Visualization of Pulpal Structures by SWIR in Endodontic Access Preparation.

Endodontic access preparation is one of the initial steps in root canal treatments and can be hindered by the obliteration of pulp canals and formation of tertiary dentin. Until now, methods for direct intraoperative visualization of the 3-dimensional anatomy of teeth have been missing. Here, we evaluate the use of shortwave infrared radiation (SWIR) for navigation during stepwise access preparation. Nine teeth (3 anteriors, 3 premolars, and 3 molars) were explanted en bloc with intact periodontium including alveolar bone and mucosa from the upper or lower jaw of human body donors. Analysis was performed at baseline as well as at preparation depths of 5 mm, 7 mm, and 9 mm, respectively. For reflection, SWIR was used at a wavelength of 1,550 nm from the occlusal direction, whereas for transillumination, SWIR was passed through each sample at the marginal gingiva from the buccal as well as oral side at a wavelength of 1,300 nm. Pulpal structures could be identified as darker areas approximately 2 mm before reaching the pulp chamber using SWIR transillumination, although they were indistinguishable under normal circumstances. Furcation areas in molars appeared with higher intensity than areas with canals. The location of pulpal structures was confirmed by superimposition of segmented micro-computed tomography (µCT) images. By radiomic analysis, significant differences between pulpal and parapulpal areas could be detected in image features. With hierarchical cluster analysis, both segments could be confirmed and associated with specific clusters. The local thickness of µCTs was calculated and correlated with SWIR transillumination images, by which a linear dependency of thickness and intensity could be demonstrated. Lastly, by in silico simulations of light propagation, dentin tubules were shown to be a crucial factor for understanding the visibility of the pulp. In conclusion, SWIR transillumination may allow direct clinical live navigation during endodontic access preparation.

Ca2+ signalling is critical for autoantibody-induced blistering of human epidermis in pemphigus.

BACKGROUND: Pemphigus is a severe bullous autoimmune skin disease. Pemphigus foliaceus (PF) is characterized by antidesmoglein (Dsg) 1 IgG causing epidermal blistering; mucosal pemphigus vulgaris (mPV) by anti-Dsg3 IgG inducing erosions in the mucosa; and mucocutaneous pemphigus vulgaris (PV) by affecting both, with autoantibodies targeting Dsg1 and Dsg3. OBJECTIVES: To characterize the Ca2+ flux pathway and delineate its importance in pemphigus pathogenesis and clinical phenotypes caused by different antibody profiles. METHODS: Immunoprecipitation, Ca2+ flux analysis, Western blotting, immunofluorescence staining, dissociation assays and a human skin ex vivo model were used. RESULTS: PV IgG and PF IgG, but neither Dsg3-specific monoclonal antibody (AK23) nor mPV IgG, caused Ca2+ influx in primary human keratinocytes. Phosphatidylinositol 4-kinase α interacts with Dsg1 but not with Dsg3. Its downstream target - phospholipase-C-γ1 (PLC) - was activated by PV IgG and PF IgG but not AK23 or mPV IgG. PLC releases inositol 1,4,5-trisphosphate (IP3) causing IP3 receptor (IP3R) activation and Ca2+ flux from the endoplasmic reticulum into the cytosol, which stimulates Ca2+ release-activated channels (CRAC)-mediated Ca2+ influx. Inhibitors against PLC, IP3R and CRAC effectively blocked PV IgG and PF IgG-induced Ca2+ influx; ameliorated alterations of Dsg1 and Dsg3 localization, and reorganization of keratin and actin filaments; and inhibited loss of cell adhesion in vitro. Finally, inhibiting PLC or IP3R was protective against PV IgG-induced blister formation and redistribution of Dsg1 and Dsg3 in human skin ex vivo. CONCLUSIONS: Ca2+ -mediated signalling is important for epidermal blistering and dependent on the autoantibody profile, which indicates different roles for signalling complexes organized by Dsg1 and Dsg3. Interfering with PLC and Ca2+ signalling may be a promising approach to treat epidermal manifestations of pemphigus.

A new ex vivo human oral mucosa model reveals that p38MAPK inhibition is not effective in preventing autoantibody-induced mucosal blistering in pemphigus.

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune disease characterized by blister formation in the epidermis and oral mucosa due to loss of keratinocyte cohesion. Autoantibodies present in patients with PV (PV-IgG) are known to primarily target desmoglein (Dsg)1 and Dsg3 in desmosomes. The mucosal-dominant subtype of PV (mdPV) is caused by PV-IgG autoantibodies against the cadherin-type adhesion molecule Dsg3. p38 mitogen-activated protein kinase (p38MAPK) signalling has been characterized as an important pathway downstream of PV-IgG binding and its inhibition is protective in ex vivo human skin. However, the role of p38MAPK signalling in mdPV is unknown as no experimental model has been available. OBJECTIVES: To establish a human ex vivo oral mucosa culture, and evaluate the p38MAPK dependency of blister formation and of ultrastructural alterations of desmosomes induced by mdPV-IgG. METHODS: Human labial mucosa was injected with mdPV-IgG as well as AK23, a pathogenic mouse monoclonal Dsg3 antibody, in the presence or absence of p38MAPK inhibitors. Viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labelling assay. Blister score was determined following haematoxylin and eosin staining and Dsg3 distribution by immunostaining. Samples were processed for transmission electron microscopy to analyse desmosome ultrastructure. RESULTS: Both AK23 and mdPV-IgG induced blisters and caused reduction in desmosome size and number in labial mucosa. Inhibition of p38MAPK was not effective in preventing these alterations. CONCLUSIONS: In contrast with human epidermis, PV-IgG and AK23 induce blisters and desmosome ultrastructural changes in labial mucosa via a mechanism not dependent on p38MAPK. What's already known about this topic? Pemphigus vulgaris IgG (PV-IgG) induces blistering as well as a reduction in desmosome number and size mediated by p38 mitogen-activated protein kinase (p38MAPK) signalling in ex vivo human skin. What does this study add? This study establishes a new human ex vivo mucosa model to test pathomechanisms mediated by PV-IgG. The study demonstrates that both AK23 and mucosal-dominant PV induce blisters and associated ultrastructural changes in labial mucosa via a mechanism not dependent on p38MAPK signalling. What is the translational message? This study highlights the respective tissue-specific responses of oral mucosa and skin related to PV pathogenesis, similar to the patient situation.

Mind the gap: mechanisms regulating the endothelial barrier.

The endothelial barrier consists of intercellular contacts localized in the cleft between endothelial cells, which is covered by the glycocalyx in a sievelike manner. Both types of barrier-forming junctions, i.e. the adherens junction (AJ) serving mechanical anchorage and mechanotransduction and the tight junction (TJ) sealing the intercellular space to limit paracellular permeability, are tethered to the actin cytoskeleton. Under resting conditions, the endothelium thereby builds a selective layer controlling the exchange of fluid and solutes with the surrounding tissue. However, in the situation of an inflammatory response such as in anaphylaxis or sepsis intercellular contacts disintegrate in post-capillary venules leading to intercellular gap formation. The resulting oedema can cause shock and multi-organ failure. Therefore, maintenance as well as coordinated opening and closure of interendothelial junctions is tightly regulated. The two principle underlying mechanisms comprise spatiotemporal activity control of the small GTPases Rac1 and RhoA and the balance of the phosphorylation state of AJ proteins. In the resting state, junctional Rac1 and RhoA activity is enhanced by junctional components, actin-binding proteins, cAMP signalling and extracellular cues such as sphingosine-1-phosphate (S1P) and angiopoietin-1 (Ang-1). In addition, phosphorylation of AJ components is prevented by junction-associated phosphatases including vascular endothelial protein tyrosine phosphatase (VE-PTP). In contrast, inflammatory mediators inhibiting cAMP/Rac1 signalling cause strong activation of RhoA and induce AJ phosphorylation finally leading to endocytosis and cleavage of VE-cadherin. This results in dissolution of TJs the outcome of which is endothelial barrier breakdown.

Inhibition of p38MAPK signalling prevents epidermal blistering and alterations of desmosome structure induced by pemphigus autoantibodies in human epidermis.

BACKGROUND: Pemphigus vulgaris (PV) is a skin blistering disease caused by autoantibodies targeting the desmosomal adhesion proteins desmoglein (Dsg) 3 and 1. The mechanisms underlying pemphigus skin blistering are not fully elucidated but p38 mitogen-activated protein kinase (p38MAPK) activation is one of the signalling events necessary for full loss of cell cohesion. However, it is unclear whether ultrastructural hallmarks of desmosome morphology as observed in patients' lesions are mediated by p38MAPK signalling. OBJECTIVES: In this study, we tested the relevance of p38MAPK for blister formation and the ultrastructural changes induced by PV autoantibodies in human skin. METHODS: Human skin samples were injected with IgG fractions of one patient suffering from mucocutaneous PV (mcPV-IgG), one from mucosal-dominant PV (mdPV-IgG) or AK23, a pathogenic monoclonal Dsg3 antibody derived from a pemphigus mouse model. Samples were processed for histological and electron microscopy analyses. RESULTS: mcPV-IgG and AK23 but not mdPV-IgG reduced desmosome size, caused interdesmosomal widening and formation of split desmosomes, and altered keratin filament insertion. In contrast, full epidermal blister formation and lower desmosome number were evident in tissue samples exposed to mcPV-IgG only. Pharmacological inhibition of p38MAPK blunted the reduction of desmosome number and size, ameliorated interdesmosomal widening and loss of keratin insertion and prevented mcPV-IgG-induced blister formation. CONCLUSIONS: Our data demonstrate that blistering can be prevented by inhibition of p38MAPK in the human epidermis. Moreover, typical morphological alterations induced by mcPV-IgG such as interdesmosomal widening and the reduction of desmosome size at least in part require p38MAPK signalling.

Endoscopic sinus surgery training courses: benefit and problems - a multicentre evaluation to systematically improve surgical training.

BACKGROUND: The aim of this multicentre study was to systematically analyse the strengths and weaknesses in the surgical training for endoscopic sinus surgery (ESS) and identify measures that may improve training. METHODOLOGY: Using a structured questionnaire, 133 participants of ESS courses in seven centres in Germany, Switzerland and Australia were asked about their experiences during their dissection courses and how they perceived their course could be improved. RESULTS: Gaining confidence in handling of instruments and endoscopes was only a problem for participants with little experience in ESS. The majority of the participants, independent from their level of training, considered infundibulotomy and anterior ethmoidectomy as the easiest dissection steps, whilst surgery of the frontal sinus posed a considerable challenge for many surgeons even those with a higher level of training. Participants with and without ESS experience thought that emphasis on anatomy was the most important improvement that could be made during their surgical training. Virtually all participants stated that the course improved their anatomical knowledge, their surgical skills and their confidence when performing ESS. CONCLUSIONS: ESS dissection courses are considered beneficial by surgical trainees. Participants felt that more emphasis on sinus anatomy in conjunction with private study is essential to maximize their skills in surgical dissection. For beginners with ESS, an infundibulotomy and anterior ethmoidectomy were thought to be the best initial procedures to help develop endoscopic surgical skills.

[Supination external rotation lesions of the ankle joint in osteoporotic lower leg specimens. Experimental induction and review of the literature]. / Die Supinations-Eversions-Verletzung des OSG in osteoporotischen Unterschenkelpräparaten. Experimentelle Induktion und Literaturübersicht.

BACKGROUND: Knowledge of the pathomechanism and the detailed extent of ankle joint lesions determines adequate therapy and success of treatment. MATERIAL AND METHODS: Supination external rotation lesions were induced in 29 human cadavera with a testing apparatus; 27 of these specimens were from elderly women. Bone mineral density was measured. The literature review includes experimental studies of this fracture entity. RESULTS: We induced stage II in 42%; applying an additional lateral force on the fibula raised the incidence. The syndesmosis stayed intact in 50% although the fibula fractured at the level of the tibial plafond. Stage IV lesions were registered in 25%. The overall low bone mineral density showed a positive correlation to the angle at which the fibula fracture occurred. CONCLUSIONS: We reproduced supination external rotation lesions according to Lauge-Hansen in osteoporotic ankles. There is a certain discrepancy between the obligatory lesion of the inferior anterior tibiofibular syndesmosis at stage II according to Lauge-Hansen, as we observed an intact syndesmosis in 50% at stage II. Stage IV defining medial malleolar fractures were reproduced after Lauge-Hansen and Michelson et al.

Mutant PIK3CA licenses TRAIL and CD95L to induce non-apoptotic caspase-8-mediated ROCK activation.

Constitutively active PI3K catalytic subunit alpha (PIK3CA) interfered with apoptosis induction downstream of death receptor-signaling complex formation allowing robust caspase-8 activation without triggering the execution steps of apoptosis. In mutant PIK3CA-expressing cells, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95L stimulated nuclear factor kappaB (NFkappaB) activation, invasion, and transition to an amoeboid-like morphology. NFkappaB activation and adoption of amoeboid shape were inhibited by caspase-8 knockdown or FLIP-S expression, but only the cell morphology alterations required caspase-8 activity. Furthermore, we identified caspase-8-mediated, caspase-3-independent cleavage of the protein kinase rho-associated, coiled-coil containing protein kinase 1 as a novel mechanism for acquiring amoeboid shape and enhanced invasiveness in response to TRAIL and CD95L. Taken together, we provide evidence that mutated PIK3CA converts the 'tumor surveillance' activity of cancer cell-expressed death receptors and caspase-8 toward tumor promotion.

Role of Rac 1 and cAMP in endothelial barrier stabilization and thrombin-induced barrier breakdown.

Barrier stabilizing effects of cAMP as well as of the small GTPase Rac 1 are well established. Moreover, it is generally believed that permeability-increasing mediators such as thrombin disrupt endothelial barrier functions primarily via activation of Rho A. In this study, we provide evidence that decrease of both cAMP levels and of Rac 1 activity contribute to thrombin-mediated barrier breakdown. Treatment of human dermal microvascular endothelial cells (HDMEC) with Rac 1-inhibitor NSC-23766 decreased transendothelial electrical resistance (TER) and caused intercellular gap formation. These effects were reversed by addition of forskolin/rolipram (F/R) to increase intracellular cAMP but not by the cAMP analogue 8-pCPT-2'-O-Methyl-cAMP (O-Me-cAMP) which primarily stimulates protein kinase A (PKA)-independent signaling via Epac/Rap 1. However, both F/R and O-Me-cAMP did not increase TER above control levels in the presence of NSC-23766 in contrast to experiments without Rac 1 inhibition. Because Rac 1 was required for maintenance of barrier functions as well as for cAMP-mediated barrier stabilization, we tested the role of Rac 1 and cAMP in thrombin-induced barrier breakdown. Thrombin-induced drop of TER and intercellular gap formation were paralleled by a rapid decrease of cAMP as revealed by fluorescence resonance energy transfer (FRET). The efficacy of F/R or O-Me-cAMP to block barrier-destabilizing effects of thrombin was comparable to Y27632-induced inhibition of Rho kinase but was blunted when Rac 1 was inactivated by NSC-23766. Taken together, these data indicate that decrease of cAMP and Rac 1 activity may be an important step in inflammatory barrier disruption.

cAMP induced Rac 1-mediated cytoskeletal reorganization in microvascular endothelium.

It is well established that cAMP stabilizes endothelial barrier functions, in part by regulation of VE-cadherin via EPAC/Rap 1. The aim of the present study was to investigate whether cAMP activates Rac 1 in microvascular endothelium. In human dermal microvascular endothelial cells (HDMEC), treatment with forskolin/rolipram (F/R) to increase cAMP by as well as the Epac/Rap 1-stimulating cAMP analogue 8-pCPT-2'-O-methyl-cAMP (O-Me-cAMP) stabilized endothelial barrier properties as revealed by raised transendothelial electrical resistance (TER). Under these conditions, immunostaining of VE-cadherin and claudin 5 were increased and linearized. This was paralleled by activation of Rac 1 by 153 +/- 16% (F/R) or 281 +/- 65% (O-Me-cAMP) whereas activity of Rho A was unchanged. F/R and O-Me-cAMP increased the peripheral actin belt and recruited the Rac 1 effector cortactin to cell junctions, similar to direct activation of Rac 1 by CNF-1. Thrombin was used to further test the physiologic relevance of cAMP-mediated Rac 1 activation. Thrombin-induced drop of TER was paralleled by intercellular gap formation, inactivation of Rac 1 and activation of Rho A at 5 and 15 min whereas baseline conditions where re-established following 60 min. Both, F/R and O-Me-cAMP completely blocked the thrombin-induced barrier breakdown. F/R completely abolished thrombin-induced Rac 1 inactivation and Rho A activation whereas O-Me-cAMP only partially blocked Rac 1 inactivation. Taken together, these results indicate that Rac 1 activation likely contributes to the barrier-stabilizing effects of cAMP in microvascular endothelium and that these effects may in part be mediated by Epac/Rap 1.

Differential role of Rho GTPases in endothelial barrier regulation dependent on endothelial cell origin.

From studies using macrovascular endothelium, it was concluded that Rho A activation generally leads to endothelial barrier breakdown. Here, we characterized the role of Rho GTPases in endothelial barrier regulation in four different cell lines, both microvascular and macrovascular. Rho A activation by cytotoxic necrotizing factor y (CNFy) induced stress fiber formation in all cell lines. This was paralleled by gap formation and barrier breakdown in microvascular mesenteric endothelial cells (MesEnd), human dermal microvascular endothelial cells (HDMEC) as well as in macrovascular pulmonary artery endothelial cells (PAEC) but not in microvascular myocardial endothelial cells (MyEnd). In MyEnd cells, activation of Rac 1 and Cdc42 by CNF-1 strengthened barrier properties whereas in MesEnd, HDMEC and PAEC all three GTPases were activated which increased permeability in PAEC but not in MesEnd and HDMEC. In PAEC, CNF-1-induced decrease of barrier properties was blocked by the Rho kinase inhibitor Y27632 indicating that co-activation of Rho A dominated the barrier response. Inactivation of Rac 1 by toxin B or by lethal toxin (LT) compromised barrier properties in all cell lines. Taken together, Rac 1 requirement for endothelial barrier maintenance but not the destabilizing role of Rho A seems to be ubiquitous.