A dominant function of p38 mitogen-activated protein kinase signaling in receptor activator of nuclear factor-κB ligand expression and osteoclastogenesis induction by Aggregatibacter actinomycetemcomitans and Escherichia coli lipopolysaccharide

Lipopolysaccharide from gram-negative bacteria is one of the microbial-associated molecular patterns that initiate the immune/inflammatory response, leading to the tissue destruction observed in periodontitis. The aim of this study was to evaluate the role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in lipopolysaccharide-induced receptor activator of nuclear factor-κB ligand (RANKL) expression by murine periodontal ligament cells. Material and Methods:  Expression of RANKL and osteoprotegerin mRNA was studied by reverse transcription-polymerase chain reaction upon stimulation with lipopolysaccharide from Escherichia coli and Aggregatibacter actinomycetemcomitans . The biochemical inhibitor SB203580 was used to evaluate the contribution of the p38 MAPK signaling pathway to lipopolysaccharide-induced RANKL and osteoprotegerin expression. Stable cell lines expressing dominant-negative forms of MAPK kinase (MKK)-3 and MKK6 were generated to confirm the role of the p38 MAPK pathway. An osteoclastogenesis assay using a coculture model of the murine monocytic cell line RAW 264.7 was used to determine if osteoclast differentiation induced by lipopolysaccharide-stimulated periodontal ligament was correlated with RANKL expression. Results:  Inhibiting p38 MAPK prior to lipopolysaccharide stimulation resulted in a significant decrease of RANKL mRNA expression. Osteoprotegerin mRNA expression was not affected by lipopolysaccharide or p38 MAPK. Lipopolysaccharide-stimulated periodontal ligament cells increased osteoclast differentiation, an effect that was completely blocked by osteoprotegerin and significantly decreased by inhibition of MKK3 and MKK6, upstream activators of p38 MAPK. Conditioned medium from murine periodontal ligament cultures did not increase osteoclast differentiation, indicating that periodontal ligament cells produced membrane-bound RANKL. Conclusion:  Lipopolysaccharide resulted in a significant increase of RANKL in periodontal ligament cells. The p38 MAPK pathway is required for lipopolysaccharide-induced membrane-bound RANKL expression in these cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65788/1/j.1600-0765.2007.01013.x.pd

Current concepts in periodontal bioengineering

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73112/1/j.1601-6343.2005.00352.x.pd

Human cultured dendritic cells show differential sensitivity to chemotherapy agents as assessed by the MTS assay

Assessment of the chemosensitivity of dendritic cells (DC) may allow more rational development of combined chemotherapy and immunotherapy protocols. Human monocyte-derived DC generated reproducible results in the MTS (Owen’s reagent) assay, which was then used to study DC survival after treatment with four different chemotherapy agents. DC preparations from three different donors were used per drug. DC were sensitive to doxorubicin (concentration range 0.1–50 μM) with variation in sensitivity between donors (IC50 244–1100 nM). The most extreme variation was seen for vinblastine (concentration range 250–0.025 μM with IC50 0.15–17.25 μM). In contrast, there was relative resistance to etoposide (concentration range 0.2–200 μM) and 5-fluorouracil (concentration range 0.7–7700 μM) with no toxicity seen until 50 μM and 770 μM respectively. The function of DC in allogeneic mixed leucocyte reactions closely paralleled results from the MTS assays. The differential sensitivity to chemotherapy agents did not appear to be due to expression of P-glycoprotein. These results suggest that etoposide or 5-fluorouracil is less likely to reduce the immunotherapeutic potential of DC and may be valuable in the design of prodrug activation therapy. © 1999 Cancer Research Campaig

Cytolytic DNA vaccine encoding lytic perforin augments the maturation of- and antigen presentation by- dendritic cells in a time-dependent manner

The use of cost-effective vaccines capable of inducing robust CD8+ T cell immunity will contribute significantly towards the elimination of persistent viral infections and cancers worldwide. We have previously reported that a cytolytic DNA vaccine encoding an immunogen and a truncated mouse perforin (PRF) protein significantly augments anti-viral T cell (including CD8+ T cell) immunity. Thus, the current study investigated whether this vaccine enhances activation of dendritic cells (DCs) resulting in greater priming of CD8+ T cell immunity. In vitro data showed that transfection of HEK293T cells with the cytolytic DNA resulted in the release of lactate dehydrogenase, indicative of necrotic/lytic cell death. In vitro exposure of this lytic cell debris to purified DCs from naïve C57BL/6 mice resulted in maturation of DCs as determined by up-regulation of CD80/CD86. Using activation/proliferation of adoptively transferred OT-I CD8+ T cells to measure antigen presentation by DCs in vivo, it was determined that cytolytic DNA immunisation resulted in a time-dependent increase in the proliferation of OT-I CD8+ T cells compared to canonical DNA immunisation. Overall, the data suggest that the cytolytic DNA vaccine increases the activity of DCs which has important implications for the design of DNA vaccines to improve their translational prospects.Danushka K. Wijesundara, Wenbo Yu, Ben J. C. Quah, Preethi Eldi, John D. Hayball, Kerrilyn R. Diener, Ilia Voskoboinik, Eric J. Gowans, and Branka Grubor-Bau

Tinnitus Intensity Dependent Gamma Oscillations of the Contralateral Auditory Cortex

Non-pulsatile tinnitus is considered a subjective auditory phantom phenomenon present in 10 to 15% of the population. Tinnitus as a phantom phenomenon is related to hyperactivity and reorganization of the auditory cortex. Magnetoencephalography studies demonstrate a correlation between gamma band activity in the contralateral auditory cortex and the presence of tinnitus. The present study aims to investigate the relation between objective gamma-band activity in the contralateral auditory cortex and subjective tinnitus loudness scores. In unilateral tinnitus patients (N = 15; 10 right, 5 left) source analysis of resting state electroencephalographic gamma band oscillations shows a strong positive correlation with Visual Analogue Scale loudness scores in the contralateral auditory cortex (max r = 0.73, p<0.05). Auditory phantom percepts thus show similar sound level dependent activation of the contralateral auditory cortex as observed in normal audition. In view of recent consciousness models and tinnitus network models these results suggest tinnitus loudness is coded by gamma band activity in the contralateral auditory cortex but might not, by itself, be responsible for tinnitus perception

Impact of leg lengthening on viscoelastic properties of the deep fascia

<p>Abstract</p> <p>Background</p> <p>Despite the morphological alterations of the deep fascia subjected to leg lengthening have been investigated in cellular and extracellular aspects, the impact of leg lengthening on viscoelastic properties of the deep fascia remains largely unknown. This study aimed to address the changes of viscoelastic properties of the deep fascia during leg lengthening using uniaxial tensile test.</p> <p>Methods</p> <p>Animal model of leg lengthening was established in New Zealand white rabbits. Distraction was initiated at a rate of 1 mm/day and 2 mm/day in two steps, and preceded until increases of 10% and 20% in the initial length of tibia had been achieved. The deep fascia specimens of 30 mm × 10 mm were clamped with the Instron 1122 tensile tester at room temperature with a constant tensile rate of 5 mm/min. After 5 load-download tensile tests had been performed, the specimens were elongated until rupture. The load-displacement curves were automatically generated.</p> <p>Results</p> <p>The normal deep fascia showed typical viscoelastic rule of collagenous tissues. Each experimental group of the deep fascia after leg lengthening kept the properties. The curves of the deep fascia at a rate of 1 mm/day with 20% increase in tibia length were the closest to those of normal deep fascia. The ultimate tension strength and the strain at rupture on average of normal deep fascia were 2.69 N (8.97 mN/mm²) and 14.11%, respectively. The increases in ultimate tension strength and strain at rupture of the deep fascia after leg lengthening were statistically significant.</p> <p>Conclusion</p> <p>The deep fascia subjected to leg lengthening exhibits viscoelastic properties as collagenous tissues without lengthening other than increased strain and strength. Notwithstanding different lengthening schemes result in varied viscoelastic properties changes, the most comparable viscoelastic properties to be demonstrated are under the scheme of a distraction rate of 1 mm/day and 20% increase in tibia length.</p

The reference frame for encoding and retention of motion depends on stimulus set size

YesThe goal of this study was to investigate the reference frames used in perceptual encoding and storage of visual motion information. In our experiments, observers viewed multiple moving objects and reported the direction of motion of a randomly selected item. Using a vector-decomposition technique, we computed performance during smooth pursuit with respect to a spatiotopic (nonretinotopic) and to a retinotopic component and compared them with performance during fixation, which served as the baseline. For the stimulus encoding stage, which precedes memory, we found that the reference frame depends on the stimulus set size. For a single moving target, the spatiotopic reference frame had the most significant contribution with some additional contribution from the retinotopic reference frame. When the number of items increased (Set Sizes 3 to 7), the spatiotopic reference frame was able to account for the performance. Finally, when the number of items became larger than 7, the distinction between reference frames vanished. We interpret this finding as a switch to a more abstract nonmetric encoding of motion direction. We found that the retinotopic reference frame was not used in memory. Taken together with other studies, our results suggest that, whereas a retinotopic reference frame may be employed for controlling eye movements, perception and memory use primarily nonretinotopic reference frames. Furthermore, the use of nonretinotopic reference frames appears to be capacity limited. In the case of complex stimuli, the visual system may use perceptual grouping in order to simplify the complexity of stimuli or resort to a nonmetric abstract coding of motion information

HP1 Recruits Activity-Dependent Neuroprotective Protein to H3K9me3 Marked Pericentromeric Heterochromatin for Silencing of Major Satellite Repeats

H3 lysine 9 trimethylation (H3K9me3) is a histone posttranslational modification (PTM) that has emerged as hallmark of pericentromeric heterochromatin. This constitutive chromatin domain is composed of repetitive DNA elements, whose transcription is differentially regulated. Mammalian cells contain three HP1 proteins, HP1α, HP1β and HP1γ These have been shown to bind to H3K9me3 and are thought to mediate the effects of this histone PTM. However, the mechanisms of HP1 chromatin regulation and the exact functional role at pericentromeric heterochromatin are still unclear. Here, we identify activity-dependent neuroprotective protein (ADNP) as an H3K9me3 associated factor. We show that ADNP does not bind H3K9me3 directly, but that interaction is mediated by all three HP1 isoforms in vitro. However, in cells ADNP localization to areas of pericentromeric heterochromatin is only dependent on HP1α and HP1β. Besides a PGVLL sequence patch we uncovered an ARKS motif within the ADNP homeodomain involved in HP1 dependent H3K9me3 association and localization to pericentromeric heterochromatin. While knockdown of ADNP had no effect on HP1 distribution and heterochromatic histone and DNA modifications, we found ADNP silencing major satellite repeats. Our results identify a novel factor in the translation of H3K9me3 at pericentromeric heterochromatin that regulates transcription