Anti-inflammatory activity of small-molecule antagonists of Toll-like receptor 2 (TLR2) in mice.

Toll-like receptor 2 (TLR2) is currently investigated as a potential therapeutic target in diseases with underlying inflammation like sepsis and arthritis. We reported the discovery, by virtual screening and biological testing, of eight TLR2 antagonists (AT1-AT8) which showed TLR2-inhibitory activity in human cells (Murgueitio et al., 2014). In this study, we have deepened in the mechanism of action and selectivity (TLR2/1 or TLR2/6) of those compounds in mouse primary cells and in vivo. The antagonists reduced, in a dose-dependent way the TNFα production (e.g. AT5 IC50 7.4 µM) and also reduced the nitric oxide (NO) formation in mouse bone marrow-derived macrophages (BMDM). Treatment of BMDM with the antagonists showed that downstream of TLR2, MAPKs phosphorylation and IkBα degradation was reduced. Notably, in a mouse model of tri-acylated lipopeptide (Pam3CSK4)-induced inflammation, AT5 attenuated the TNFα and IL-6 inflammatory response. Further, the effect of AT5 in the stimulation of BMDM by the endogenous alarmin HMGB1 was investigated. Our results indicate that AT4-AT7 and, particularly AT5 appear as good starting points for the development of inhibitors targeting TLR2 in inflammatory disorders.

Progressive loss of phasic, but not tonic, GABAA receptor-mediated inhibition in dentate granule cells in a model of post-traumatic epilepsy in rats.

Traumatic brain injury (TBI) is a risk factor for the development of epilepsy, which can occur months to years after the insult. The hippocampus is particularly vulnerable to the pathophysiological effects of TBI. Here, we determined whether there are long-term changes in inhibition in the dentate gyrus that could contribute to the progressive susceptibility to seizures after TBI. We used severe lateral-fluid percussion brain injury to induce TBI in rats. In this model, spontaneous seizure activity, which involves the hippocampus, appears after a long latent period, resembling the human condition. We demonstrate that synaptic GABA(A) receptor-mediated inhibition is profoundly reduced in ipsilateral dentate granule cells 1 month after TBI. Moreover, synaptic inhibition decreases over time, and by 6 months after TBI, it is also significantly decreased contralaterally. Progressive loss of synaptic inhibition is paralleled by a decline in the number of parvalbumin-positive interneurons, but, in contrast to status epilepticus models, GABA(A) receptor subunit expression is largely unaltered. At both time points, the magnitude of tonic GABA(A) receptor-mediated currents after TBI is maintained, indicating a preservation of the inhibitory constraint of granule cells through tonic inhibition. Our results extend the time window during which strategies to target epileptogenesis may be effective.

Parvalbumin interneurons and calretinin fibers arising from the thalamic nucleus reuniens degenerate in the subiculum after kainic acid-induced seizures.

The subiculum is the major output area of the hippocampus. It is closely interconnected with the entorhinal cortex and other parahippocampal areas. In animal models of temporal lobe epilepsy (TLE) and in TLE patients it exerts increased network excitability and may crucially contribute to the propagation of limbic seizures. Using immunohistochemistry and in situ-hybridization we now investigated neuropathological changes affecting parvalbumin and calretinin containing neurons in the subiculum and other parahippocampal areas after kainic acid-induced status epilepticus. We observed prominent losses in parvalbumin containing interneurons in the subiculum and entorhinal cortex, and in the principal cell layers of the pre- and parasubiculum. Degeneration of parvalbumin-positive neurons was associated with significant precipitation of parvalbumin-immunoreactive debris 24 h after kainic acid injection. In the subiculum the superficial portion of the pyramidal cell layer was more severely affected than its deep part. In the entorhinal cortex, the deep layers were more severely affected than the superficial ones. The decrease in number of parvalbumin-positive neurons in the subiculum and entorhinal cortex correlated with the number of spontaneous seizures subsequently experienced by the rats. The loss of parvalbumin neurons thus may contribute to the development of spontaneous seizures. On the other hand, surviving parvalbumin neurons revealed markedly increased expression of parvalbumin mRNA notably in the pyramidal cell layer of the subiculum and in all layers of the entorhinal cortex. This indicates increased activity of these neurons aiming to compensate for the partial loss of this functionally important neuron population. Furthermore, calretinin-positive fibers terminating in the molecular layer of the subiculum, in sector CA1 of the hippocampus proper and in the entorhinal cortex degenerated together with their presumed perikarya in the thalamic nucleus reuniens. In addition, a significant loss of calretinin containing interneurons was observed in the subiculum. Notably, the loss in parvalbumin positive neurons in the subiculum equaled that in human TLE. It may result in marked impairment of feed-forward inhibition of the temporo-ammonic pathway and may significantly contribute to epileptogenesis. Similarly, the loss of calretinin-positive fiber tracts originating from the nucleus reuniens thalami significantly contributes to the rearrangement of neuronal circuitries in the subiculum and entorhinal cortex during epileptogenesis.

Delayed luminescence of high homeopathic potencies on sugar globuli.

Delayed luminescence signals of Arg.met. CMf (100Mf), Canth. CMf, Bov. CMf absorbed onto sugar globuli was observed by exciting them at their known resonance frequency of 2.060 MHz. Arn. CMf also showed delayed luminescence when excited at 2.060 MHz and at 1.828 MHz. Alc. LMK (50MK) could not be excited by 2.060 MHz and showed properties of control globuli. Canth. LMK could not be excited at 2.006 MHz. The delayed luminescence signals were characterized by the coefficient B(2) typical of the delayed luminescence of non-living complex systems, and by the coefficient B(0) typical of living systems. Both coefficients can be considered as indicative of holistic quantum structures in homeopathic potencies.

Effect of colorpuncture on spontaneous photon emission in a subject suffering from multiple sclerosis.

Spontaneous photon signals from four sites of a human subject suffering from multiple sclerosis were detected in 3600 bins of 50 milliseconds by a photo multiplier sensitive in 160-630 nm, before and after a session of colorpuncture treatment. Measurements were made in 22 sessions over a period of 9 months. Each signal was analyzed to determine if it was a quantum signal in a squeezed state. The analysis first generates 10 signals of bin sizes (50 to 500 milliseconds at 50 millisecond intervals) by merging the counts at contiguous bins of the observed signal and then estimates three squeezed state parameters (r, theta and phi) in these ten signals and nine other combinations of signals. All estimations yield r=2.72.10(-10), theta = 101.91 degrees and phi = 69.53 degrees for TolX=10(-8) in every signal of a healthy subject. These are normal values of the parameters. Other values of parameters in a signal of any estimation indicate some ailment. The deviation from the squeezed state description of a signal is quantified by a new property, "coherency index", which appears to be a good indicator of health. A session of colorpuncture treatment changed coherency indices of signals from different sides and provided relief to the subject suffering from multiple sclerosis. The changes in coherency indices and relief were temporary. Changes in coherency indices lasting for longer periods occurred after many sessions of treatment.

Influence of hypoxia and of hypoxemia on the development of cardiac activity in zebrafish larvae.

Cardiac activity and anaerobic metabolism were analyzed in zebrafish larvae raised under normoxia (PO(2) = 20 kPa) and under chronic hypoxia (PO(2) = 10 kPa) at three different temperatures (25, 28, and 31 degrees C). Heart rate increased with development and with temperature. Under normoxia, cardiac output increased significantly at high temperature (31 degrees C), but not at 28 or at 25 degrees C. Under chronic hypoxia, however, heart rate as well as cardiac output increased at all temperatures in larvae at about hatching time or shortly thereafter. Cardiac activity of larvae raised for 2 wk after fertilization with a reduced hemoglobin oxygen-carrying capacity in their blood (hypoxemia; due to the presence of CO or of phenylhydrazine in the incubation water) was not different from control animals. Whole body lactate content of these animals did not increase. Thus there was no indication of a stimulated anaerobic energy metabolism. The increase in cardiac activity observed during hypoxia suggests that at about hatching time receptors are present that sense hypoxic conditions, and this information can be used to induce a stimulation of convective oxygen transport to compensate for a reduction in bulk oxygen diffusion in the face of a reduced oxygen gradient between environmental water and tissues. Under normoxia, however, the PO(2) gradient between environmental water and tissues and diffusional oxygen transport assure sufficient oxygen supply even if hemoglobin oxygen transport in the blood is severely impaired. Thus, under normoxic conditions and with a normal metabolic rate of the tissues, convective oxygen transport is not required until approximately 2 wk after fertilization.

Mode isolation: a new algorithm for modal parameter identification.

Multiple degree of freedom (MDOF) algorithms are the dominant methods for extracting modal parameters from measured data. These methods are founded on the notion that because the response of a linear dynamic system is the sum of many modal contributions, the extraction technique must deal with all of the modal parameters in a simultaneous fashion. The Mode Isolation Algorithm (MIA) described here is a frequency domain formulation that takes an alternative viewpoint. It extracts the modal parameters of each mode in an iterative search, and then refines the estimation of each mode by isolating its effect from the other modal contributions. The first iteration estimates modes in a hierarchy of their dominance. As each mode is estimated, its contribution is subtracted from the data set, until all that remains is noise. The second and subsequent iterations subtract the current estimates for all other modes to identify the properties of the mode under consideration. The various operations are described in detail, and then illustrated using data from a four-degree-of-freedom system that was previously used to assess the Eigensystem Realization Algorithm (ERA) and Enhanced ERA. Eigenvalues and mode shapes are compared for each algorithm. Another example analyzes simulated data for a cantilever beam with three suspended one-degree-of-freedom subsystems, in which the parameters are adjusted to bring two natural frequencies into close proximity. The results suggest that MIA is more accurate, and more robust in the treatment of noisy data, than either ERA version, and that it is able to identify modes whose bandwidth is comparable to the difference of adjacent natural frequencies.

[Epidemiologic studies of tinnitus aurium]. / Epidemiologische Untersuchungen zum Tinnitus Aurium.

The prevalence of tinnitus was examined in a group of 270 persons from an average cross-section of the population of Ulm. For statistical reasons we selected eight groups aged exactly 10, 20, 30, 40, 50, 60, 70, and 80 years. Thirty-one percent of those examined have already noted the presence of tinnitus at some point in time. Temporary tinnitus lasting not longer than 5 min. was reported in 19.5% and longer lasting tinnitus in 11.5% of cases. The highest incidence of 55% was found at the age of 20. The previously assumed age dependent increase in the incidence of tinnitus was seen only in the longer lasting type (5 min). The age dependent incidence of tinnitus was statistically correlated to audiogram, blood pressure, grade of physical activity, noise stress, and vertigo. The low incidence of tinnitus in presbyacusis (11%) differs from previous studies. Hypertension was associated with a lower incidence of tinnitus than normotension or hypotension.