Analgesic and disease modifying effects of interferential current in psoriatic arthritis.

Interferential current (IFC) was suggested to improve the skin manifestations of psoriasis vulgaris, possibly by enhancing the intracellular concentration of cyclic AMP. We assessed the efficacy of IFC on psoriatic arthritis (PsA). Nine consecutive patients were analyzed at baseline and after 16 weeks of IFC therapy. Bipolar IFC was applied twice daily to hands, feet plus all affected joints. IFC improved SF-36 assessed body pain, but not other SF-36 subscales. Morning stiffness, tender joint counts, and physician assessed disease activity improved. In contrast, visual analogue scale assessed pain, CRP and ESR measurements were unchanged. MRI of the most affected hand or foot documented a tendency towards worsened tendinitis, soft tissue swelling, and new joint space narrowing and erosions. Bone scintigraphy showed a trend towards deterioration. New joints became inflamed within treated sites. Thus IFC has analgesic effects in PsA, but does not have a satisfactory disease modifying effect.

Neuronal outgrowth of PC-12 cells after combined treatment with nerve growth factor and a magnetic field: influence of the induced electric field strength.

In view of possible therapeutic applications of magnetic fields, the effect of an enhancement of neuronal outgrowth at higher figures of flux density and induced field strength was investigated. On the average sinusoidal magnetic field treatment at 100 microTrms/50 Hz did not change nerve growth factor (NGF) induced neurite outgrowth to a statistically significant extent. These results suggest that further increasing the induced field strength by using either higher flux densities and/or more sophisticated wave forms might be necessary to cause the neuronal response of PC-12 cells, as seen in other experiments.

Quantification of NGF-dependent neuronal differentiation of PC-12 cells by means of neurofilament-L mRNA expression and neuronal outgrowth.

We demonstrate that the degree of neuronal development of PC-12 cell differentiation can be quantified by the expression of neurofilament-L (NF-L) mRNA, when an optimal concentration of NGF (50 ng/ml) is used. During the first 7 days of NGF treatment, the relative amount of NF-L mRNA was found to increase continuously and to correlate with the outgrowth of neurites in a statistically significant way. Thus, mRNA expression is, under these conditions, a suitable means for reliably monitoring the differentiation of PC-12 cells as early as after 3 days of NGF treatment. The results obtained with 5 ng/ml NGF differ from those with 50 ng/ml: during the first 3 days of NGF treatment, neuronal outgrowth was less than with 50 ng/ml, although the NF-L mRNA levels did not depend significantly on NGF concentration. Beyond day 3, NF-L mRNA levels did not increase further at 5 ng/ml as opposed to 50 ng/ml NGF. These differences point to different signal transduction processes involved in neuronal differentiation at high and low NGF concentration. Expression of NF-L protein in response to NGF treatment was also demonstrated. In summary, our results stress that stable and sustained differentiation of PC-12 cells can only be achieved with 50 ng/ml NGF.

Interferential current is effective in palmar psoriaris: an open prospective trial.

Interferential current (IFC) has been shown to improve psoriasis in a small case series. So far no formal clinical trial had been conducted. As IFC is associated with slight prickling sensations a blinded study design was not feasible. Therefore, an open type prospective study was conducted with the assumption of 18% spontaneous remission rate. A response rate of 50% or less was judged as indicating no effect (hypothetical control), while 80% or more was considered as success (alternative hypothesis). In this "quasi-controlled" study 12 patients with therapy resistant palmar psoriasis received local treatment with IFC during a 12 week period. Treatment was performed at low current density in two daily sessions, each of 6 minutes duration. Erythema, scaling, induration, fissures and pustules were recorded in separate scores every 4 weeks. Response of a patient was judged positive when the total score of these criteria was reduced at least by two points at the end of treatment. After 12 weeks of treatment, 11 of 12 patients were cured or showed marked remission with the median overall score reduced by 4 points. An interim analysis was performed in order to decide whether the results had already reached significance (a < 0.05). The analysis revealed a statistically significant response rate of 90% (95% confidence interval 62-99%). These results are highly encouraging and should focus attention on this new therapy modality, which, in contrast to other treatments is not associated with side effects and discomfort.

Induction of sister chromatid exchanges in fibroblasts from normal donors and from patients with xeroderma pigmentosum after combined treatment with ultraviolet radiation and modulated low frequency electric currents.

Dermal fibroblasts derived from normal donors as well as from patients with xeroderma pigmentosum (XP) were exposed in vitro to ultraviolet radiation of 254 nm wavelength and to weak electric 4, 000 Hz currents modulated in amplitude with 50 Hz. Treatment with this so-called interferential current (IFC) increased the rate of sister chromatid exchanges (SCE) in all fibroblast types up to roughly 50% above the spontaneous level. When IFC was applied in combination with UV-radiation, it lowered UV-induced increase of the SCE-rate. A reduction of UV-induced SCEs was seen when XP-cells were exposed to IFC prior to UV-treatment, compared to the reverse order of treatment. The order of exposure has only been analysed in 5 XP patients, not in normal donors. The relationships between different combinations of exposure parameters (IFC-amperage, UV-dose, cell pathology, order of treatment) were examined by analysis of variance (ANOVA).

Response of cytosolic calcium, cyclic AMP, and cyclic GMP in dimethylsulfoxide-differentiated HL-60 cells to modulated low frequency electric currents.

The action of interferential current (IFC), an amplitude-modulated 4000 kHz current used in therapeutic applications, upon intracellular calcium, adenosine 3':5'-cyclic monophosphate (cAMP), and guanosine 3':5'-cyclic monophosphate (cGMP) was investigated. Human promyelocytes (HL-60) were differentiated to granulocytes by dimethylsulfoxide (DMSO) treatment and exposed for 5 min at 25, 250, and 2500 microA/cm2 current density. No significant changes in cytosolic free calcium were detected as a function of modulation frequency of the IFC. However, intracellular cAMP reacted in a complex way to modulation frequency, resulting in stimulations and depressions within the range of frequencies studied (0-125 Hz). The "windows" of modulation frequency, where statistically significant increases or decreases in cAMP were noted, coincided with those published earlier for mouse fibroblasts. Cellular cGMP content was always lowered by IFC treatment. Furthermore, no significant influence of IFC current density upon the three second messengers was noted. These results, which also include data relating to treatment with sinusoidal 50 Hz current, contribute to a more detailed understanding of the primary biophysical mechanisms of signal transduction by time-varying electric fields.

Action of a 50 Hz magnetic field on proliferation of cells in culture.

Proliferation of SV40-3T3 mouse fibroblasts and human HL-60 promyelocytes was studied after treatment with a sinusoidal 2 mT 50 Hz magnetic field. A single exposure of 60 minutes caused quasicyclic changes in the number of SV40-3T3 cultures as function of time after treatment, which was interpreted to be due to the induction of chronobiological mechanisms by the field. Moreover, small variations in cell cycle distribution were measured during postexposure incubation for both cell lines. To discriminate between the effect of the magnetic vector and the induced electric field, HL-60 cell exposure was also performed on organ culture dishes. These dishes consist of two coaxially centered, isolated compartments in which different electric field levels are induced in the medium during treatment. Cell growth was affected in the outer compartment only where the induced electric field ranged from 8 to 12 mVpeak/meter at 2 mT, but it was not affected in the inner compartment (field range 0-4 mVpeak/meter). This suggests that the effects on cell growth are due to the induced electric field and are expressed only above a threshold of between 4 and 8 mVpeak/meter.

Action of 50 Hz magnetic fields on cyclic AMP and intercellular communication in monolayers and spheroids of mammalian cells.

To investigate the influence of physiological parameters such as cell density and three-dimensional cell contact on the biological action of a 2 mT/50 Hz magnetic field, mouse fibroblasts were exposed as monolayers and as multicellular spheroids. Changes in cyclic AMP content of cells and alterations in gap junction-mediated intercellular communication were measured immediately after 5 min of exposure to the field. In monolayers of intermediate cell density (1 x 10(5) cells/cm2), the field treatment caused an increase in cAMP to 121% of the control level, whereas, at 3 x 10(5) cells/cm2 (near confluence), a decrease to 88% of the unexposed cells was observed. Furthermore, field exposure stimulated gap-junction communication to 160% of the control level as determined by Lucifer yellow dye exchange. In spheroids, alterations in the radial profile of cellular cAMP were observed that were due both to field-induced local cAMP changes and to increased gap-junction permeability for this second messenger, the latter causing radial cAMP gradients to be flattened. The results indicate a strong dependence of field action on physiological parameters of the system exposed.

Stochastic resonance as a possible mechanism of amplification of weak electric signals in living cells.

The most important but still unresolved problem in bioelectromagnetics is the interaction of weak electromagnetic fields (EMFs) with living cells. Thermal and other types of noise pose restrictions in cell detection of weak signals. As a consequence, some extant experimental results that indicate low-intensity field effects cannot be accounted for, and this renders the results themselves questionable. One way out of this dead end is to search for possible mechanisms of signal amplification. In this paper, we discuss a general mechanism in which a weak signal is amplified by system noise itself. This mechanism was discovered several years ago in physics and is known, in its simplest form, as a stochastic resonance. It was shown that signal amplification may exceed a factor of 1000, which renders existing estimations of EMF thresholds highly speculative. The applicability of the stochastic resonance concept to cells is discussed particularly with respect to the possible role of the cell membrane in the amplification process.

Cyclic AMP response in cells exposed to electric fields of different frequencies and intensities.

The action on intracellular cyclic AMP (cAMP) of therapeutically used 4000-Hz electric fields was investigated and compared with 50-Hz data. Cultured mouse fibroblasts were exposed for 5 minutes to 4000-Hz sine wave internal electric fields between 3 mV/m and 30 V/m applied within culture medium. A statistically significant decrease in cellular cAMP concentration relative to unexposed cells was observed for fields higher than 10 mV/m. The drop in cAMP was most pronounced at lower field strengths (71% of controls at 30 mV/m) and tended to disappear at higher field strengths. An increase of cAMP content was observed with 50-Hz electric fields, as was also the case when 4000-Hz fields were modulated with certain low frequencies.

The DNA content of some mammalian cells measured by flow cytometry and its influence on radiation sensitivity.

The DNA content of nine mammalian cell lines was determined by flow cytometry. Using radiobiological data from this and other laboratories a correlation between DNA mass and 1/D0 for X-rays, alpha-particles, and heavy ions could be established when the quantities were plotted on a log-log scale. The slopes of the regression lines amounted to 0.65 (X-rays), 0.64 (alpha-particles) and 0.74 (heavy ions). A similar correlation was found between DNA content and mean inactivation dose. The rather uniform slopes close to 2/3 suggest that radiosensitivity may depend on the surface area of the sensitive target, (cell nucleus) indicating a possible non-uniform distribution of radiosensitive sites within the nucleus.

Radioresistance of rat glioma cell lines cultured as multicellular spheroids. Correlation with electrical cell-to-cell-coupling.

Two chemically induced rat glioblastomas, RG2 and F98, were cultured as monolayers and as multicellular spheroids and subjected to Co-gamma-irradiation. In parallel, intercellular communication between cells was determined as electrical coupling between neighbouring cells using micro-electrode techniques. A third glioblastoma with known radiobiological response (9L) was assayed with respect to intercellular communication and included into this analysis. Electrical coupling was low for RG2, intermediate for F98, and high for 9L. Radioresistance of spheroids, as expressed in terms of the mean inactivation dose computed from the survival curves increased in the same direction (RG2: 2.4 Gy; F98: 5.1 Gy; 9L: 6.5 Gy). A comparison of these parameters demonstrates a correlation between solid tumor radioresistance and gap-junctional cell-to-cell communication, at least for the class of glioblastomas analysed in this study.

Mean inactivation dose: a useful concept for intercomparison of human cell survival curves.

The mean inactivation dose (D) is calculated for published in vitro survival curves obtained from cell lines of both normal and neoplastic human tissues. Cells belonging to different histological categories (melanomas, carcinomas, etc.) are shown to be characterized by distinct values of D which are related to the clinical radiosensitivity of tumors from these categories. Compared to other ways of representing in vitro radiosensitivity, e.g., by the multitarget parameters D0 and n, the parameter D has several specific advantages: (i) D is representative for the whole cell population rather than for a fraction of it; (ii) it minimizes the fluctuations of the survival curves of a given cell line investigated by different authors; (iii) there is low variability of D within each histological category; (iv) significant differences in radiosensitivity between the categories emerge when using D. D appears to be a useful concept for specifying intrinsic radiosensitivity of human cell lines.

Relationship between intercellular communication and radiosensitivity of human tumor xenografts.

Micro-electrode techniques permit the detection of electrical coupling between adjacent cells if they are connected by intercellular junctions (gap junctions). This technique was applied to four human tumors xenografted onto nude mice. In three of the tumors, which showed a 'contact resistance' after irradiation in vivo, electrical coupling could be established. No coupling was found in the other tumor, which did not exhibit contact resistance. These results are similar to those obtained recently with cultured spheroids of mammalian cell lines in which only the electrically coupled cell types developed contact resistance to ionizing radiation.

Radiosensitivity of four human tumor xenografts. Influence of hypoxia and cell-cell contact.

Contact effect (CE) and hypoxia have been studied in human tumor cell lines transplanted in athymic nude mice. Four cell lines--one melanoma (Bell) and three colorectal adenocarcinomas (HT29, HRT18, and HCT8)--were studied. Cell survival was determined with an in vivo to in vitro colony-forming assay. Survival curves were obtained under three different conditions: (1) tumor cells irradiated in air-breathing mice, (2) tumor cells irradiated in animals asphyxiated for 10 min, and (3) tumor cells plated and irradiated either immediately or 5 hr later. For all cell lines, radiosensitivity appeared to be lower when cells were irradiated in vivo than when they were irradiated in vitro. Only in the case of the HCT8 tumor did the relative in vivo radioresistance seem to be linked to hypoxia; in the other cell lines, hypoxia alone could not account for the lower in vivo radiosensitivity. Our results suggest that a CE plays an important role in the response of human tumor xenografts to irradiation.

Is there a relationship between hypoxia, contact resistance, and intercellular communication?

This investigation addresses the shape of radiation survival curves of cells cultured as multicell spheroids. It is shown that spheroids of cells capable of intercellular communication by gap-junctions display survival curves lacking a radioresistant fraction of hypoxic cells. Compared to the corresponding monolayers, these spheroid survival curves exhibit a uniform increase in radioresistance due to the "contact effect". In contrast, biphasic survival curves indicative of hypoxic cells are obtained with non-communicating spheroids, however, without indication of a contact effect. Evidence is presented that this relationship between intercellular communication, hypoxia, and contact effect may possibly also hold for survival curves of solid tumors.

Increased radioresistance of cells in cultured multicell spheroids. II. Kinetic and cytogenetic studies.

Using the Chinese hamster cell line B14 FAF28, several specific properties of the contact effect (CE) of radiation action in spheroids were investigated. CE was found to "protect" the spheroid cells against several types of radiation-induced cytogenetic misfunctions such as blockage in S and G2+M-phase, mutagenesis, and chromosome damage. However, repair of DNA strand-breaks was the same in monolayers and spheroids. Furthermore, CE is a property of the single cell and does not depend on the proliferative status (cycling or non-cycling) of the cells. We conclude that CE is the result of a physiological modification of the cells occurring during growth in the three-dimensional spheroid matrix and requiring metabolic cooperation and cyclic AMP for its induction.

Intercellular communication, three-dimensional cell contact and radiosensitivity.

When electrically coupled mammalian cells are cultured as spherical clones (spheroids) and exposed to ionizing radiation they are less radiosensitive than monolayers of the same cell line. Investigations into the possible role of coupling (gap junctions) and three-dimensional contact in the expression of this phenomenon revealed 1) a correlation between cell coupling and the activity of adenylate cyclase in monolayers, 2) a sharp drop of cyclase activity in spheroids of coupled cells compared to monolayers, and 3) a decrease of coupling with age ("maturation") of the spheroids. These results suggest profound physiological alterations in communicating cells induced under conditions of tight three-dimensional contact as a possible cause for the reduced radiosensitivity of spheroids.