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Prior studies show that purified T cell receptors (TCRs) and antibodies catalyze the oxidation of water to H2O2 in the presence of singlet oxygen, but the comparative efficiencies of TCRs and antibodies in this process have not been reported. Since H2O2 has been shown to activate TCRs and selectively regulate redox sensitive TCR signaling pathways, it is important to understand the physiological significance of these recently uncovered processes. This new information might be used to develop new therapeutic tools for immune and inflammatory diseases. In this paper, we present data showing that under equivalent conditions Jurkat T cell membranes produce H2O2 at a rate of 457 pM/min/mg protein/muW/cm2 while IgG antibodies produce H2O2 at a rate of 192 pM/min/mg protein/muW/cm2. Taking into account the number of catalytic sites in a milligram of T cell membranes and IgGs, we calculate that TCRs catalyze H2O2 production at a specific rate that is about 10(6) times greater than the rate of IgGs. Based on these observations and calculations, we conclude that the comparatively high rate of H2O2 production by TCRs makes it more likely that this is a physiologically relevant process than the H2O2 production by IgGs. In addition, the catalytic rate for H2O2 production by TCRs is comparable to the rates of other physiologically important processes, such as catalysis by enzymes. This suggests that singlet oxygen-dependent, TCR mediated, H2O2 production is likely to be physiologically important, perhaps as H2O2 being a small molecule regulator of TCR signal transduction or a modulator of T cell gene transcription.

Rapidly accumulating evidence indicates that inflammatory T cells sensitively respond to their redox environment by activating signal transduction pathways. The hypothesis that T-cell receptors have the potential to catalytically transform singlet oxygen into H(2)O(2) attracted our attention since the biophysical regulation of this process would provide a new tool for therapeutically directing T cells down a preferred signaling pathway. Light-dependent production of H(2)O(2) was first described in antibodies, and we reproduced these findings. Using a real-time H(2)O(2) sensor we extended them by showing that the reaction proceeds in a biphasic way with a short-lived phase that is fast compared to the slow second phase of the reaction. We then showed that Jurkat T cells biophotonically produce about 30nM H(2)O(2)/min/mg protein when pretreated with NaN(3). This activity was concentrated 4 to 5 times in T-cell membrane preparations. The implications of these observations for the development of new therapeutic tools for inflammatory diseases are discussed.

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.

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.

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.

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., (Copyright 2002 Wiley-Liss, Inc.)

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., (Copyright 2002 Wiley-Liss, Inc.)

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.

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 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.

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.

This report outlines a simple mechanism, based on the Hall Effect, by which static and low frequency (50-60 Hz) pulsed electromagnetic fields (PEMFs) can modify cation flow across biological membranes and alter cell metabolism. We show that magnetic fields commonly found in the environment can be expected to cause biologically significant interactions between transported cations and basic domains of cation channel proteins. We calculate that these interactions generate forces of a magnitude similar to those created by normal transmembrane voltage changes known to gate cation channels. Thus PEMFs are shown to have the potential of regulating flow through cation channels, changing the steady state concentrations of cellular cations and thus the metabolic processes dependent on cation concentrations.

Malonaldehyde formed by lipid oxidation is regarded as a main crosslinker in the formation of natural age pigment. To elucidate the mechanism of pigment formation the reaction of malonaldehyde with biomembranes using fluorescence spectroscopy has been studied. Rat liver mitochondrial ghosts or bovine serum albumin were reacted with malonaldehyde. In both cases two main fluorescence changes were observed: protein fluorescence decreased to 50% of its initial value in about two hours; aminoiminopropene fluorescence reached a maximum at a much slower rate. The kinetics support a two-step reaction hypothesis. First, malonaldehyde reacts with protein quenching its fluorescence. Next fluorescent interprotein aminoiminopropene (AIP) crosslinks are formed. The fluorescence lifetime value of the induced AIP fluorophore was shown to be similar to the lifetime of naturally occurring age pigment previously reported for mitochondrial ghosts prepared from aged animals (5.4 ns +/- 0.3 and 5.9 ns +/- 0.6, respectively).

Mitochondrial DNA (mtDNA) was isolated from liver mitochondria of rats between 2 and 24 months of age. The mtDNA was purified by cesium chloride--ethidium bromide isopycnic density gradient centrifugation. In the gradients, in addition to the two expected bands of ethidium--DNA complex, there was observed a third, more dense band (d = 1.69 g/cm3). This novel band, rarely observed in preparations from younger animals, was present in most preparations from older animals. The latter was characterized using the diphenylamine assay(s) and ascertained to contain DNA and carbohydrate components. Agarose gel electrophoresis revealed the DNA of the novel band to have a migration identical to form I mtDNA. Digestion of the novel band with the restriction endonuclease Bam HI yielded products identical to those obtained upon treatment of form I mtDNA with Bam HI. The observation of mtDNA at a density of 1.69 g/cm3 indicates the presence, predominantly in older animals, of a subclass of mtDNA molecules with altered ethidium binding properties. The significance of this mtDNA and its position in the gradient is unclear at this time.