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.