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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]

Under steady-state conditions, aged neutrophils are removed from the circulation in bone marrow, liver, and spleen, thereby maintaining myeloid cell homeostasis. The fate of these aged immune cells under inflammatory conditions, however, remains largely obscure. Here, we demonstrate that in the acute inflammatory response during endotoxemia, aged neutrophils cease returning to the bone marrow and instead rapidly migrate to the site of inflammation. Having arrived in inflamed tissue, aged neutrophils were found to exhibit a higherphagocytic activity as compared with the subsequently recruited non aged neutrophils. This distinct behavior of aged neutrophils under inflammatory conditions is dependent on specific age-related changes in their molecular repertoire that enable these "experienced" immunecells to instantly translateinflammatory signalsintoimmuneresponses. In particular, aged neutrophils engage Toll-like receptor-4- and p38 MAPK-dependent pathways to induce conformational changes in β2 integrins that allow thesephagocytes to effectively accomplish their mission in the front line of the inflammatory response. Hence, ageing in the circulation might represent a critical process for neutrophils that enables these immune cells to properly unfold their functional properties for host defense. [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]

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]

In vitro studies suggest that leukocytes locomote in an ameboid fashion independently of pericellular proteolysis. Whether this motility pattern applies for leukocyte migration in inflamed tissue is still unknown. In vivo microscopy on the inflamed mouse cremaster muscle revealed that blockade of serine proteases or of matrix metalloproteinases (MMPs) significantly reduces intravascular accumulation and transmigration of neutrophils. Using a novel in vivo chemotaxis assay, perivenular microinjection of inflammatory mediators induced directional interstitial migration of neutrophils. Blockade of actin polymerization, but not of actomyosin contraction abolished neutrophil interstitial locomotion. Multiphoton laser scanning in vivo microscopy showed that the density of the interstitial collagen network increases in inflamed tissue, thereby providing physical guidance to infiltrating neutrophils. Although neutrophils locomote through the interstitium without pericellular collagen degradation, inhibition of MMPs, but not of serine proteases, diminished their polarization and interstitial locomotion. In this context, blockade of MMPs was found to modulate expression of adhesion/signaling molecules on neutrophils. Collectively, our data indicate that serine proteases are critical for neutrophil extravasation, whereas these enzymes are dispensable for neutrophil extravascular locomotion. By contrast, neutrophil interstitial migration strictly relies on actin polymerization and does not require the pericellular degradation of collagen fibers but is modulated by MMPs [ABSTRACT FROM AUTHOR]

Background-Urokinase-type plasminogen activator (uPA) has recently been implicated m the pathogene is of ischemia-reperfusion (1/R) injury. The underlying mechanisms remain largely unclear. Methods and Results-Using in vivo microscopy on the mouse cremaster muscle, 1/R-elicited finn adherence and tran migration of neutrophils were found to be significantly diminished in uP A-deficient mice and in mice treated with the uPA inhibitor WX-340, but not in uPA receptor (uPAR)-deficient mice. Interestingly, postischemic leukocyte response were significantly reduced on blockade of the integrin CD11b/Mac-1, which also erves as uPAR receptor. Using a cell transfer technique, postischemic adherence and transmigration of wild-type leukocytes were significantly decreased in uPA-deficient animals, whereas uPA-deficient leukocytes exhibited a electively reduced transmigration in wild-type animal . On 1/R or stimulation with recombinant uPA, > 90% of firmly adherent leukocytes colocalized with CD31-immunoreactive endothelial junctions as detected by in vivo fluorescence micro copy. In a model of hepatic I/R, treatment with WX-340 significantly attenuated postischemic neutrophil infiltration and tissue injury. Conclusions-Our data suggest that endothelial uPA promotes intravascular adherence, whereas leukocyte uPA facilitates the .Subsequent paracellular tran migration of neutrophil during I/R. This process is regulated via CD11b/Mac-1 , and does not require uPAR. Pharmacological blockade of uPA interfere with these events and effectively attenuate postischemic tissue injury. [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]

CD4+ T cells recruited to the liver play a key role in the pathogenesis of ischemia/reperfusion (I/R) injury. The mechanism of their activation during alloantigen-independent I/R is not completely understood. We hypothesized that liver-resident dendritic cells (DCs) interact with CD4+ T cells in the postischemic liver and that modulation of DCs or T-cell-DC interactions attenuates liver inflammation. In mice, warm hepatic I/R (90/120-240 min) was induced. Tolerogenic DCs were generated in situ by pretreatment of animals with the vitamin D analog paricalcitol. A mAb-CD44 was used for blockade of CD4+ T-cell-DC interactions. As shown by 2-photon in vivo microscopy as well as confocal microscopy, CD4+ T cells were closely colocalized with DCs in the postischemic liver. Pretreatment with paricalcitol attenuated I/R-induced maturation of DCs (flow cytometry), CD4+ T-cell recruitment into the liver (intravital microscopy), and hepatocellular/microvascular damage (intravital microscopy, alanine aminotransferase/aspartate aminotransferase, histology). However, interruption of T-cell-DC interaction increased proinflammatory DC maturation and even enhanced tissue damage. Simultaneous treatment with an anti-CD44mAb completely abolished the beneficial effect of paricalcitol on T-cell migration and tissue injury. Our study demonstrates for the first time that hepatic DCs interact with CD4+ T cells in the postischemic liver in vivo; modulation of DCs and/or generation of tolerogenic DCs attenuates intrahepatic CD4+ T-cell recruitment and reduces I/R injury; and interruption of CD44-dependent CD4+ T-cell-DC interactions enhances tissue injury by preventing the modulatory effect of hepatic DCs on T cells, especially type 1 T helper effector cells. Thus, hepatic DCs are strongly involved in the promotion of CD4+ T-cell-dependent postischemic liver inflammation. [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]

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]

Ischemia-reperfusion activates innate immunity and sterile inflammation, resulting in acute kidney injury. Since pentraxin 3 (PTX3) regulates multiple aspects of innate immunity and tissue inflammation, we tested whether PTX3 would be involved in renal ischemia-reperfusion injury. Renal pedicle clamping increased PTX3 serum levels, as well as PTX3 expression, inside the kidney but predominantly in CD45/CD11c+ cells, a subpopulation of intrarenal mononuclear phagocytes. Lack of PTX3 aggravated postischemic acute kidney injury as evidenced by massive tubular necrosis, and TNF and IL-6 release, as well as massively increased neutrophil and macrophage infiltrates at 24 h. This was followed by tubular atrophy, interstitial fibrosis, and kidney shrinking 10 weeks later. In vivo microscopy uncovered increased leukocyte adhesion and transmigration in postischemic microvessels of Ptx3-deficient mice. Furthermore, injection of recombinant PTX3 up to 6 h after reperfusion prevented renal leukocyte recruitment and postischemic kidney injury. Thus, local PTX3 release from a subpopulation of intrarenal mononuclear phagocytes or delayed PTX3 treatment limits postischemic renal inflammation. Conversely, Ptx3 loss-of-function mutations predispose to postischemic acute kidney injury and subsequent chronic kidney disease. [ABSTRACT FROM AUTHOR]

Initial observations suggested that C-C motif chemokines exclusively mediate Chemotaxis of mononuclear cells. In addition, recent studies also implicated these chemotactic cytokines in the recruitment of neutrophils. The underlying mechanisms remained largely unknown. Using in vivo microscopy on the mouse cremas-ter muscle, intravascular adherence and subsequent paracellular transmigration of neutrophils elicited by the chemokine (C-C motif) ligand 3 (CCL3, synonym MIP-1&agr;) were significantly diminished in mice with a deficiency of the chemokine (C-C motif) receptor 1 (Ccr1-/-) or 5 (Ccr5-/-). Using cell-transfer techniques, neutrophil responses required leukocyte CCR1 and nonleukocyte CCR5. Furthermore, neutrophil extravasation elicited by CCL3 was almost completely abolished on inhibition of G protein-receptor coupling and PI3K&ggr;-dependent signaling, while neutrophil recruitment induced by the canonical neutrophil attractants chemokine (C-X-C motif) ligand 1 (CXCL1, synonym KC) or the lipid mediator platetelet-activating factor (PAF) was only partially reduced. Moreover, Ab blockade of &bgr;2 integrins, of «, integrins, or of their putative counter receptors ICAM-1 and VCAM-1 significantly attenuated CCL3-, CXCL1-, or PAF-elicited intravascular adherence and paracellular transmigration of neutrophils. These data indicate that the C-C motif chemokine CCL3 and canonical neutrophil attractants exhibit both common and distinct mechanisms for the regulation of intravascular adherence and transmigration of neutrophils. [ABSTRACT FROM AUTHOR]

Abstract: The increasing interest in biomedical applications of semiconductor quantum dots (QDs) is closely linked to the use of surface modifications to target specific sites of the body. The immense surface area of vascular endothelium is a possible interaction platform with systemically administered QDs. Therefore, the aim of this study was to investigate the microvascular distribution of neutral, cationic, and anionic QDs in vivo. QDs with carboxyl-, amine- and polyethylene glycol surface coatings were injected into the blood circulation of mice. In vivo microscopy of the cremaster muscle, two-photon microscopy of skeletal and heart muscle, as well as quantitative fluorescence measurements of blood, excreta, and tissue samples were performed. Transmission electron microscopy was used to detect QDs at the cellular level. The in vitro association of QDs with cultured endothelial cells was investigated by flow cytometry and confocal microscopy. Anionic QDs exhibited a very low residence time in the blood stream, preferably accumulated in organs with a prominent mononuclear phagocytic component, but were also found in other tissues with low phagocytic properties where they were predominantly associated with capillary endothelium. This deposition behavior was identified as a new, phagocyte-independent principle contributing to the rapid clearance of anionic QDs from the circulation. [Copyright &y& Elsevier]

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]