A photochemical microreactor used to analyze hydrogen peroxide (H2O2) production of T lymphocytes.

In this report we describe a new photochemical reactor and its use in the study of ultraviolet-B light (UVB) dependent H2O2 production by T lymphocytes. In the reactor multiple biological samples rotate around a luminescent tube and thus simultaneously absorb a uniform light-flux. The reactor was developed to expand our earlier studies where we showed that UVB activates T lymphocytes so that 10(7) cells produce about 60 nmol H2O2 per minute. H2O2 has increasingly become recognized as a cell signaling molecule regulating immune reactions mediated by T lymphocytes. Our laboratory is researching the potential of such immune regulators as potential future therapeutic agents. To study photochemical H2O2 production in small samples such as suspensions of T lymphocyte cultures or cell extracts, we designed a reactor in which 12 samples (each 50 - 500 microliters) can be exposed to light under temperature-controlled conditions. The samples are mounted on a rotating platform equidistant from the axis of rotation, where the light source of the photoreactor is located. Rotating the samples helps assure that all samples receive a uniform amount of light energy over time. A cooling fan is integrated in the base of the reactor to help minimize convective heat transfer between the lamp and the samples. We simultaneously operate two identical systems to be able to compare data that are obtained from light exposed samples under control and experimental conditions. Data on the influence of therapeutically relevant electromagnetic fields on H2O2 production of T lymphocytes are presented. H2O2 was quantified using the Amplex Red dye.

Catheter-tip sensor to monitor production of hydrogen peroxide in small biosamples.

Recently, it was shown that antibodies in the presence of ultraviolet (UV) light give rise to singlet oxygen which ultimately leads to the production of hydrogen peroxide (H2O2). In this research, we are interested in understanding the role of H2O2 in T-cell activity during inflammation. Since the T-cell receptor has been shown to have the same oxidative catalytic potential as antibodies, we started experiments measuring H2O2 production in antibodies and T cells. After showing that a positively polarized Clark oxygen electrode can be used in measuring H2O2 production in antibodies and T-cells, it is the goal of the current study to characterize the use of a catheter-tip sensor under similar conditions. Our catheter has a platinum ring which acts as the anode and a silver/silver chloride tip which acts as the cathode. Although this newly designed amperometric biosensor works on the same principles of electrochemistry, its compact size equips us with the potential for in vivo use and small sample testing. Operating at a polarizing voltage of 0.7 Volts v/s Ag/AgCl, the bare sensor produced a current of 8 +/- 2 nA per microM H2O2 with a 10 seconds response time, over a range of 0-50 microM H2O2. For use with biosamples, we added a hydrophilic H2O2 permeable membrane, which reduced the electrode current to 0.48 +/- 0.1 nA/microM H2O2 and increased the response time to 2 minutes. On the other hand, the addition of the membrane improved the signal to noise ratio and the selectivity of the sensor. Using this sensor, we reproduced the light mediated H2O2 production which was recorded at the rate of 20 nM per minute for 1 milliliter of 6.7 microM rat IgG solution. We further discuss the usefulness, limitation and the future scope of this real time monitoring system for H2O2 research using small biosamples.

Effect of hydrogen peroxide on proliferation, apoptosis and interleukin-2 production of Jurkat T cells.

Hydrogen peroxide (H2O2) is well known as a cell damaging agent that is produced during normal cell metabolism of aerobic organisms. An excessive production of oxygen metabolites such as H2O2 leads to oxidative stress and disease. On the other hand, it recently was discovered that H2O2 is not only a deleterious oxidant for cells but can also play an important role as a beneficial signaling molecule in certain cells such as T lymphocytes. T lymphocytes are major regulatory cells in the inflammatory cascade and can act by releasing either toxins or beneficial signaling molecules. Understanding how to regulate the actions of H2O2 in T cells will allow for the creation of novel ways to improve the treatment of inflammatory diseases. The current study presents baseline information on the effects of H2O2 on Jurkat cells, a T cell line that we use as a T cell model for therapeutic research. We first determined the half-life of 0-80 mumolar H2O2 added to Jurkat cultures using a realtime H2O2 monitoring system. We then exposed Jurkat cells to such H2O2 concentrations and found that 50 +/- 10 mumolar H2O2 promoted interleukin-2 production in cells activated with anti-CD3 at the T cell receptor plus phorbol myristate acetate as a co-stimulatory signal. These effects were not seen in non-activated, normal Jurkat cells, where H2O2 inhibited cell proliferation and induced apoptosis in a dose-dependent way without affecting interleukin-2 production. Our data indicate that Jurkat cells can model both healthy and inflammatory T cells that respond differently to oxidative metabolites such as H2O2.

Noninvasive treatment of inflammation using electromagnetic fields: current and emerging therapeutic potential.

Magnets, electric current and time varying magnetic fields always have played a role in human medicine. Natural magnetic stones were used in ancient cultures to induce a therapeutic effect and modern clinical practice would be far less effective without nuclear magnetic resonance imaging, cardiac pacemakers, and bone growth stimulators. This paper presents a summary of natural and artificial electromagnetic field (EMF) characteristics that are currently in use or under investigation for other therapeutic applications. This background understanding provides a basis for discussion on the success and possible risks of emerging and novel EMF therapies. Although interest in energy medicine has existed for centuries in some parts of the world, in recent years this is an area of heightened interest for western healthcare practitioners. This awareness has been triggered by the growing body of knowledge on how EMFs interact with cellular systems. EMF therapy for the treatment of pain, cancer, epilepsy, and inflammatory diseases like psoriasis, tendinitis and rheumatoid arthritis is currently being explored. The long-term success of this new area of medicine is still unknown. On the one hand, it remains to be seen whether positive human outcomes with EMF therapy could be explained by enhancement of the placebo effect. Optimistically, EMF therapy has the potential to revolutionize medicine, which is currently dominated by pharmaceutical and surgical interventions. In this case, new therapeutic tools may be developed for future clinicians to provide noninvasive treatments with low risk of side effects and no problem with drug interactions.

Comparison of a triaxial fluxgate magnetometer and Toftness sensometer for body surface EMF measurement.

INTRODUCTION: The use of magnetic fields to treat disease has intrigued mankind since the time of the ancient Greeks. More recently it has been shown that electromagnetic field (EMF) treatment aids bone healing, and repetitive transcranial magnetic stimulation (rTMS) appears to be beneficial in treating schizophrenia and depression. Since external EMFs influence internal body processes, we hypothesized that measurement of body surface EMFs might be used to detect disease states and direct the course of subsequent therapy. However, measurement of minute body surface EMFs requires use of a sensitive and well documented magnetometer. In this study we evaluated the sensitivity and frequency response of a fluxgate magnetometer with a triaxial probe for use in detecting body surface EMF and we compared the magnetometer readings with a signal from a Toftness Sensometer, operated by an experienced clinician, in the laboratory and in a clinical setting. METHODS: A Peavy Audio Amplifier and variable power output Telulex signal generator were used to develop 50 microT EMFs in a three coil Merritt coil system. A calibrated magnetometer was used to set a 60 Hz 50 microT field in the coil and an ammeter was used to measure the current required to develop the 50 microT field. At frequencies other than 60 Hz, the field strength was maintained at 50 microT by adjusting the Telulex signal output to keep the current constant. The field generated was monitored using a 10 turn coil connected to an oscilloscope. The oscilloscope reading indicated that the field strength was the same at all frequencies tested. To determine if there was a correspondence between the signals detected by a fluxgate magnetometer (FGM1) and the Toftness Sensometer both devices were placed in the Merritt coil and readings were recorded from the FGM1 and compared with the ability of a highly experienced Toftness operator to detect the 50 microT field. Subsequently, in a clinical setting, FGM1 readings made by an FGM1 technician and Sensometer readings were made by 4 Toftness Sensometer operators, having various degrees of experience with this device. Each examiner obtained instrument readings from 5 different volunteers in separate chiropractic adjusting rooms. Additionally, one of the Toftness Sensometers was equipped with an integrated fluxgate magnetometer (FGM2) and this magnetometer was used to obtain a second set of EMF readings in the clinical setting. RESULTS: The triaxial fluxgate magnetometer was determined to be moderately responsive to changes in magnetic field frequency below 10 Hz. At frequencies above 10 Hz the readings corresponded to that of the ambient static geofield. The practitioner operating the Toftness Sensometer was unable to detect magnetic fields at high frequencies (above 10 Hz) even at very high EMFs. The fluxgate magnetometer was shown to be essentially a DC/static magnetic field detector and like all such devices it has a limited frequency range with some low level of sensitivity at very low field frequencies. The interexaminer reliability of four Toftness practitioners using the Sensometer on 5 patients showed low to moderate correlation. CONCLUSIONS: The fluxgate magnetometer although highly sensitive to static (DC) EMFs has only limited sensitivity to EMFs in the range of 1 to 10 Hz and is very insensitive to frequencies above 10 Hz. In laboratory comparisons of the Sensometer and the fluxgate magnetometer there was an occasional correspondence between the two instruments in detecting magnetic fields within the Merritt coil but these occasions were not reproducible. In the clinical studies there was low to moderate agreement between the clinicians using the Sensometer to diagnosing spinal conditions and there was little if any agreement between the Sensometer and the fluxgate magnetometer in detecting EMFs emanating from the volunteers body surface.

Autoradiographic evaluation of electromagnetic field effects on serotonin (5HT1A) receptors in rat brain.

Serotonin (5HT1A) is a chemical mediator of inflammation and the largest single neurotransmitter system of the brain. Its secretion and physiological actions mediate stress and pain, affecting both immune and nervous system functions through the hypothalamic-pituitary-adrenal axis. Serotonin receptor dysfunction is well-characterized in mental disturbances like depression and anxiety. Transcranial magnetic stimulation has been used therapeutically to treat refractory disorders like non-responsive depression and may act in part through its effect on 5HT1A receptors. Previously we have shown that in vitro, 5HT1A receptor binding to a radioactive agonist can be modulated by specific intensity and frequency electromagnetic fields (EMFs). In the present report we have used quantitative receptor autoradiography to evaluate 5HT1A receptor density in rat brain and the impact of pulsed EMF exposure on receptor binding in key brain regions. Rats used in this study had whole body exposures to either a geofield control or to pulsed EMFs to evaluate the treatment for chemically-induced tendinitis. Since the brains were exposed coincidentally as a consequence of the main experiment, we investigated the potential for EMF-induced changes in areas such as the hippocampus. This pilot study should provide a detailed understanding of magnetic field effects on stress-responsive brain regions and will lead to a more coordinated approach to the use of such modalities for therapeutic intervention in humans.

Use of proteomics methodology to evaluate inflammatory protein expression in tendinitis.

In previous studies we established a rat model of acute tendinitis including functional and mechanical measures of healing. Achilles' tendinitis was induced by injection of collagenase, an enzyme that produces localized fiber digestion and edema formation. As quantitative measures of tissue inflammation, hypercellularity and edema were evaluated in injured tendons in comparison with controls. Using the rat tendinitis model, we have applied isotope-coded affinity tag analysis (ICAT) methodology to indicate localized tendon healing by quantitating protein expression. This novel proteomics method allows detection of subtle differences in protein levels that provide a detailed picture of tendinitis healing. The method involves a new class of chemical linkers used to differentially label cysteine residues from similar peptides in control and treated protein samples with heavy (deuterium off of backbone) and light (hydrogen off of backbone) ICAT reagents that are otherwise chemically identical. Proteins were extracted under liquid nitrogen from control untreated or injured Achilles' tendons 72 hours after collagenase-injection. These proteins were digested with endoproteinase Glu-C and trypsin and the resulting peptide mixtures were evaluated using reverse-phase C18 HPLC and Tristricine SDS-polyacrylamide gel electrophoresis. The two ICAT-modified peptide populations were mixed, affinity-purified and analyzed using microcapillary liquid chromatography and electrospray ionization tandem mass-spectroscopy. The process resulted in relative abundance and charge-to-mass ratio data used in conjunction with database searching to identify proteins expressed differentially in the two treatment groups. By analyzing different time periods in the healing process, an accurate model of the healing rat tendon can be made.

Characterization of a real time H2O2 monitor for use in studies on H2O2 production by antibodies and cells.

It was recently shown that antibodies catalyze a reaction between water and ultraviolet light (UV) creating singlet oxygen and ultimately H2O2. Although the in vivo relevance of these antibody reactions is unclear, it is interesting that among a wide variety of non-antibody proteins tested, the T cell receptor is the only protein with similar capabilities. In clinical settings UV is believed to exert therapeutic effects by eliminating inflammatory epidermal T cells and we hypothesized that UV-triggered H2O2 production is involved in this process. To test the hypothesis we developed tools to study production of H2O2 by T cell receptors with the long-term goal of understanding, and improving, UV phototherapy. Here, we report the development of an inexpensive, real time H2O2 monitoring system having broad applicability. The detector is a Clark oxygen electrode (Pt, Ag/AgCl) modified to detect UV-driven H2O2 production. Modifications include painting the electrode black to minimize UV effects on the Ag/AgCl electrode and the use of hydrophilic, large pore Gelnots electrode membranes. Electrode current was converted to voltage and then amplified and recorded using a digital multimeter coupled to a PC. A reaction vessel with a quartz window was developed to maintain constant temperature while permitting UV irradiation of the samples. The sensitivity and specificity of the system and its use in cell-free and cell-based assays will be presented. In a cellfree system, production of H2O2 by CD3 antibodies was confirmed using our real time H2O2 monitoring method. Additionally we report the finding that splenocytes and Jurkat T cells also produce H2O2 when exposed to UV light.

Human embryonic stem cell research: an ethical controversy in the US & Germany.

Stem cells refer to a broad class of cells with potential for prolonged self-renewal and the potential to differentiate. Human embryonic stem (ES) cells are derived from a fertilized human egg, before it has been implanted into a womb. Recently published research has used human embryos, produced by in-vitro fertilization for clinical purposes, to develop lines of pluripotent human ES cells. This review discusses the legal status and ethical implications of ES cell research with special attention paid to the situation in the USA and Germany.

Quantitative characterization of rat tendinitis to evaluate the efficacy of therapeutic interventions.

Tendinitis is a painful soft tissue pathology that accounts for almost half of all occupational injuries in the United States. It is often caused by repeated movements and may result in loss of work and income. Current treatments for tendinitis are aimed at reducing inflammation, the major cause of the pain. Although anti-inflammatory drugs and various alternative therapies are capable of improving tendinitis, there are no quantitative scientific data available regarding their impact on inflammation. The objective of this study is to determine the time course for healing of rat tendinitis without intervention to be able to assess the efficacy of tendinitis treatments. We are interested in evaluating the therapeutic use of pulsed electromagnetic fields (PEMFs), a therapeutic modality that has been found to be beneficial for healing soft tissue injuries. Tendinitis was induced in Harlan Sprague Dawley rats by collagenase injections into the Achilles tendon, and tendons were collected for four weeks post-injury. To determine the amount of edema, we used caliper measurements of the rat ankles and quantified the tendon water content. To determine the extent of inflammation, we estimated the number of inflammatory cells on histological sections applying stereological methods. The data reveal that edema is maximal 24 hours after injury accompanied by a massive infiltration of inflammatory cells. Inflammatory cells are then gradually replaced by fibroblasts, which are responsible for correcting damage to the extracellular matrix. This natural time course of tendon healing will be used to evaluate the use of PEMFs as a possible therapeutic modality.

Effect of short duration electromagnetic field exposures on rat mass.

Daily preexposure and postexposure mass measurements of 65 rats (young males and females, old males) a proprietary pulsed wound healing field, pulsed electromagnetic field, (PEMF), or their control fields for 4 h/day for 21 days. Statistical analysis of mass changes over time showed that young rats exposed to PEMF lost more mass and recovered it more slowly compared to controls (2-4% more loss) than did older PEMF exposed rats or any 60 Hz exposed rats. We conclude that daily preexposure and postexposure mass measurements are needed to adequately assess the effects of electromagnetic fields on body mass.