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Purpose: The aim of the present study was to assess the efficacy of the Ronch®AP palatal device in treating patients with moderate and severe forms of obstructive sleep apnea syndrome. Methods: In a randomized controlled trial 22 patients were examined with the Ronch®AP palatal device after 4 weeks of usage. Their results were compared to a control group of 30 patients who did not receive any treatment during this time. All patients included did not tolerate CPAP therapy. Among other parameters the apnea–hypopnea index (AHI) was measured using nocturnal cardiorespiratory polysomnography. Daytime sleepiness was assessed using Epworth Sleepiness Scale. Pittsburgh Sleep Quality Index was used to analyze sleep quality. Results: Using the Ronch®AP palatal device AHI was reduced from an average of 35.34 ± 14.9/h to 19.18 ± 14.93/h, whereas the control group only showed a minimal mean reduction from 31.32 ± 12.76/h to 29.37 ± 17.11/h. The difference in reduction between the two randomized groups was highly significant (d = − 14.2, 95% CI 5.9–22.6, t = 3.4, df = 49.9, p = 0.001). Epworth Sleepiness Scale score was lowered from 9.18 ± 4.73 to 7.82 ± 4.14 on average and sleep quality improved by − 1.91 ± 2.31. Both changes were also statistically relevant (p < 0.005). Conclusions: The Ronch®AP device is an effective alternative treatment option for patients suffering from moderate and severe forms of obstructive sleep apnea syndrome and not tolerating CPAP therapy. Trial registration number: 407-16 with approval from the local ethical committee (Ethikkommission der Medizinischen Fakultät der LMU München). [ABSTRACT FROM AUTHOR]

Obstructive sleep apnea (OSA) is a common sleep-related breathing disorder characterized by recurrent episodes of upper airway obstruction and subsequent hypoxia. In patients with OSA, severity and number of these hypoxic events positively correlate with the extent of associated cardiovascular pathology. The molecular mechanisms underlying intermittent hypoxia (IH)-driven cardiovascular disease in OSA, however, remain poorly understood—partly due to the lack of adequate experimental models. Here, we present a novel experimental approach that utilizes primary human endothelial cells cultivated under shear stress. Oxygen partial pressure dynamics were adopted in our in vitro model according to the desaturation-reoxygenation patterns identified in polysomnographic data of severe OSA patients (n = 10, with 892 severe desaturations, SpO2<80%). Using western blot analysis, we detected a robust activation of the two major inflammatory pathways ERK and NF-κB in endothelial cells, whereas no HIF1α and HIF2α protein stabilization was observed. In line with these findings, mRNA and protein expression of the pro-inflammatory adhesion and signaling molecule ICAM-1 and the chemokine CCL2 were significantly increased. Hence, we established a novel in vitro model for deciphering OSA-elicited effects on the vascular endothelium. First data obtained in this model point to the endothelial activation of pro-inflammatory rather than hypoxia-associated pathways in OSA. Future studies in this model might contribute to the development of targeted strategies against OSA-induced, secondary cardiovascular disease. [ABSTRACT FROM AUTHOR]

Background: The microvascular endothelium inherently controls nutrient delivery, oxygen supply, and immune surveillance of malignant tumors, thus representing both biological prerequisite and therapeutic vulnerability in cancer. Recently, cellular senescence emerged as a fundamental characteristic of solid malignancies. In particular, tumor endothelial cells have been reported to acquire a senescence-associated secretory phenotype, which is characterized by a pro-inflammatory transcriptional program, eventually promoting tumor growth and formation of distant metastases. We therefore hypothesize that senescence of tumor endothelial cells (TEC) represents a promising target for survival prognostication and prediction of immunotherapy efficacy in precision oncology. Methods: Published single-cell RNA sequencing datasets of different cancer entities were analyzed for cell-specific senescence, before generating a pan-cancer endothelial senescence-related transcriptomic signature termed EC.SENESCENCE.SIG. Utilizing this signature, machine learning algorithms were employed to construct survival prognostication and immunotherapy response prediction models. Machine learning-based feature selection algorithms were applied to select key genes as prognostic biomarkers. Results: Our analyses in published transcriptomic datasets indicate that in a variety of cancers, endothelial cells exhibit the highest cellular senescence as compared to tumor cells or other cells in the vascular compartment of malignant tumors. Based on these findings, we developed a TEC-associated, senescence-related transcriptomic signature (EC.SENESCENCE.SIG) that positively correlates with pro-tumorigenic signaling, tumor-promoting dysbalance of immune cell responses, and impaired patient survival across multiple cancer entities. Combining clinical patient data with a risk score computed from EC.SENESCENCE.SIG, a nomogram model was constructed that enhanced the accuracy of clinical survival prognostication. Towards clinical application, we identified three genes as pan-cancer biomarkers for survival probability estimation. As therapeutic perspective, a machine learning model constructed on EC.SENESCENCE.SIG provided superior pan-cancer prediction for immunotherapy response than previously published transcriptomic models. Conclusions: We here established a pan-cancer transcriptomic signature for survival prognostication and prediction of immunotherapy response based on endothelial senescence. [ABSTRACT FROM AUTHOR]

Penetrating traumas of the head are generally life-threatening injuries, whose management poses a substantial challenge for emergency department teams. These injuries are characteristically violence-associated and frequently accompanied by damage of essential organs including brain, meninges, large vessels, cranial nerves, eyes, viscerocranium, internal ear, and/or labyrinth. Here, we present an exceptional case of head trauma caused by a knife blade, which was stuck deep inside in the petrous bone. After the extraction of the knife, the patient had very few immediate and no long-term post-traumatic complications. In conclusion, high-end technical equipment as well as an interdisciplinary team of specialized physicians is recommended for the management of penetrating head trauma to optimize the outcome. [ABSTRACT FROM AUTHOR]

The assessment of leukocyte activation in vivo is mainly based on surrogate parameters, such as cell shape changes and migration patterns. Consequently, additional parameters are required to dissect the complex spatiotemporal activation of leukocytes during inflammation. Here, we showed that intravital microscopy of myeloid leukocyte Ca2+ signals with Ca2+ reporter mouse strains combined with bioinformatic signal analysis provided a tool to assess their activation in vivo. We demonstrated by two-photon microscopy that tissue-resident macrophages reacted to sterile inflammation in the cremaster muscle with Ca2+ transients in a distinct spatiotemporal pattern. Moreover, through high-resolution, intravital spinning disk confocal microscopy, we identified the intracellular Ca2+ signaling patterns of neutrophils during the migration cascade in vivo. These patterns were modulated by the Ca2+ channel Orai1 and Gαi-coupled GPCRs, whose effects were evident through analysis of the range of frequencies of the Ca2+ signal (frequency spectra), which provided insights into the complex patterns of leukocyte Ca2+ oscillations. Together, these findings establish Ca2+ frequency spectra as an additional dimension to assess leukocyte activation and migration during inflammation in vivo. Decoding Ca2+ signals: The immune responses to infection and sterile inflammation are complex, involving the coordinated recruitment and activation of multiple cell types. To better understand the activation states of immune cells in the context of inflammation, Mehari et al. used various in vivo imaging techniques with transgenic Ca2+ reporter mice to measure the intensity and frequency of intracellular Ca2+ signals in macrophages and neutrophils as they responded to a laser-induced tissue injury. Analysis of these data revealed the kinetics of recruitment of tissue-resident macrophages and identified distinct patterns of intracellular Ca2+ signals in neutrophils that characterized the different stages of their recruitment from the blood to sites of injury. These findings suggest that in vivo monitoring of Ca2+ signals provides insights into the functional states of immune cells. [ABSTRACT FROM AUTHOR]

High intratumoral levels of urokinase‐type plasminogen activator (uPA)‐plasminogen activator inhibitor‐1 (PAI‐1) heteromers predict impaired survival and treatment response in early breast cancer. The pathogenetic role of this protein complex remains obscure. Here, we demonstrate that heteromerization of uPA and PAI‐1 multiplies the potential of the single proteins to attract pro‐tumorigenic neutrophils. To this end, tumor‐released uPA‐PAI‐1 utilizes very low‐density lipoprotein receptor and mitogen‐activated protein kinases to initiate a pro‐inflammatory program in perivascular macrophages. This enforces neutrophil trafficking to cancerous lesions and skews these immune cells toward a pro‐tumorigenic phenotype, thus supporting tumor growth and metastasis. Blockade of uPA‐PAI‐1 heteromerization by a novel small‐molecule inhibitor interfered with these events and effectively prevented tumor progression. Our findings identify a therapeutically targetable, hitherto unknown interplay between hemostasis and innate immunity that drives breast cancer progression. As a personalized immunotherapeutic strategy, blockade of uPA‐PAI‐1 heteromerization might be particularly beneficial for patients with highly aggressive uPA‐PAI‐1high tumors. Synopsis: Uhl et al report that heteromerization of the serine protease urokinase‐type plasminogen activator (uPA) and the serpin plasminogen activator inhibitor‐1 (PAI‐1) enforces the trafficking of pro‐tumorigenic neutrophils to malignant lesions in highly aggressive subtypes of breast cancer. High levels of uPA‐PAI‐1 heteromers in human breast cancer were related to extensive neutrophil infiltration of the malignant tumors and a poor clinical outcome.uPA‐PAI‐1 heteromers induced the release of the cytokine TNF in perivascular macrophages via VLDLr, in turn activating microvascular endothelial cells.Endothelially‐deposited uPA‐PAI‐1 heteromers subsequently supported intravascular adhesion and extravasation of circulating neutrophils by promoting β2 integrin clustering on their cell surface.uPA‐PAI‐1‐primed neutrophils unfolded pro‐tumorigenic properties, potently stimulating breast cancer cell proliferation by the release of neutrophil elastase.Pharmacological blockade of uPA‐PAI‐1 heteromerization by a novel small molecule inhibitor prevented neutrophil infiltration and tumor progression in experimental breast cancer. [ABSTRACT FROM AUTHOR]

Microvascular dysfunction plays a fundamental role in the pathogenesis of salivary gland disorders. Restoring and preserving microvascular integrity might therefore represent a promising strategy for the treatment of these pathologies. The mechanisms underlying microvascular dysfunction in salivary glands, however, are still obscure, partly due to the unavailability of adequate in vivo models. Here, we present a novel experimental approach that allows comprehensive in vivo analyses of the salivary gland microvasculature in mice. For this purpose, we employed different microscopy techniques including multi-photon in vivo microscopy to quantitatively analyze interactions of distinct immune cell subsets in the submandibular gland microvasculature required for their infiltration into the surrounding parenchyma and their effects on microvascular function. Confocal microscopy and multi-channel flow cytometry in tissue sections/homogenates complemented these real-time analyses by determining the molecular phenotype of the participating cells. To this end, we identified key adhesion and signaling molecules that regulate the subset- and tissue-specific trafficking of leukocytes into inflamed glands and control the associated microvascular leakage. Hence, we established an experimental approach that allows in vivo analyses of microvascular processes in healthy and diseased salivary glands. This enables us to delineate distinct pathogenetic factors as novel therapeutic targets in salivary gland diseases. [ABSTRACT FROM AUTHOR]

Uromodulin (UMOD) is produced and secreted by tubular epithelial cells. Secreted UMOD polymerizes (pUMOD) in the tubular lumen, where it regulates salt transport and protects the kidney from bacteria and stone formation. Under various pathological conditions, pUMOD accumulates within the tubular lumen and reaches extratubular sites where it may interact with renal interstitial cells. Here, we investigated the potential of extratubular pUMOD to act as a damage associated molecular pattern (DAMP) molecule thereby creating local inflammation. We found that intrascrotal and intraperitoneal injection of pUMOD induced leukocyte recruitment in vivo and led to TNF-α secretion by F4/80 positive macrophages. Additionally, pUMOD directly affected vascular permeability and increased neutrophil extravasation independent of macrophage-released TNF-α. Interestingly, pUMOD displayed no chemotactic properties on neutrophils, did not directly activate β2 integrins and did not upregulate adhesion molecules on endothelial cells. In obstructed neonatal murine kidneys, we observed extratubular UMOD accumulation in the renal interstitium with tubular atrophy and leukocyte infiltrates. Finally, we found extratubular UMOD deposits associated with peritubular leukocyte infiltration in kidneys from patients with inflammatory kidney diseases. Taken together, we identified extratubular pUMOD as a strong inducer of leukocyte recruitment, underlining its critical role in mounting an inflammatory response in various kidneys pathologies. [ABSTRACT FROM AUTHOR]

Radiotherapy is an essential part of multi-modal cancer therapy. Nevertheless, for certain cancer entities such as colorectal cancer (CRC) the indications of radiotherapy are limited due to anatomical peculiarities and high radiosensitivity of the surrounding normal tissue. The development of molecularly targeted, combined modality approaches may help to overcome these limitations. Preferably, such strategies should not only enhance radiation-induced tumor cell killing and the abrogation of tumor cell clonogenicity, but should also support the stimulation of anti-tumor immune mechanisms – a phenomenon which moved into the center of interest of preclinical and clinical research in radiation oncology within the last decade. The present study focuses on inhibition of heat shock protein 90 (HSP90) whose combination with radiotherapy has previously been reported to exhibit convincing therapeutic synergism in different preclinical cancer models. By employing in vitro and in vivo analyses, we examined if this therapeutic synergism also applies to the priming of anti-tumor immune mechanisms in model systems of CRC. Our results indicate that the combination of HSP90 inhibitor treatment and ionizing irradiation induced apoptosis in colorectal cancer cells with accelerated transit into secondary necrosis in a hyperactive Kras-dependent manner. During secondary necrosis, dying cancer cells released different classes of damage-associated molecular patterns (DAMPs) that stimulated migration and recruitment of monocytic cells in vitro and in vivo. Additionally, these dying cancer cell-derived DAMPs enforced the differentiation of a monocyte-derived antigen presenting cell (APC) phenotype which potently triggered the priming of allogeneic T cell responses in vitro. In summary, HSP90 inhibition – apart from its radiosensitizing potential – obviously enables and supports the initial steps of anti-tumor immune priming upon radiotherapy and thus represents a promising partner for combined modality approaches. The therapeutic performance of such strategies requires further in-depth analyses, especially for but not only limited to CRC. [ABSTRACT FROM AUTHOR]

Aim: To optimize the design of nanoparticles for diagnosis or therapy of vascular diseases, it is mandatory to characterize the determinants of nano-bio interactions in vascular lesions. Materials & methods: Using ex vivo and in vivo microscopy, we analyzed the interactive behavior of quantum dots with different surface functionalizations in atherosclerotic lesions of ApoE-deficient mice. Results: We demonstrate that quantum dots with different surface functionalizations exhibit specific interactive behaviors with distinct molecular and cellular components of the injured vessel wall. Moreover, we show a role for fibrinogen in the regulation of the spatio-temporal interaction dynamics in atherosclerotic lesions. Conclusion: Our findings emphasize the relevance of surface chemistry-driven nano-bio interactions on the differential in vivo behavior of nanoparticles in diseased tissue. [ABSTRACT FROM AUTHOR]

Aim: To assess the role of the endothelial glycocalyx (eGCX) for the uptake of nanoparticles by endothelial cells. Methods: The expression of the eGCX on cultured human umbilical vein endothelial cells was determined by immunostaining of heparan sulfate. Enzymatic degradation of the eGCX was achieved by incubating the cells with eGCX-shedding enzymes. The uptake of 50-nm polystyrene nanospheres was quantified by confocal microscopy. Results: Human umbilical vein endothelial cells expressed a robust eGCX when cultured for 10 days. The uptake of both carboxylated and aminated polystyrene nanospheres was significantly increased in cells in which the glycocalyx was enzymatically degraded, while it remained at a low level in cells with an intact glycocalyx. Conclusion: The eGCX constitutes a barrier against the internalization of blood-borne nanoparticles by endothelial cells. [ABSTRACT FROM AUTHOR]

Effective immune responses require the directed migration of leukocytes from the vasculature to the site of injury or infection. How immune cells “find” their site of extravasation remains largely obscure. Here, we identified a previously unrecognized role of platelets as pathfinders guiding leukocytes to their exit points in the microvasculature: upon onset of inflammation, circulating platelets were found to immediately adhere at distinct sites in venular microvessels enabling these cellular blood components to capture neutrophils and, in turn, inflammatory monocytes via CD40-CD40L-dependent interactions. In this cellular crosstalk, ligation of PSGL-1 by P-selectin leads to ERK1/2 MAPK-dependent conformational changes of leukocyte integrins, which promote the successive extravasation of neutrophils and monocytes to the perivascular tissue. Conversely, blockade of this cellular partnership resulted in misguided, inefficient leukocyte responses. Our experimental data uncover a platelet-directed, spatiotemporally organized, multicellular crosstalk that is essential for effective trafficking of leukocytes to the site of inflammation. [ABSTRACT FROM AUTHOR]

ABSTRACT Although carbon-based nanomaterials (CBNs) have been shown to exert prothrombotic effects in microvessels, it is poorly understood whether CBNs also have the potential to interfere with the process of leukocyte-endothelial cell interactions and whether the shape of CBNs plays a role in these processes. Thus, the aim of this study was to compare the acute effects of two differently shaped CBNs, fiber-shaped single-walled carbon nanotubes (SWCNT) and spherical ultrafine carbon black (CB), on thrombus formation as well as on leukocyte-endothelial cell interactions and leukocyte transmigration in the murine microcirculation upon systemic administration in vivo. Systemic administration of both SWCNT and CB accelerated arteriolar thrombus formation at a dose of 1 mg kg-1 body weight, whereas SWCNT exerted a prothrombotic effect also at a lower dose (0.1 mg kg-1 body weight). In vitro, both CBNs induced P-selectin expression on human platelets and formation of platelet-granulocyte complexes. In contrast, injection of fiber-shaped SWCNT or of spherical CB did not induce leukocyte-endothelial cell interactions or leukocyte transmigration. In vitro, both CBNs slightly increased the expression of activation markers on human monocytes and granulocytes. These findings suggest that systemic administration of CBNs accelerates arteriolar thrombus formation independently of the CBNs' shape, but does not induce leukocyte-endothelial cell interactions or leukocyte transmigration. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background and Purpose: Roscovitine, a cyclin-dependent kinase (CDK) inhibitor that induces tumour cell death, is under evaluation as an anti-cancer drug. By triggering leukocyte apoptosis, roscovitine can also enhance the resolution of inflammation. Beyond death-inducing properties, we tested whether roscovitine affects leukocyte-endothelial cell interaction, a vital step in the onset of inflammation.Experimental Approach: Leukocyte-endothelial cell interactions were evaluated in venules of mouse cremaster muscle, using intravital microscopy. In primary human endothelial cells, we studied the influence of roscovitine on adhesion molecules and on the nuclear factor-κB (NF-κB) pathway. A cellular kinome array, in vitro CDK profiling and RNAi methods were used to identify targets of roscovitine.Key Results: In vivo, roscovitine attenuated the tumour necrosis factor-α (TNF-α)-induced leukocyte adherence to and transmigration through, the endothelium. In vitro, roscovitine strongly inhibited TNF-α-evoked expression of endothelial adhesion molecules (E-selectin, intercellular cell adhesion molecule, vascular cell adhesion molecule). Roscovitine blocked NF-κB-dependent gene transcription, but not the NF-κB activation cascade [inhibitor of κB (IκB) kinase activity, IκB-α degradation, p65 translocation]. Using a cellular kinome array and an in vitro CDK panel, we found that roscovitine inhibited protein kinase A, ribosomal S6 kinase and CDKs 2, 5, 7 and 9. Experiments using kinase inhibitors and siRNA showed that the decreased endothelial activation was due solely to blockade of CDK5 and CDK9 by roscovitine.Conclusions and Implications: Our study highlights a novel mode of action for roscovitine, preventing endothelial activation and leukocyte-endothelial cell interaction by inhibition of CDK5 and 9. This might expand its usage as a promising anti-inflammatory compound. [ABSTRACT FROM AUTHOR]

Clinical trials revealed beneficial effects of the broad-spectrum serine protease inhibitor aprotinin on the prevention of ischemia-reperfusion (I/R) injury. The underlying mechanisms remained largely unclear. Using in vivo microscopy on the cremaster muscle of male C57BL/6 mice, aprotinin as well as inhibitors of the serine protease plasmin including tranexamic acid and e-aminocaproic acid were found to significantly diminish I/R-elicited intravascular firm adherence and (subsequent) transmigration of neutrophils. Remodeling of collagen IV within the postischemic perivenular basement membrane was almost completely abrogated in animals treated with plasmin inhibitors or aprotinin. In separate experiments, incubation with plasmin did not directly activate neutrophils. Extravascular, but not intravascular administration of plasmin caused a dose-dependent increase in numbers of firmly adherent and transmigrated neutrophils. Blockade of mast cell activation as well as inhibition of leukotriene synthesis or antagonism of the platelet-activating-factor receptor significantly reduced plasmin-dependent neutrophil responses. In conclusion, our data suggest that extravasated plasmin(ogen) mediates neutrophil recruitment in vivo via activation of perivascular mast cells and secondary generation of lipid mediators. Aprotinin as well as the plasmin inhibitors tranexamic acid and e-aminocaproic acid interfere with this inflammatory cascade and effectively prevent postischemic neutrophil responses as well as remodeling events within the vessel wall. [ABSTRACT FROM AUTHOR]

In advanced inflammatory disease, microvascular thrombosis leads to the interruption of blood supply and provokes ischemic tissue injury. Recently, intravascularly adherent leukocytes have been reported to shape the blood flow in their immediate vascular environment. Whether these rheological effects are relevant for microvascular thrombogenesis remains elusive. Employing multi-channel in vivo microscopy, analyses in microfluidic devices, and computational modeling, we identified a previously unanticipated role of leukocytes for microvascular clot formation in inflamed tissue. For this purpose, neutrophils adhere at distinct sites in the microvasculature where these immune cells effectively promote thrombosis by shaping the rheological environment for platelet aggregation. In contrast to larger (lower-shear) vessels, this process in high-shear microvessels does not require fibrin generation or extracellular trap formation, but involves GPIbα-vWF and CD40-CD40L-dependent platelet interactions. Conversely, interference with these cellular interactions substantially compromises microvascular clotting. Thus, leukocytes shape the rheological environment in the inflamed venular microvasculature for platelet aggregation thereby effectively promoting the formation of blood clots. Targeting this specific crosstalk between the immune system and the hemostatic system might be instrumental for the prevention and treatment of microvascular thromboembolic pathologies, which are inaccessible to invasive revascularization strategies. [ABSTRACT FROM AUTHOR]