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Abstract Background The majority of patients with recurrent or metastasized head and neck squamous cell carcinoma (HNSCC) do not benefit from immune checkpoint blockade (ICB) while several patients experience severe and persistent immune-mediated side effects. Therefore, predictive biomarkers are urgently needed to allow for a personalized treatment. In this study, we investigated DNA methylation of the immune checkpoint gene CTLA4 with regard to its predictive value. Methods We analyzed CTLA4 promoter methylation in tumors of HNSCC patients (N = 29) treated with ICB at the University Medical Center Bonn with regard to response to ICB and progression-free survival. We further analyzed a second cohort (N = 138) of patients that did not receive ICB with regard to CTLA4 promoter methylation, CTLA-4 protein expression, and immune cell infiltrates. Finally, we tested inducibility of CTLA-4 protein expression in HNSCC cells using the DNA methyltransferase inhibitor decitabine. Results Lower CTLA4 promoter methylation correlated with response to ICB and prolonged progression-free survival. We could show that not only tumor infiltrating immune cells, but also HNSCC cells harbor cytoplasmic and nuclear CTLA-4 expression. CTLA4 promoter methylation inversely correlated with infiltrates of CD3+, CD4+, CD8+, and CD45+ immune cells. CTLA4 methylation did not correlate with protein expression in tumors, however, decitabine treatment led to decreased CTLA4 methylation and an induction of CTLA4 mRNA and CTLA-4 protein expression in HNSCC cell lines. Conclusions Our results indicate that CTLA4 DNA hypomethylation is a predictive biomarker for response to ICB in HNSCC. Our study warrants further analyses of the predictive value of CTLA4 DNA methylation in clinical trials of anti-PD-1 and/or anti-CTLA-4 immunotherapy in HNSCC.

Abstract Background Inducible T cell costimulator ICOS is an emerging target in immuno-oncology. The aim of this study was to investigate the epigenetic regulation of ICOS in melanoma by DNA methylation. Methods We comprehensively investigate ICOS DNA methylation of specific CpG sites and expression pattern within the melanoma microenvironment with regard to immune correlates, differentiation, clinical outcomes, and immune checkpoint blockade (ICB) response. Results Our study revealed a sequence-contextual CpG methylation pattern consistent with an epigenetically regulated gene. We found a cell type-specific methylation pattern and locus-specific correlations and associations of CpG methylation with ICOS mRNA expression, immune infiltration, melanoma differentiation, prognosis, and response to ICB. High ICOS mRNA expression was identified as a surrogate for enriched immune cell infiltration and was associated with favorable overall survival (OS) in non-ICB-treated patients and predicted response and a prolonged progression-free survival (PFS) following ICB therapy initiation. ICOS hypomethylation, however, significantly correlated with poor OS in non-ICB patients but predicted higher response and prolonged PFS and OS in ICB-treated patients. Moreover, we observed cytoplasmic and sporadically nuclear tumor cell-intrinsic ICOS protein expression. Tumor cell-intrinsic ICOS protein and mRNA expression was inducible by pharmacological demethylation with decitabine. Conclusion Our study identified ICOS DNA methylation and mRNA expression as promising prognostic and predictive biomarkers for immunotherapy in melanoma and points towards a hitherto undescribed role of ICOS in tumor cells.

ABSTRACTPD-L1 status assessed by immunohistochemistry (IHC) has failed to reliably predict outcomes for patients with metastatic urothelial carcinoma (mUC) on immune checkpoint blockade (ICB). PD-L1 promoter methylation is an epigenetic mechanism that has been shown to regulate PD-L1 mRNA expression in various malignancies. The aim of our present study was to evaluate the predictive potential of PD-L1 promoter methylation status (mPD-L1) in ICB-treated mUC compared to conventional IHC-based PD-L1 assessment. We quantified mPD-L1 in formalin-fixed and paraffin-embedded tissue sections using an established quantitative methylation-specific PCR assay (qMSP) in a well-characterized multicenter ICB-treated cohort comprising N = 107 patients with mUC. Additionally, PD-L1 protein expression in tumor tissues was assessed using regulatory approved IHC protocols. The effect of pharmacological hypomethylation by the DNA methyltransferase inhibitor decitabine in combination with interferon-γ stimulation in urothelial carcinoma cell lines was investigated by IHC and FACS. mPD-L1 hypomethylation predicted objective response rate at the first staging on ICB. Patients with tumors categorized as PD-L1 hypomethylated (lower quartile) showed significantly prolonged progression-free (PFS) and overall survival (OS) after ICB initiation. In contrast, PD-L1 protein expression status neither correlated with response nor survival. In multivariable Cox regression analyses, PD-L1 promoter hypermethylation remained an independent predictor of unfavorable PFS and OS. In urothelial carcinoma cell lines, pharmacological demethylation led to an upregulation of membranous PD-L1 expression and an enhanced inducibility of PD-L1 expression by interferon γ. Hypomethylation of the PD-L1 promoter is a promising predictive biomarker for response to ICB in patients with mUC.

Abstract Background Idiopathic recurrent cervical swelling may be caused by lymphatic abnormalities. Methods Ten patients (9 females, mean age 51.2 ± 7) with idiopathic recurrent cervical swelling underwent MR‐lymphangiography (MRL). MR‐lymphangiograms were evaluated regarding lymphatic anatomy and flow. Individualized treatment was recommended according to MRL‐findings. Results 8/10 patients presented with left‐sided, 2/10 with right‐sided swelling. Pathological lymph‐flow was identified in all cases: thoracic duct dilatation in patients with left‐sided and right lymphatic duct dilatation in right‐sided swelling, accessory thoracic lymphatics in 7/10 and reflux in 8/10 cases. In two cases, a lymphatic thrombus was identified. After treatment, symptoms resolved completely in 6/10 cases and partially in 1/10 cases. The remaining three patients have intermittent swellings but have no treatment wish. Conclusion Idiopathic recurrent cervical swelling can be caused by lymphatic anomalies. MRL displays impaired lymphatic drainage, lymphatic vessel dilatation, and chylolymphatic reflux as hallmarks of this condition and may aid in targeted treatment planning.

IntroductionPreservation of residual hearing remains a great challenge during cochlear implantation. Cochlear implant (CI) electrode array insertion induces changes in the microvasculature as well as nitric oxide (NO)-dependent vessel dysfunction which have been identified as possible mediators of residual hearing loss after cochlear implantation.MethodsA total of 24 guinea pigs were randomized to receive either a CI (n = 12) or a sham procedure (sham) by performing a cochleostomy without electrode array insertion (n = 12). The hearing threshold was determined using frequency-specific compound action potentials. To gain visual access to the stria vascularis, a microscopic window was created in the osseous cochlear lateral wall. Cochlear blood flow (CBF) and cochlear microvascular permeability (CMP) were evaluated immediately after treatment, as well as after 1 and 2 h, respectively. Finally, cochleae were resected for subsequent immunohistochemical analysis of the iNOS expression.ResultsThe sham control group showed no change in mean CBF after 1 h (104.2 ± 0.7%) and 2 h (100.8 ± 3.6%) compared to baseline. In contrast, cochlear implantation resulted in a significant continuous decrease in CBF after 1 h (78.8 ± 8.1%, p < 0.001) and 2 h (60.6 ± 11.3%, p < 0.001). Additionally, the CI group exhibited a significantly increased CMP (+44.9% compared to baseline, p < 0.0001) and a significant increase in median hearing threshold (20.4 vs. 2.5 dB SPL, p = 0.0009) compared to sham after 2 h. Intriguingly, the CI group showed significantly lower iNOS-expression levels in the organ of Corti (329.5 vs. 54.33 AU, p = 0.0003), stria vascularis (596.7 vs. 48.51 AU, p < 0.0001), interdental cells (564.0 vs. 109.1 AU, p = 0.0003) and limbus fibrocytes (119.4 vs. 18.69 AU, p = 0.0286).ConclusionMechanical and NO-dependent microvascular dysfunction seem to play a pivotal role in residual hearing loss after CI electrode array insertion. This may be facilitated by the implantation associated decrease in iNOS expression. Therefore, stabilization of cochlear microcirculation could be a therapeutic strategy to preserve residual hearing.

Exposure to nanoparticles is inevitable as they become widely used in industry, cosmetics, and foods. However, knowledge of their (patho)physiological effects on biological entry routes of the human body and their underlying molecular mechanisms is still fragmented. Here, we examined the molecular effects of amorphous silica nanoparticles (aSiNPs) on cell lines mimicking the alveolar-capillary barrier of the lung. After state-of-the-art characterization of the used aSiNPs and the cell model, we performed cell viability-based assays and a protein analysis to determine the aSiNP-induced cell toxicity and underlying signaling mechanisms. We revealed that aSiNPs induce apoptosis in a dose-, time-, and size-dependent manner. aSiNP-induced toxicity involves the inhibition of pro-survival pathways, such as PI3K/AKT and ERK signaling, correlating with reduced expression of the anti-apoptotic protein Survivin on the protein and transcriptional levels. Furthermore, induced Survivin overexpression mediated resistance against aSiNP-toxicity. Thus, we present the first experimental evidence suggesting Survivin as a critical cytoprotective resistor against silica-based nanotoxicity, which may also play a role in responses to other NPs. Although Survivin’s relevance as a biomarker for nanotoxicity needs to be demonstrated in vivo, our data give general impetus to investigate the pharmacological modulation of Survivin`s functions to attenuate the harmful effects of acute or chronic inhalative NP exposure.

Transportation noise is recognized as an important cardiovascular risk factor. Key mechanisms are noise-triggered vascular inflammation and oxidative stress with subsequent endothelial dysfunction. Here, we test for adaptation or tolerance mechanisms in mice in response to chronic noise exposure. C57BL/6J mice were exposed to aircraft noise for 0, 4, 7, 14 and 28d at a mean sound pressure level of 72 dB(A) and peak levels of 85 dB(A). Chronic aircraft noise exposure up to 28d caused persistent endothelial dysfunction and elevation of blood pressure. Likewise, reactive oxygen species (ROS) formation as determined by dihydroethidium (DHE) staining and HPLC-based measurement of superoxide formation in the aorta/heart/brain was time-dependently increased by noise. Oxidative burst in the whole blood showed a maximum at 4d or 7d of noise exposure. Increased superoxide formation in the brain was mirrored by a downregulation of neuronal nitric oxide synthase (Nos3) and transcription factor Foxo3 genes, whereas Vcam1 mRNA, a marker for inflammation was upregulated in all noise exposure groups. Induction of a pronounced hearing loss in the mice was excluded by auditory brainstem response audiometry. Endothelial dysfunction and inflammation were present during the entire 28d of aircraft noise exposure. ROS formation gradually increases with ongoing exposure without significant adaptation or tolerance in mice in response to chronic noise stress at moderate levels. These data further illustrate health side effects of long-term noise exposure and further strengthen a consequent implementation of the WHO noise guidelines in order to prevent the development of noise-related future cardiovascular disease.

Vitamin D (VitD) and its receptor (VDR) have been intensively investigated in many cancers. As knowledge for head and neck cancer (HNC) is limited, we investigated the (pre)clinical and therapeutic relevance of the VDR/VitD-axis. We found that VDR was differentially expressed in HNC tumors, correlating to the patients’ clinical parameters. Poorly differentiated tumors showed high VDR and Ki67 expression, whereas the VDR and Ki67 levels decreased from moderate to well-differentiated tumors. The VitD serum levels were lowest in patients with poorly differentiated cancers (4.1 ± 0.5 ng/mL), increasing from moderate (7.3 ± 4.3 ng/mL) to well-differentiated (13.2 ± 3.4 ng/mL) tumors. Notably, females showed higher VitD insufficiency compared to males, correlating with poor differentiation of the tumor. To mechanistically uncover VDR/VitD’s pathophysiological relevance, we demonstrated that VitD induced VDR nuclear-translocation (VitD < 100 nM) in HNC cells. RNA sequencing and heat map analysis showed that various nuclear receptors were differentially expressed in cisplatin-resistant versus sensitive HNC cells including VDR and the VDR interaction partner retinoic acid receptor (RXR). However, RXR expression was not significantly correlated with the clinical parameters, and cotreatment with its ligand, retinoic acid, did not enhance the killing by cisplatin. Moreover, the Chou–Talalay algorithm uncovered that VitD/cisplatin combinations synergistically killed tumor cells (VitD < 100 nM) and also inhibited the PI3K/Akt/mTOR pathway. Importantly, these findings were confirmed in 3D-tumor-spheroid models mimicking the patients’ tumor microarchitecture. Here, VitD already affected the 3D-tumor-spheroid formation, which was not seen in the 2D-cultures. We conclude that novel VDR/VitD-targeted drug combinations and nuclear receptors should also be intensely explored for HNC. Gender-specific VDR/VitD-effects may be correlated to socioeconomic differences and need to be considered during VitD (supplementation)-therapies.

Epidemiological studies showed that traffic noise has a dose-dependent association with increased cardiovascular morbidity and mortality. Whether microvascular dysfunction contributes significantly to the cardiovascular health effects by noise exposure remains to be established. The connection of inflammation and immune cell interaction with microvascular damage and functional impairment is also not well characterized. Male C57BL/6J mice or gp91phox−/y mice with genetic deletion of the phagocytic NADPH oxidase catalytic subunit (gp91phox or NOX-2) were used at the age of 8 weeks, randomly instrumented with dorsal skinfold chambers and exposed or not exposed to aircraft noise for 4 days. Proteomic analysis (using mass spectrometry) revealed a pro-inflammatory phenotype induced by noise exposure that was less pronounced in noise-exposed gp91phox−/y mice. Using in vivo fluorescence microscopy, we found a higher number of adhesive leukocytes in noise-exposed wild type mice. Dorsal microvascular diameter (by trend), red blood cell velocity, and segmental blood flow were also decreased by noise exposure indicating microvascular constriction. All adverse effects on functional parameters were normalized or improved at least by trend in noise-exposed gp91phox−/y mice. Noise exposure also induced endothelial dysfunction in cerebral microvessels, which was associated with higher oxidative stress burden and inflammation, as measured using video microscopy. We here establish a link between a pro-inflammatory phenotype of plasma, activation of circulating leukocytes and microvascular dysfunction in mice exposed to aircraft noise. The phagocytic NADPH oxidase was identified as a central player in the underlying pathophysiological mechanisms.

Abstract Background Reports of head and neck ultrasound examinations are frequently written by hand as free texts. This is a serious obstacle to the learning process of the modality due to a missing report structure and terminology. Therefore, there is a great inter-observer variability in overall report quality. Aim of the present study was to evaluate the impact of structured reporting on the learning process as indicated by the overall report quality of head and neck ultrasound examinations within medical school education. Methods Following an immersion course on head and neck ultrasound, previously documented images of three common pathologies were handed out to 58 medical students who asked to create both standard free text reports (FTR) and structured reports (SR). A template for structured reporting of head and neck ultrasound examinations was created using a web-based approach. FTRs and SRs were evaluated with regard to overall quality, completeness, required time to completion and readability by two independent raters (Paired Wilcoxon test, 95% CI). Ratings were assessed for inter-rater reliability (Fleiss’ kappa). Additionally, a questionnaire was utilized to evaluate user satisfaction. Results SRs received significantly better ratings in terms of report completeness (97.7% vs. 53.5%, p

Abstract Background Reports of head and neck ultrasound examinations are frequently written by hand as free texts. Naturally, quality and structure of free text reports is variable, depending on the examiner’s individual level of experience. Aim of the present study was to compare the quality of free text reports (FTR) and structured reports (SR) of head and neck ultrasound examinations. Methods Both standard FTRs and SRs of head and neck ultrasound examinations of 43 patients were acquired by nine independent examiners with comparable levels of experience. A template for structured reporting of head and neck ultrasound examinations was created using a web-based approach. FTRs and SRs were evaluated with regard to overall quality, completeness, required time to completion, and readability by four independent raters with different specializations (Paired Wilcoxon test, 95% CI) and inter-rater reliability was assessed (Fleiss’ kappa). A questionnaire was used to compare FTRs vs. SRs with respect to user satisfaction (Mann-Whitney U test, 95% CI). Results By comparison, completeness scores of SRs were significantly higher than FTRs’ completeness scores (94.4% vs. 45.6%, p

This article contains supporting information on data collection for the research article entitled “Aircraft noise exposure drives the activation of white blood cells and induces microvascular dysfunction in mice” by Eckrich et al. We found that noise-induced stress triggered microvascular dysfunction via involvement of innate immune-derived reactive oxygen species. In this article, we present the instrumentation of mice with dorsal skinfold chambers for in vivo microscopic imaging of blood flow, interaction of leukocytes with the vascular wall (also by fluorescent labelling of blood cells) and vessel diameter. In addition, we explain the preparation of cerebral arterioles for measurement of vascular reactivity in vitro. • visualization of noise-dependent effects in dorsal skinfold chamber. • in vivo microscopy of noise-dependent activation of white blood cells. • analysis of noise-dependent microvascular dysfunction in dorsal skinfold chamber and cannulated cerebral arterioles.

Background and Objectives: Tonsillar infections are a common reason to see a physician and lead to a reduction in the patients’ health-related quality of life (HRQoL). HRQoL may be an important criterion in decision science and should be taken into account when deciding when to perform tonsillectomy, especially for chronic tonsillitis. The aim of this study was to determine the health utility for different states of tonsillar infections. Materials and Methods: Hospitalized patients with acute tonsillitis or a peritonsillar abscess were asked about their HRQoL with the 15D questionnaire. Patients who had undergone tonsillectomy were reassessed six months postoperatively. Results: In total, 65 patients participated in the study. The health states of acute tonsillitis and peritonsillar abscess had both a utility of 0.72. Six months after tonsillectomy, the mean health utility was 0.95. Conclusions: Our study confirms a substantial reduction in utility due to tonsillar infections. Tonsillectomy significantly improves the utility and therefore HRQoL six months after surgery.

Background: The co-receptor lymphocyte-activation gene-3 (LAG3, LAG-3, CD223) is a potential target for immune checkpoint inhibition immunotherapies. However, little is known about the biological and clinical significance of LAG3 DNA methylation in melanoma and its microenvironment. Methods: We evaluated LAG3 promoter and gene body methylation in a cohort of N = 470 melanoma patients obtained from The Cancer Genome Atlas (TCGA cohort), an independent cohort of N = 120 patients from the University Hospital Bonn, and in subsets of peripheral blood leukocytes, melanocytes, and melanoma cell lines. We validated the association of LAG3 methylation with mRNA expression in vitro in the melanoma cell line A375 treated with the hypomethylating agent 5-azacytidine and stimulated with interferon-γ. Finally, we investigated correlations between LAG3 methylation and progression-free survival in patients treated with immune checkpoint blockade (ICB cohort, N = 118). Findings: Depending on the analysed locus (promoter, gene body) we found region-dependent significant LAG3 methylation differences between monocytes, B cells, CD8+ and CD4+ T cells, regulatory T cells, melanocytes, and melanoma cell lines. In tumor tissues, methylation correlated significantly with LAG3 mRNA expression, immune cell infiltrates (histopathologic lymphocyte score and RNA-Seq signatures of distinct immune infiltrates), and an interferon-γ signature. Finally, LAG3 methylation was associated with overall survival in the TCGA cohort and progression-free survival in the ICB cohort. We detected basal LAG3 mRNA expression in the melanoma cell A375 and an interferon-γ inducible expression after demethylation with 5-azacytidine. Interpretation: Our study points towards an epigenetic regulation of LAG3 via promoter methylation and suggests a prognostic and predictive significance of LAG3 methylation in melanoma. Our results give insight in the tumor cell-intrinsic transcriptional regulation of LAG3 in melanoma. In perspective, our results might pave the way for investigating LAG3 methylation as a predictive biomarker for response to anti-LAG3 immune checkpoint blockage. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Computed tomography (CT) scans represent the gold standard in the planning of functional endoscopic sinus surgeries (FESS). Yet, radiologists and otolaryngologists have different perspectives on these scans. In general, residents often struggle with aspects involved in both reporting and operation planning. The aim of this study was to compare the completeness of structured reports (SR) of preoperative CT images and structured operation planning (SOP) to conventional reports (CR) and conventional operation planning (COP) to potentially improve future treatment decisions on an individual level. In total, 30 preoperative CT scans obtained for surgical planning of patients scheduled for FESS were evaluated using SR and CR by radiology residents. Subsequently, otolaryngology residents performed a COP using free texts and a SOP using a specific template. All radiology reports and operation plannings were evaluated by two experienced FESS surgeons regarding their completeness for surgical planning. User satisfaction of otolaryngology residents was assessed by using visual analogue scales. Overall radiology report completeness was significantly higher using SRs regarding surgically important structures compared to CRs (84.4 vs. 22.0%, p

Abstract Head and neck squamous cell carcinoma (HNSCC) often metastasize to lymph nodes resulting in poor prognosis for patients. Unfortunately, the underlying molecular mechanisms contributing to tumour aggressiveness, recurrences, and metastasis are still not fully understood. However, such knowledge is key to identify biomarkers and drug targets to improve prognosis and treatments. Consequently, we performed genome-wide expression profiling of 15 primary HNSSCs compared to corresponding lymph node metastases and non-malignant tissue of the same patient. Differentially expressed genes were bioinformatically exploited applying stringent filter criteria, allowing the discrimination between normal mucosa, primary tumours, and metastases. Signalling networks involved in invasion contain remodelling of the extracellular matrix, hypoxia-induced transcriptional modulation, and the recruitment of cancer associated fibroblasts, ultimately converging into a broad activation of PI3K/AKT-signalling pathway in lymph node metastasis. Notably, when we compared the diagnostic and prognostic value of sequencing data with our expression analysis significant differences were uncovered concerning the expression of the receptor tyrosine kinases EGFR and ERBB2, as well as other oncogenic regulators. Particularly, upregulated receptor tyrosine kinase combinations for individual patients varied, implying potential compensatory and resistance mechanisms against specific targeted therapies. Collectively, we here provide unique transcriptional profiles for disease predictions and comprehensively analyse involved signalling pathways in advanced HNSCC.

Background: Porous polyethylene (PPE) implants are used for the reconstruction of tissue defects but have a risk of rejection in case of insufficient ingrowth into the host tissue. Various growth factors can promote implant ingrowth, yet a long-term gradient is a prerequisite for the mediation of these effects. As modification of the implant surface with nanocarriers may facilitate a long-term gradient by sustained factor release, implants modified with crosslinked albumin nanocarriers were evaluated in vivo. Methods: Nanocarriers from murine serum albumin (MSA) were prepared by an inverse miniemulsion technique encapsulating either a low- or high-molar mass fluorescent cargo. PPE implants were subsequently coated with these nanocarriers. In control cohorts, the implant was coated with the homologue non-encapsulated cargo substance by dip coating. Implants were consequently analyzed in vivo using repetitive fluorescence microscopy utilizing the dorsal skinfold chamber in mice for ten days post implantation. Results: Implant-modification with MSA nanocarriers significantly prolonged the presence of the encapsulated small molecules while macromolecules were detectable during the investigated timeframe regardless of the form of application. Conclusions: Surface modification of PPE implants with MSA nanocarriers results in the alternation of release kinetics especially when small molecular substances are used and therefore allows a prolonged factor release for the promotion of implant integration.

ObjectiveFitting cochlear implants, especially the precise determination of electrical hearing thresholds, is a time-consuming and complex task for patients as well as audiologists. Aim of the study was to develop a method that enables cochlear implant (CI) patients to determine their electrical hearing thresholds precisely and independently. Applicability and impact of this method on speech perception in noise at soft speech levels were evaluated.MethodAn adaptive psychoacoustic procedure for precise hearing threshold determination (precT) was implemented using MatLab (MathWorks) and a graphical user interface was created. Sound signals were calibrated with a CIC4-Implant-Decoder. Study design: A prospective study including 15 experienced adult cochlear implant users was conducted. Electrical hearing thresholds were determined using the automated precT procedure (auto-precT). Speech perception in noise at 50 dB SPL presentation levels was measured for three conditions: (P1) T-levels kept at the previously established T-levels; (P2) T-levels set to the hearing thresholds determined using auto-precT application; (P3) T-levels set 10 cu below the values determined with auto-precT.ResultsAll subjects were able to perform the auto-precT application independently. T-levels were altered on average by an absolute value of 10.5 cu using auto-precT. Median speech reception thresholds were significantly improved from 2.5 dB SNR (P1) to 1.6 dB SNR (P2, p = 0.02). Speech perception was lowest using the globally lowered T-levels, median 2.9 dB SNR (P3, not significant compared to P1 and P2).ConclusionThe applicability of the developed auto-precT application was confirmed in the present clinical study. Patients benefited from adjusting previously established T-levels to the threshold levels determined by the auto-precT application. The integration of the application in the clinical fitting routine as well as a remote fitting software approach is recommended. Furthermore, future possibilities of auto-precT include the implementation of the application on tablets or smart phones.

The selenoenzyme glutathione peroxidase 4 (GPx4) has been described to control specific cyclooxygenases (COXs) and lipoxygenases (LOXs) that exert substantiated functions in tumor growth and angiogenesis. Therefore, we hypothesized a putative regulatory role of GPx4 during tumor progression and created transformed murine embryonic fibroblasts with inducible disruption of GPx4. GPx4 inactivation caused rapid cell death in vitro, which could be prevented either by lipophilic antioxidants or by 12/15-LOX-specific inhibitors, but not by inhibitors targeting other LOX isoforms or COX. Surprisingly, transformed GPx4+/- cells did not die when grown in Matrigel but gave rise to tumor spheroids. Subcutaneous implantation of tumor cells into mice resulted in knockout tumors that were indistinguishable in volume and mass in comparison to wild-type tumors. However, further analysis revealed a strong vascular phenotype. We observed an increase in microvessel density as well as a reduction in the number of large diameter vessels covered by smooth muscle cells. This phenotype could be linked to increased 12/15-LOX activity that was accompanied by an up-regulation of basic fibroblast growth factor and down-regulation of vascular endothelial growth factor A protein expression. Indeed, pharmacological inhibition of 12/15-LOX successfully reversed the tumor phenotype and led to “normalized” vessel morphology. Thus, we conclude that GPx4, through controlling 12/15-LOX activity, is an important regulator of tumor angiogenesis as well as vessel maturation.

Rapid fibrovascularization is a prerequisite for successful biomaterial engraftment. In addition to their well-known roles in fibrinolysis, urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA) or their inhibitor plasminogen activator inhibitor-1 (PAI-1) have recently been implicated as individual mediators in non-fibrinolytic processes, including cell adhesion, migration, and proliferation. Since these events are critical for fibrovascularization of biomaterial, we hypothesized that the components of the plasminogen activation system contribute to biomaterial engraftment. Employing in vivo and ex vivo microscopy techniques, vessel and collagen network formation within porous polyethylene (PPE) implants engrafted into dorsal skinfold chambers were found to be significantly impaired in uPA-, tPA-, or PAI-1-deficient mice. Consequently, the force required for mechanical disintegration of the implants out of the host tissue was significantly lower in the mutant mice than in wild-type controls. Conversely, surface coating with recombinant uPA, tPA, non-catalytic uPA, or PAI-1, but not with non-catalytic tPA, accelerated implant vascularization in wild-type mice. Thus, uPA, tPA, and PAI-1 contribute to the fibrovascularization of PPE implants through common and distinct effects. As clinical perspective, surface coating with recombinant uPA, tPA, or PAI-1 might provide a novel strategy for accelerating the vascularization of this biomaterial.

ObjectiveBetahistine is a histamine H(1)-receptor agonist and H(3)-receptor antagonist that is administered to treat Menière's disease. Despite widespread use, its pharmacological mode of action has not been entirely elucidated. This study investigated the effect of betahistine on guinea pigs at dosages corresponding to clinically used doses for cochlear microcirculation.MethodsThirty healthy Dunkin-Hartley guinea pigs were randomly assigned to five groups to receive betahistine dihydrochloride in a dose of 1,000 mg/kg b. w. (milligram per kilogram body weight), 0.100 mg/kg b. w., 0.010 mg/kg b. w., 0.001 mg/kg b. w. in NaCl 0.9% or NaCl 0.9% alone as placebo. Cochlear blood flow and mean arterial pressure were continuously monitored by intravital fluorescence microscopy and invasive blood pressure measurements 3 minutes before and 15 minutes after administration of betahistine.ResultsWhen betahistine was administered in a dose of 1.000 mg/kg b. w. cochlear blood flow was increased to a peak value of 1.340 arbitrary units (SD: 0.246; range: 0.933-1.546 arb. units) compared to baseline (pConclusionsBetahistine has a dose-dependent effect on the increase of blood flow in cochlear capillaries. The effects of the dosage range of betahistine on cochlear microcirculation corresponded well to clinically used single dosages to treat Menière's disease. Our data suggest that the improved effects of higher doses of betahistine in the treatment of Menière's disease might be due to a corresponding increase of cochlear blood flow.