Measurements of low oxygen tension in vitro and response of macrophages to levels applicable to peri-and postoperative treatment of traumatic brain injury.

Established clinical guidelines for treatment of severe traumatic brain injury aim at maintaining intracranial and cerebral perfusion pressures. Recently, it has been shown that additional regulation of cerebral oxygen delivery helps to decrease patient mortality and leads to improved 6-month quality-of-life scores. However, eubaric oxygen-guided therapy is still controversial since it is well known that hyperoxia can cause unwanted secondary brain injury. Research studies are warranted to better understand the range of oxygen pressures that positively influence brain cell behavior. We perform such studies using a two-enzyme in vitro system that allows exposing tissue culture cells to various steady-state, or rapidly changing, oxygen pressures. Here, we present a mathematical model of the system and its validation by real-time monitoring of oxygen tensions. We additionally present preliminary evidence that human brain macrophages have a different oxygen tolerance compared to systemic macrophages and propose improvements to our in vitro system to make it applicable for data collection that aim at refining oxygen-guided therapy for patients with traumatic brain injury.

Macrophages under low oxygen culture conditions respond to ion parametric resonance magnetic fields - biomed 2011.

Macrophages, when entering inflamed tissue, encounter low oxygen tension due to the impairment of blood supply and/or the massive infiltration of cells that consume oxygen. Previously, we showed that such macrophages release more bacteriotoxic hydrogen peroxide (H2O2) when exposed in vitro to low oxygen than when cultured at usual ambient oxygen conditions. In this study, we use this low-oxygen, inflammatory macrophage model to test the macrophages’ response to low-frequency magnetic fields. Low-frequency fields are clinically used for bone and wound healing and are emerging as therapy for inflammatory diseases. The acceptance of these non-invasive therapies is slow due to the lack of knowledge of the cellular targets for magnetic fields. One possible target is biologically relevant ions. The Ion Parametric Resonance (IPR) concept predicts that specific externally applied AC and DC magnetic fields will resonate with the cyclotron motion of ions. This concept is supported by experimental evidence, especially on a neuronal cell line. Using our macrophage model, we tested AC and DC magnetic fields at the amplitude ratio and frequency predicted by the IPR model for resonance with hydrogen, magnesium and manganese ions. Under these conditions, we found a significant increase in H2O2 release compared to control cells. Magnetic field exposure conditions in which parameters differed from the predictions of the IPR model showed no, or a smaller difference, with respect to the control cultures. These data indicate that magnetic fields can enhance H2O2 release of inflammatory macrophages, which is consistent with the predictions of the IPR model.

Macrophage activity in response to steady-state oxygen and hydrogen peroxide concentration - biomed 2010.

Macrophages are important players of the immune system to fight infections since they eliminate microorganisms. During this process they often encounter a wide range of oxygen concentrations, from roughly 13% O2 (PO2 = 760 x 0.13 mmHg) in arterial blood to less than 1% O2 in inflamed tissue. Macrophages contribute to the elimination of microorganisms by releasing oxygen derivates such as hydrogen peroxide. The objective of this study was to test macrophage activity under various O2 and H2O2 concentrations such as present during infection. We exposed macrophages to steady-state O2 concentrations between 21% and 1% O2 and steady-state H2O2 concentrations between 0 and 20 M using a novel enzymatic system. The system uses glucose oxidase (Gox) and catalase (Cat) in standard, open-system, cell culture vessels. Macrophage activity was determined as change in phagocytosis and as release of antimicrobial H2O2 into extracellular medium. We show that O2 concentrations below 7% enhance the activity of macrophages of the THP-1 cell line in a dose-dependent way; with doubled activity at 1% O2 compared to 21% O2 conditions. We further show that macrophages are able to function in an environment of high H2O2concentrations and are even stimulated by H2O2 concentrations below 20 M. The activity of hypoxic macrophages is up to 3-fold enhanced in the presence of H2O2 as compared to the activity triggered by low O2 conditions alone. Our data show that hypoxia and H2O2, as present in infectious conditions, strongly enhance macrophage activity. The data further demonstrate the usefulness and versatility of the Gox/Cat system for studies of infectious and inflammatory diseases.

Monitoring temperature and light exposure of biosamples exposed to ultraviolet and low energy radiation.

We previously showed that T-lymphocytes produce catalytic amounts of hydrogen peroxide (H2O2) in a membrane-associated process when irradiated with narrowband ultraviolet B (UVB) light. This form of phototherapy is thought to be highly effective for treatment of inflammatory skin diseases such as psoriasis, but also includes the potential for severe burning and development of skin cancer. Consequently, information on the therapeutic mechanism of narrowband UVB phototherapy and its regulation is warranted. Our laboratory is researching the mechanistic involvement of T-cell H2O2 production and its potential regulation by low energy electromagnetic field (EMF) radiation, which has been shown to beneficially influence inflammatory diseases such as psoriasis. To study photochemical H2O2 production in small samples such as suspensions of T-lymphocyte cell extracts, we use a reactor in which 12 microliter-sized samples are exposed to UVB. We simultaneously operate two identical systems, one for experimental, the other for control samples, within a walk-in environmental chamber maintained at 37 degrees C. The current paper addresses the control of UVB light exposure and temperature in our experimental setup. We quantified UVB light b y radiometric sp ot measurements and by chemical potassium ferrioxalate actinometry. We modified the actinometer so that UVB light of 5-hour experiments could be detected. Temperature was controlled by air convection and remained constant within 0.5 degrees C in air and liquid samples. Preliminary data of the effect of low energy EMF radiation on T-cell H2O2 production are presented.

Quantitative studies on biological water oxidation: a novel mechanism of T cells and antibodies.

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.

Biophotonic hydrogen peroxide production by antibodies, T cells, and T-cell membranes.

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.

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.

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.

Investigation of the relationship between arch height and maximum pronation angle during running.

A three-dimensional motion analysis study is performed on 8 subjects running on a treadmill. Medical and lateral longitudinal arch height is measured from a weight-bearing x-ray. A statistical analysis is performed to search for a relationship between arch height and maximum angle of pronation. The data analysis showed a significant relationship (sig. level = 0.0252) between the medial longitudinal arch height and the maximum eversion angle. The data analysis also showed a significant relationship between the lateral longitudinal arch height and the maximum eversion angle (sig. level = 0.0358).