and LTE(4), are lipid mediators that have been implicated in the pathogenesis of several inflammatory processes, including asthma. The human LTD(4) receptor, CysLT(1)R, was recently cloned and characterized. We had previously shown that HL-60 cells differentiated toward the eosinophilic lineage (HL-60/eos) developed specific functional LTD(4) receptors. The present work was undertaken to study the potential modulation of CysLT(1)R expression in HL-60/eos by IL-5, an important regulator of eosinophil function. Here, we report that IL-5 rapidly up-regulates CysLT(1)R mRNA expression, with consequently enhanced CysLT(1)R protein expression and function in HL-60/eos. CysLT(1)R mRNA expression was augmented 2- to 15-fold following treatment with IL-5 (1-20 ng/ml). The effect was seen after 2 h, was maximal by 4 h, and maintained at 8 h. Although CysLT(1)R mRNA was constitutively expressed in undifferentiated HL-60 cells, its expression was not modulated by IL-5 in the absence of differentiation. Differentiated HL-60/eos cells pretreated with IL-5 (10 ng/ml) for 24 h showed enhanced CysLT(1)R expression on the cell surface, as assessed by flow cytometry using a polyclonal anti-CysLT(1)R Ab. They also showed enhanced responsiveness to LTD(4), but not to LTB(4) or platelet-activating factor, in terms of Ca(2+) mobilization, and augmented the chemotactic response to LTD(4). Our findings suggest a possible mechanism by which IL-5 can modulate eosinophil functions and particularly their responsiveness to LTD(4), and thus contribute to the pathogenesis of asthma and allergic diseases.     

A new mechanical stimulator for cultured bone cells using piezoelectric actuator

In this study, a new mechanical stimulator using the piezoelectric actuator was developed to give cultured bone cells mechanical strains with more physiologic magnitude, frequency components, and waveform. This stimulator provides bone cells in a three-dimensional collagen gel block culture mechanical strains with magnitude of 200-40,000 microstrain and frequency of DC-100 Hz, which sufficiently covers physiological strains on bone. Furthermore, the stimulator can generate not only common strain waveforms like sine and rectangular waves, but also arbitrary strain waveforms synthesized on a personal computer. In particular, the controllability of strain frequency and waveform is an advance over that of previous stimulators. Thus, this device can facilitate new findings regarding bone cell responses to mechanical stimuli.     

No effect of pulsed electromagnetic fields on PC12 and HL-60 cells

The effect of pulsed electromagnetic fields (PEMF) similar to those used in transcranial magnetic stimulation (TMS) on two tumour cell lines, the human promyelocytic leukaemia cell line (HL-60) and the rat pheochromocytoma cell line (PC12), was investigated. The two cell lines were exposed to non-homogeneous pulsed electromagnetic fields (about 0.25–4.5 T peak magnetic field strength; 1–8 exponential pulses, 0.25 Hz) at different positions on the coil (2×25 mm). After exposure with various intensities, various numbers of pulses and at different coil positions, cell viability and the intracellular cyclic AMP content were determined in the two cell lines. Additionally, in HL-60 cells the intracellular Hsp72 content and in PC12 cells the release of the neurotransmitters dopamine, noradrenaline and acetylcholine were measured after PEMF treatment. The results of these analyses do not hint at alterations in the cell viability or in the content of cAMP, Hsp72, dopamine, noradrenaline, and acetylcholine in the two tumour cell lines after PEMF exposure under various conditions.     

Interleukin-8 primes oxidative burst in neutrophil-like HL-60 through changes in cytosolic calcium

In response to a variety of stimuli, neutrophils release large amount of reactive oxygen species (ROS) generated by NADPH oxidase. This process known as the respiratory burst is dependent on cytosolic free calcium concentration ([Ca(2+)](i)). Proinflammatory cytokines such as interleukin-8 (IL-8) may modulate ROS generation through a priming phenomenon. The aim of this study was to determine the effect of human IL-8 on ROS production in neutrophil-like dimethylsulfoxide-differentiated HL-60 cells (not equalHL-60 cells) and further to examine the role of Ca(2+) mobilization during the priming. IL-8 at 10 nM induced no ROS production but a [Ca(2+)](i) rise (254 +/- 36 nM). IL-8 induced a strongly enhanced (2 fold) ROS release during stimulation with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF). This potentiation of ROS production is dependent of extracellular Ca(2+) (17.0+/-4.5 arbitrary units (A.U.) in the absence of Ca(2+) versus 56.6 +/- 3.9 A.U. in the presence of 1.25 mM of Ca(2+)). Also, IL-8 enhanced fMLF-stimulated increase in [Ca(2+)](i) (375 +/- 35 versus 245 +/- 21 nM, 0.1 microM of fMLF). IL-8 had no effect on not equalHL-60 cells in response to 1 microM of thapsigargin (472 +/- 66 versus 470 +/- 60 nM). In conclusion, Ca(2+) influx is necessary for a full induction of neutrophil priming by IL-8.     

High-frequency chest compression: effect of the third generation compression waveform

High-frequency chest compression (HFCC) therapy has become the prevailing form of airway clearance for patients with cystic fibrosis (CF) in the United States. The original square waveform was replaced in 1995 with a sine waveform without published evidence of an equality of effectiveness. The recent development of a triangle waveform for HFCC provided the opportunity to compare the functional and therapeutic effects of different waveforms. Clinical testing was done in patients at home with therapy times recorded with all sputum collected in preweighed sealable vials. The eight study patients with CF were regular users of a sine waveform device. They produced sputum consistently and were clinically stable. They used their optimum frequencies for therapy for each waveform and, for one week for each waveform, collected all sputum during their twice-daily timed HFCC therapies. After collection, these vials were reweighed, desiccated, and reweighed to calculate wet and dry weights of sputum per minute of therapy time. Frequency associated vest pressures transmitted to the mouth, and induced airflows at the mouth were measured in healthy volunteers. The pressure waveforms produced in the vest were, in shape, faithfully demonstrable at the mouth. In the healthy subject the transmission occurred in 2 ms and was attenuated to about 75% of the vest pressure for the triangle waveform and 60% for the sine waveform. All patients produced more sputum with the triangle waveform than with the sine waveform. The mean increase was 20%+ range of 4% to 41%. P value was <.001. Future studies of HFCC should investigate the other effects of the sine and triangle waveforms, as well as the neglected square waveform, on mucus clearance and determine the best frequencies for each waveform, disease, and patient.     

Bioeffects induced by exposure to microwaves are mitigated by superposition of ELF noise

We have previously demonstrated that microwave fields, amplitude modulated (AM) by an extremely low-frequency (ELF) sine wave, can induce a nearly twofold enhancement in the activity of ornithine decarboxylase (ODC) in L929 cells at SAR levels of the order of 2.5 W/kg. Similar, although less pronounced, effects were also observed from exposure to a typical digital cellular phone test signal of the same power level, burst modulated at 50 Hz. We have also shown that ODC enhancement in L929 cells produced by exposure to ELF fields can be inhibited by superposition of ELF noise. In the present study, we explore the possibility that similar inhibition techniques can be used to suppress the microwave response. We concurrently exposed L929 cells to 60 Hz AM microwave fields or a 50 Hz burst-modulated DAMPS (Digital Advanced Mobile Phone System) digital cellular phone field at levels known to produce ODC enhancement, together with band-limited 30–100 Hz ELF noise with root mean square amplitude of up to 10 μT. All exposures were carried out for 8 h, which was previously found to yield the peak microwave response. In both cases, the ODC enhancement was found to decrease exponentially as a function of the noise root mean square amplitude. With 60 Hz AM microwaves, complete inhibition was obtained with noise levels at or above 2 μT. With the DAMPS digital cellular phone signal, complete inhibition occurred with noise levels at or above 5 μT. These results suggest a possible practical means to inhibit biological effects from exposure to both ELF and microwave fields. Bioelectromagnetics 18:422–430, 1997. © 1997 Wiley-Liss, Inc.     

Electric field-induced changes in agonist-stimulated calcium fluxes of human HL-60 leukemia cells

The mechanism of biological effects of extremely-low-frequency electric and magnetic fields may involve induced changes of Ca²⁺ transport through plasma membrane ion channels. In this study we investigated the effects of externally applied, low-intensity 60 Hz electric (E) fields (0.5 V/m, current density 0.8 A/m²⁺) on the agonist-induced Ca²⁺ fluxes of HL-60 leukemia cells. The suspensions of HL-60 cells received E-field or sham exposure for 60 min and were simultaneously stimulated either by 1 μM ATP or by 100 μM histamine or were not stimulated at all. After E-field or sham exposure, the responses of the intracellular calcium levels of the cells to different concentrations of ATP (0.2–100 μM) were assessed. Compared with control cells, exposure of ATP-activated cells to an E-field resulted in a 20–30% decrease in the magnitude of [Ca²⁺]_i elevation induced by a low concentration of ATP (<1 μM). In contrast, exposure of histamine-activated HL-60 cells resulted in a 20–40% increase of ATP-induced elevation of [Ca²⁺]_i. E-field exposure had no effect on non-activated cells. Kinetic analysis of concentration-response plots also showed that compared with control cells, exposure to the E-field resulted in increases of the Michaelis constant, K_m, value in ATP-treated cells and of the maximal [Ca²⁺]_i peak rise in histamine-treated HL-60 cells. The observed effects were reversible, indicating the absence of permanent structural damages induced by acute 60 min exposure to electric fields. These results demonstrate that low-intensity electric fields can alter calcium distribution in cells, most probably due to the effect on receptor-operated Ca²⁺ and/or ion channels. Bioelectromagnetics 19:366–376, 1998. © 1998 Wiley-Liss, Inc.     

Electrosensory phase sensitivity in the weakly electric fish Eigenmannia in the detection of signals similar to its own

The electric organ discharge (EOD) of the South American knifefish Eigenmannia sp. is a permanently present wave signal of usually constant amplitude and frequency (similar to a sine wave). A fish perceives discharges of other fish as a modulation of its own. At frequency identity (F = 0 Hz) the phase difference between a fish's own electric discharge and that of another fish affects the superimposed waveform. It was unclear whether or not the electrosensory stimulus-intensity threshold as behaviourally determined depends on the phase difference between a fish's own EOD and a sine-wave stimulus (at F = 0 Hz). Also the strength of the jamming avoidance response (JAR), a discharge frequency shift away from a stimulus that is sufficiently close to the EOD frequency, as a function of phase difference was studied. Sine-wave stimuli were both frequency-clamped and phase-locked to a fish's discharge frequency (F = 0 Hz). In food-rewarded fish, the electrosensory stimulus-intensity threshold depended significantly on the phase difference between a fish's discharge and the stimulus. Stimulus-intensity thresholds were low (down to 3 V/cm, peak-to-peak) when the superimposed complex wave changed such that the shift in zero-crossings times relative to the original EOD was large but amplitude change minimal; stimulus-intensity thresholds were high (up to 16.9 V/cm, peak-to-peak) when the shift in zero-crossings times was small but amplitude change maximal. Similar results were obtained for the non-conditioned JAR: at constant supra-threshold stimulus intensities and F = 0 Hz, the phase difference significantly affected the strength of the JAR, although variability between individuals was higher than that observed in the conditioned experiments.Abbreviations ACP active phase coupling - EOD electric organ discharge - JAR jamming avoidance response - F frequency (fish) — frequency (stimulus) [Hz] - p-p peak-to-peak     

Zinc modulates mRNA levels of cytokines

Zinc plays an important role in cell-mediated immune function. Altered cellular immune response resulting from zinc deficiency leads to frequent microbial infections, thymic atrophy, decreased natural killer activity, decreased thymic hormone activity, and altered cytokine production. In this study, we examined the effect of zinc deficiency on IL-2 and IFN-gamma in HUT-78 (Th0) and D1.1 (Th1) cell lines and TNF-alpha, IL-1 beta, and IL-8 in the HL-60 (monocyte-macrophage) cell line. The results demonstrate that zinc deficiency decreased the levels of IL-2 and IFN-gamma cytokines and mRNAs in HUT-78 after 6 h of PMA/p-phytohemagglutinin (PHA) stimulation and in D1.1 cells after 6 h of PHA/ionomycin stimulation compared with the zinc-sufficient cells. However, zinc deficiency increased the levels of TNF-alpha, IL-1 beta, and IL-8 cytokines and mRNAs in HL-60 cells after 6 h of PMA stimulation compared with zinc-sufficient cells. Actinomycin D study suggests that the changes in the levels of these cytokine mRNAs were not the result of the stability affected by zinc but might be the result of altered expression of these cytokine genes. These data demonstrate that zinc mediates positively the gene expression of IL-2 and IFN-gamma in the Th1 cell line and negatively TNF-alpha, IL-1 beta, and IL-8 in the monocyte-macrophage cell line. Our study shows that the effect of zinc on gene expression and production of cytokines is cell lineage specific.     

Electrically induced release of amino acids formed from (U14C) glucose in rat brain cortex slices,studied by a simplified dansylation procedure

The nonessential amino acids glutamate, aspartate, glutamine, -minobutyrate (GABA), alanine, glycine, and proline present in rat thin brain cortex slices were labeled by in vitro incubation of these with [U-¹⁴C]glucose, and the efflux of such endogenous radioactive amino acids and of lactate was studied in a superfused system, under control conditions or when the slices were depolarized by various procedures. When electrical stimuli known to induce selective neurotransmitter release (1 or 1.5 volt, sine wave 60 Hz) were applied for 10 sec to the slices, no significant increase in amino acid efflux was found. When more intense stimuli (4 volt, 60 Hz) were applied for 60 sec, or extracellular potassium was raised to 56 mM, both conditions being known to induce nonselective substance release, the efflux of essentially all amino acids and of lactate was markedly increased. Increases in efflux were proportionately larger for glutamate, aspartate, and -aminobutyrate, and this could be accounted for by their greater intracellular chemical (or electrochemical) potentials, but not because of a selective release mechanism for them. Amino acids were analyzed as their 1-dimethylaminonaphthalene-5-sulfonyl (dansyl) derivatives, by a modification of existing procedures in which the dansyl (DNS) derivatives were efficiently extracted from acidified incubation fluid into an organic phase. This rapidly desalted the derivatives and allowed their concentration and chromatographic separation on thin-layer silica gel sheets with little loss.