The onset risk of carcinoma in patients continuing tacrolimus topical treatment for oral lichen planus: a case report.

Oral lichen planus is a chronic inflammatory mucocutaneous disease. Topical use of steroids and other immuno-modulating therapies have been tried for this intractable condition. Nowadays, tacrolimus ointment is used more commonly as a choice for treatment. However, a number of discussions have taken place after tacrolimus was reported to be carcinogenic. This report describes a patient who applied tacrolimus ointment to the lower lip after being diagnosed with oral lichen planus in 2008, and whose lesion developed squamous cell carcinoma in 2010. Since the relationship between tacrolimus and cancer development has been reported in only a few cases, including this case report, the clinician must be careful selecting tacrolimus as a second-line treatment for oral lichen planus.

Reactive oxygen species generation in gingival fibroblasts of Down syndrome patients detected by electron spin resonance spectroscopy.

Oral manifestations of Down syndrome include high susceptibility to gingival inflammation with early onset and rapidly progressive periodontitis. The influence of reactive oxygen species (ROS) on periodontitis of Down syndrome is unclear. The aim of this study was to characterize ROS formation in Down syndrome-gingival fibroblasts (DS-GF) using electron spin resonance (ESR) spin trapping with 5,5-dimetyl-1-pyrolline-N-oxide (DMPO), and to determine whether ROS generation plays a role in the pathogenesis of periodontitis in Down syndrome patients. We observed formation of the DMPO-OH spin adduct, indicating HO* generation from cultured DS-GF and non-DS-GF. The increased HO* generation in cultured DS-GF was strongly decreased in the presence of the H2O2 scavenger, catalase, or the iron chelator, desferal. This may due to the enzymatic ability of over-expressed CuZn-superoxide dismutase in Down syndrome to catalyze the formation of H2O2 from O2*-, thereby increasing the availability of substrate H2O2 for the iron-dependent generation of HO* via the Fenton reaction, suggesting that HO* generated from DS-GF may be involved in progressive periodontitis of Down syndrome.

Biting reduces acute stress-induced oxidative stress in the rat hypothalamus.

We investigated the inhibitory effect of para-masticatory activity, namely biting, on restraint stress-induced oxidative stress. A blood brain barrier-permeable nitroxyl spin probe, 3-methoxycarbonyl-2,2,5,5,-tetramethylpyrrolidine-1-oxyl (MC-PROXYL), was administered to rats and L-band electron spin resonance (ESR) and ESR-computerized tomography (ESR-CT) imaging were used to show that the decay rate constant of MC-PROXYL in the hypothalamus of isolated brain after 30 min of restraint stress was more rapid than in unrestrained control rats, suggesting that restraint was associated with oxidative stress. Interestingly, biting during restraint stress caused the decay rate constant of MC-PROXYL in isolated brain to approach that of the control group. These observations suggest that biting suppresses oxidative stress induced by restraint stress, and that the anti-stress effect of masticatory motor activity movements, such as biting, are important for reducing the adverse effects associated with exposure to psychological stressors.

Assessment of oxidative stress in the spontaneously hypertensive rat brain using electron spin resonance (ESR) imaging and in vivo L-Band ESR.

This study examined the blood brain barrier (BBB)-permeable nitroxyl compound, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (MC-PROXYL), as a spin probe for the assessment of oxidative stress in the brain by electron spin resonance (ESR) imaging and in vivo L-band ESR. Preliminary comparisons were made by ESR imaging of MC-PROXYL in the isolated brains of normal Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and stroke prone SHR (SHRSP). The decay of the ESR images of MC-PROXYL in the isolated brains was faster in SHR than in normal WKY, but was only moderate in SHRSP. In addition, the decay rate of MC-PROXYL in the heads of live rats, as measured noninvasively by L-band ESR, was faster in SHR than in WKY, and was slower in SHR than in SHRSP. Taken together, our data suggest that the oxidative stress of SHR is not as high as that in high oxidative stress animal models such as SHRSP. This is the first study to present reconstructed 3D images of the distribution of MC-PROXYL in the isolated SHR brain. The ESR technique employed herein appears to be a powerful tool for evaluating oxidative stress and for detecting the region of oxidative stress in the brain of SHR.

Evidence of reactive oxygen species generation in synovial fluid from patients with temporomandibular disease by electron spin resonance spectroscopy.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of temporomandibular disorders. In the present study, we provide the first evidence of ROS generation in the synovial fluid from human temporomandibular disorder patients, as shown by electron spin resonance (ESR) and spin trapping. Three distinct ESR spectra of DMPO spin adducts were observed in the synovial fluid. They corresponded to three free radical species: hydroxyl radical (HO(*)), hydrogen radical (H(*)), and carbon-center radical (R(*)). Among them, the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OH spectrum was the most prominent, suggesting that HO(*) was dominantly generated in the synovial fluid from temporomandibular disorder patients. Desferrioxamine (DFO), an iron chelator, strongly depressed the DMPO-OH signal intensity in the synovial fluid from patients with temporomandibular disorders. We successfully demonstrated ROS-induced oxidative stress in the synovial fluid from temporomandibular disorder patients. ROS generation in the temporomandibular joint could lead to exacerbation of inflammation and activation of cartilage matrix degrading enzymes that proceed to degenerative change of the temporomandibular joint. Thus, iron-dependent generation of HO( *) might have a crucial role in the pathogenesis of temporomandibular disorders.

Assessment of ESR-CT imaging by comparison with autoradiography for the distribution of a blood-brain-barrier permeable spin probe, MC-PROXYL, to rodent brain.

Blood-brain-barrier (BBB)-permeable, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-yloxy (MC-PROXYL) and BBB-impermeable carbamoyl-PROXYL were used to assess the ESR imaging technique by comparing with autoradiography. For this purpose, spin probes, 14C-labeled at their five-membered ring, [14C]MC-PROXYL and [14C]carbamoyl-PROXYL, were newly synthesized. These probes were i.p. or i.v. injected into rats and autoradiograms were recorded. The autoradiograms of rat head showed that [14C]MC-PROXYL distributed well in the brain compared to [14C]carbamoyl-PROXYL. In vivo ESR spectra and two-dimensional ESR images of isolated rat brain treated with MC- or carbamoyl-PROXYL also indicated the extensive distribution of MC-PROXYL but not carbamoyl-PROXYL in the rat brain. The three-dimensional ESR images of the head of rats and mice were consistent with the fact that MC-PROXYL but not carbamoyl-PROXYL is incorporated into the brain. The ESR-CT images were better for mice than rats. However, the quality of the ESR-CT images was still not satisfactory. Although the resolution and sensitivity of the ESR-CT images were worse than those of the autoradiographic images, the former technique has unique features and advantages; e.g., functional, noninvasive and three-dimensional detection.

Real-time monitoring of nitric oxide in ischemic myocardium using an NO-selective electrode calibrated by electron spin resonance.

Using a Langendorff-perfused rat heart preparation and selective electrodes, we determined nitric oxide (NO) and oxygen levels in cardiac tissue. An NO-selective electrode that was calibrated by electron spin resonance (ESR) spectroscopy was inserted into the middle of the myocardium in the left ventricle. Simultaneously, we used an O2-selective electrode to measure the partial pressure of oxygen (pO2) in the perfusate, Krebs-Henseleit (K-H) solution, that was ejected from the heart. After 30 min of aerobic control perfusion, hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. Under ischemic conditions, with a gradually decreasing pO2, NO detected by an NO-sensitive electrode within the myocardium was gradually increased. The maximum concentration increases in NO and decreases in pO2 during global ischemia were +10.200 +/- 1.223 microM and -58.608 +/- 4.123 mmHg, respectively. NO and pO2 levels both recovered to pre-ischemia baseline values when perfusion was restarted after global ischemia (reperfusion). The presence of Nomega-nitro-L-arginine methyl ester (L-NAME, 10 mM), a NOS inhibitor, prevented ischemia/reperfusion-induced changes in NO. This study shows that an NO-selective electrode that is calibrated by ESR can provide accurate, real-time monitoring of cardiac NO in normal and ischemic myocardium.

Measurement of oxidative stress in the rodent brain using computerized electron spin resonance tomography.

The strategy of this study was to improve the electronic spin resonance (ESR) application used to detect free radical-induced oxidative stress in animal models. We have developed an in vivo ESR imaging system with high-quality ESR-computed tomography (CT) images by using a nitroxyl spin probe--BBB-permeable, 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-yloxy (MC-PROXYL)--in living small animals. We first measured the distribution of MC-PROXYL in the head region of a living mouse by using ESR-CT imaging after treatment with MC-PROXYL. In the ESR-CT experiments, it was clearly observed that MC-PROXYL was well distributed in the brain of head region of a living mouse. The ESR-CT images taken after treatment with MC-PROXYL demonstrate that the decay of MC-PROXYL in the isolated brain of a spontaneously hypertensive rat (SHR) was more rapid than that in a Wister Kyoto rat. ESR-CT analysis revealed that the region of rapid decay might be located in the cerebral cortex of the isolated brain of an SHR. These results suggest that the present study of ESR-CT imaging would be a useful tool for monitoring and detecting the locations of oxidative stress in the brains of rodent animal models.

Interactions between Lactobacillus kefiranofaciens and Saccharomyces cerevisiae in mixed culture for kefiran production.

Since a positive effect on the growth and kefiran production of Lactobacillus kefiranofaciens was observed in a mixed culture with Saccharomyces cerevisiae, the elucidation of the interactions between L. kefiranofaciens and S. cerevisiae may lead to higher productivity. Hence, the microbial interaction of each strain was investigated. Apart from the positive effect of a reduction in the amount of lactic acid by S. cerevisiae, a positive effect of S. cerevisiae on the growth and kefiran production of L. kefiranofaciens in a mixed culture was observed. Various experiments were carried out to study this effect. In this study, the observed increase in capsular kefiran in a mixed culture with inactivated S. cerevisiae correlated well to that in an anaerobic mixed culture. Differences in capsular kefiran production were observed for different initial S. cerevisiae concentrations under anaerobic conditions. From these fermentation results, it was concluded that the physical contact with S. cerevisiae mainly enhanced the capsular kefiran production of L. kefiranofaciens in a mixed culture. Therefore, in an anaerobic mixed culture, this direct contact resulted in higher capsular kefiran production than that in pure culture.

Measurement of oxidative stress in stroke-prone spontaneously hypertensive rat brain using in vivo electron spin resonance spectroscopy.

A number of researchers have reported that free radicals generated in the brain are involved in various brain dysfunctions, including ischemia-reperfusion injury, brain tumors, and neurodegenerative diseases. It has been reported that the spin probe MC-PROXYL can penetrate the blood-brain barrier and can be useful for evaluating oxidative stress in the brain. Preliminary comparisons were made by ESR imaging of the heads of live mice and isolated rat brains using the spin probe MC-PROXYL and the blood-brain-barrier impermeable probe carbamoyl-PROXYL. The results showed that MC-PROXYL, but not carbamoyl-PROXYL, was widely distributed in the brain. These methods were also applied for the imaging of brains from stroke-prone spontaneously hypertensive rats (SHRSPs). The rapid decay of 2D ESR images of MC-PROXYL in isolated SHRSP-brain was observed, compared to Wistar-Kyoto rats (WKYs), using the ESR imaging system. Furthermore, we provide evidence, by using L-band ESR non-invasively, that the decay rate of MC-PROXYL in the head region is faster in live SHRSPs than in live WKYs. Taken together, the high oxidative stress sustained by oxygen radical generation in SHRSPs may cause the alteration of MC-PROXYL metabolism in the brain. Our results suggest that in vivo ESR could be applied to the assessment of antioxidant effects on oxidative stress in the brain in animal disease models, such as the SHRSP.

Inhibition of superoxide generation from fMLP-stimulated leukocytes by high concentrations of nitric oxide or peroxynitrite: characterization by electron spin resonance spectroscopy.

This present study examined the effects of high concentrations of nitric oxide (NO*) and peroxynitrite (ONOO-) on superoxide (O2*-) production from formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated polymorphonuclear leukocytes (PMNs) by using electron spin resonance (ESR) and spin trapping with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO). We demonstrated that ONOO- (100 microM) decreased the ESR signal of DEPMPO-OOH from fMLP-activated PMNs, indicating the inhibition of O2*- generation, while it enhanced the signal of DEPMPO-OH. Inhibition of the respiratory burst was also observed when PMNs were pre-exposed to high concentrations of NO* (100 microM), generated by the NO* donor NOR-1, 30 min prior to stimulation with fMLP. NOR-1 inhibited O2*- generation more effectively under conditions in which ONOO-was formed concurrently. The ability of high concentrations of either ONOO- or NO* to inhibit O2*-generation from fMLP-stimulated PMNs is relevant to pathophysiological conditions, such as severe inflammation, in which NO* or ONOO- production can be significantly elevated.

Vascular effects of singlet oxygen (1O2) generated by photo-excitation on adrenergic neurotransmission in isolated rabbit mesenteric vein.

It has been suggested that reactive oxygen species (ROS) may be involved in the regulation of vascular tone. However, the nature of ROS effects on vascular sensitivity remains to be elucidated. The present study was designed to investigate the effects of ROS, especially 1O2, on neurotransmission at the sympathetic neurovascular junction. Basal noradrenaline (NA) release, release of NA induced by electrical stimulation (ES), and resting NA release at the sympathetic nerve terminals were determined using a superfusion technique. The amount of NA was determined by HPLC; isometric tension changes evoked by ES were also recorded simultaneously. 1O2 was generated from Rose Bengal by photo-activation. The generation of 1O2 in the superfusate was monitored by electron spin resonance (ESR) using the spin trap 2,2,6,6-tetramethyl-4-piperidinol throughout the experimental time course. The ESR results confirmed that 1O2 was generated by photo-activation of Rose Bengal via the formation of 2,2,6,6-tetramethyl-4-hydroxyl-piperidinyloxy. Exposure of helical strips of rabbit mesenteric vein to 1O2 induced a significant increase in tension and NA release during the basal period, but had no effect on ES-induced release. L-histidine, an 1O2 scavenger, significantly inhibited the observed effects on vascular tension and NA release in response to 1O2. These results suggest that 1O2 may induce NA-mediated vasoconstriction at the postjunctional site, and may be associated with Ca(2+)-independent NA release from the prejunctional site of adrenergic neurotransmission.