Optimization of extracellular vesicles preparation from saliva of head and neck cancer patients.

Small extracellular vesicles from saliva (SEVs) have high potential as biomarkers in Head and Neck cancer (HNC). However, there is no common consensus on the ideal method for their isolation. This study compared different ultracentrifugation (UC) methods (durations and + /- additional purification) with size exclusion chromatography (SEC) and investigated the potential of SEVs as diagnostic biomarkers and their biological activity on NK and CD8+ T cells. SEVs from 19 HNC patients and 8 healthy donors (HDs) were thoroughly characterized. Transmission electron microscopy confirmed the isolation of vesicles by all methods. The average size determined via nanoparticle-tracking analysis was smaller for SEVs isolated by SEC than UC. The highest particle-to-protein yield was achieved by UC (3 h + 3 h) (UCopt) and SEC. However, SEC yielded considerably fewer SEVs. Comparing the surface marker cargo, SEVs isolated by UCopt from HNC patients carried more PD-L1, FasL, and TGF-ß than SEVs from HDs. These levels correlated with tumor stage and HPV status. SEVs downregulated NKG2D expression on primary NK cells. HNC SEVs accelerated CD8+ T cell death compared to HD SEVs. This study suggests that UCopt is preferable when isolation of a high particle-to-protein load is required. Especially PD-L1 and FasL on SEVs hold substantial potential as diagnostic biomarkers.

Modulation of PD­L1 expression by standard therapy in head and neck cancer cell lines and exosomes.

Although checkpoint inhibitors (CPI) have recently extended the treatment options and improved clinical response of advanced stage head and neck squamous cell carcinoma (HNSCC), treatment success remains unpredictable. Programmed cell death ligand­1 (PD­L1) is a key player in immunotherapy. Tumor cells, and exosomes derived therefrom, are carriers of PD­L1 and efficiently suppress immune responses. The aim of the present study was to analyze the influence of established therapies on PD­L1 expression of HNSCC cell lines and their exosomes. The HNSCC cell lines, UM­SCC­11B, UM­SCC­14C and UM­SCC­22C were treated with fractionated radiotherapy (RT; 5x2 Gy), cisplatin (CT) and cetuximab (Cetux) as monotherapy, or combined therapy, chemoradiotherapy (CRT; RT and CT) or radioimmunotherapy (RT and Cetux). The expression of PD­L1 and phosphorylated (p)ERK1/2 as a mediator of radioresistance were assessed using western blotting, immunohistochemistry and an ex vivo vital tissue culture model. Additionally, exosomes were isolated from concentrated supernatants of the (un­)treated HNSCC cell lines by size exclusion chromatography. Exosomal protein expression levels of PD­L1 were detected using western blotting and semi­quantitative levels were calculated. The functional impact of exosomes from the (un­)treated HNSCC cell lines on the proliferation (MTS assay) and apoptosis (Caspase 3/7 assay) of the untreated HNSCC cell lines were measured and compared. The HNSCC cell lines UM­SCC­11B and UM­SCC­22B showed strong expression of pERK1/2 and PD­L1, respectively. RT upregulated the PD­L1 expression in UM­SCC­11B and UM­SCC­14C and in exosomes from all three cell lines. CT alone induced PD­L1 expression in all cell lines. CRT induced the expression of PD­L1 in all HNSCC cell lines and exosomes from UM­SCC­14C and UM­SCC­22B. The data indicated a potential co­regulation of PD­L1 and activated ERK1/2, most evident in UM­SCC­14C. Exosomes from irradiated UM­SCC­14C cells protected the unirradiated cells from apoptosis by Caspase 3/7 downregulation. The present study suggested a tumor cell­mediated regulation of PD­L1 upon platinum­based CRT in HNSCC and in exosomes. A co­regulation of PD­L1 and MAPK signaling response was hypothesized.

Plasma-derived small extracellular vesicles unleash the angiogenic potential in head and neck cancer patients.

BACKGROUND: In Head and neck cancer (HNC) angiogenesis is essential for tumor progression and metastasis. Small extracellular vesicles (sEVs) from HNC cell lines alter endothelial cell (EC) functions towards a pro-angiogenic phenotype. However, the role of plasma sEVs retrieved from HNC patients in this process is not clear so far. METHODS: Plasma sEVs were isolated on size exclusion chromatography columns from 32 HNC patients (early-stage UICC I/II: 8, advanced-stage UICC III/IV: 24), 12 patients with no evident disease after therapy (NED) and 16 healthy donors (HD). Briefly, sEVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), BCA protein assays and Western blots. Levels of angiogenesis-associated proteins were determined using antibody arrays. The interaction of fluorescently-labeled sEVs with human umbilical vein ECs was visualized by confocal microscopy. The functional effect of sEVs on tubulogenesis, migration, proliferation and apoptosis of ECs was assessed. RESULTS: The internalization of sEVs by ECs was visualized using confocal microscopy. Based on antibody arrays, all plasma sEVs were enriched in anti-angiogenic proteins. HNC sEVs contained more pro-angiogenic MMP-9 and anti-angiogenic proteins (Serpin F1) than HD sEVs. Interestingly, a strong inhibition of EC function was observed for sEVs from early-stage HNC, NED and HD. In contrast, sEVs from advanced-stage HNC showed a significantly increased tubulogenesis, migration and proliferation and induced less apoptosis in ECs than sEVs from HD. CONCLUSIONS: In general, plasma sEVs carry a predominantly anti-angiogenic protein cargo and suppress the angiogenic properties of ECs, while sEVs from (advanced-stage) HNC patients induce angiogenesis compared to HD sEVs. Thus, tumor-derived sEVs within the plasma of HNC patients might shift the angiogenic switch towards angiogenesis.

[Sleep medicine for the ENT specialist]. / Schlafmedizin für den Hals-Nasen-Ohren-Facharzt.

Sleep-related breathing disorders can be divided into obstructive and central sleep apnea, and hypoventilation syndromes. The diagnosis is made according to an algorithm in which clinical symptoms and a polygraphy or polysomnography usually point the way. After initial diagnosis of the most common obstructive sleep apnea, conservative therapies such as positive airway pressure therapy (PAP therapy), positional therapy, and/or mandibular advancement splint are used in many cases, supplemented by treatment of risk factors. If PAP therapy is not possible, more detailed diagnosis of airway obstruction, often with sleep videoendoscopy, is required. In general, "muscle-sparing" surgical techniques such as tonsillectomy with uvulapalatopharyngoplasty (TE-UPPP) should be considered whenever possible. This is especially important in the surgical treatment of snoring. More surgical therapy alternatives are for example barbed wire pharyngoplasty, tongue pacemaker and bimaxillary advancement. Optimal therapy alternatives should be evaluated in a sleep medicine center.

Immunization against Gonadotropin-Releasing Hormone in Female Beef Calves to Avoid Pregnancy at Time of Slaughter.

Precocious puberty in beef heifers can result in unwanted pregnancies due to accidental breeding by farm bulls. Inbreeding, premature calving followed by dystocia and a high stillbirth rate or slaughtering of pregnant heifers are the consequences of this behaviour. The aim of the study was to postpone puberty by using Improvac®, an anti-GnRH vaccine. Therefore, n = 25 calves were twice vaccinated, once at the age of 5 and then at 6.5 months. n = 24 calves served as unvaccinated case controls. The onset of puberty was assigned if progesterone analysis in the blood exceeded 1 ng/mL. Progesterone values were excluded if the corresponding serum cortisol levels were ≥60 nmol/L. Our target was met, as in the vaccinated group none of the calves exceeded a progesterone value >1 ng/mL until the scheduled age of slaughter at 11 months and only 12.5% of the animals exceeded a progesterone value of 1 ng/mL over the whole measuring period (>400 days) compared with 56.5% of the calves in the control group. In conclusion, the favourable results from our study using the vaccine Improvac® represent an animal-friendly, non-invasive and reliable way to avoid early pregnancy in heifers as well as the slaughter of pregnant cattle.

Validating a two-high-threshold measurement model for confidence rating data in recognition.

Signal Detection models as well as the Two-High-Threshold model (2HTM) have been used successfully as measurement models in recognition tasks to disentangle memory performance and response biases. A popular method in recognition memory is to elicit confidence judgements about the presumed old/new status of an item, allowing for the easy construction of ROCs. Since the 2HTM assumes fewer latent memory states than response options are available in confidence ratings, the 2HTM has to be extended by a mapping function which models individual rating scale usage. Unpublished data from 2 experiments in Bröder and Schütz (2009) validate the core memory parameters of the model, and 3 new experiments show that the response mapping parameters are selectively affected by manipulations intended to affect rating scale use, and this is independent of overall old/new bias. Comparisons with SDT show that both models behave similarly, a case that highlights the notion that both modelling approaches can be valuable (and complementary) elements in a researcher's toolbox.

Signal detection and threshold models of source memory.

Recently, several authors claimed that the curvilinear shape of rating-based source memory receiver operating characteristics (ROCs) refutes threshold models. However, rating-based ROCs are not diagnostic to disprove threshold models. Furthermore, source memory ROC-analyses ignore influences of other processes like old-new-detection and old-new-response-tendencies, so direct estimation of parameters is preferable. Five source monitoring experiments with different response bias manipulations and materials were conducted. We fitted the Two High Threshold Multinomial Model of Source Monitoring by Bayen, Murnane, and Erdfelder (1996) and the Multivariate Signal Detection Model for Selection DeCarlo (2003). We also included rating-based ROCs. The results suggest that both models are at least equally valid as measurement tools which capture bias processes in the corresponding parameters, and they can perhaps be integrated theoretically.

Recognition ROCs are curvilinear-or are they? On premature arguments against the two-high-threshold model of recognition.

Recent reviews of recognition receiver operating characteristics (ROCs) claim that their curvilinear shape rules out threshold models of recognition. However, the shape of ROCs based on confidence ratings is not diagnostic to refute threshold models, whereas ROCs based on experimental bias manipulations are. Also, fitting predicted frequencies to actual data is a more sensitive method for model comparisons than ROC regressions. In a reanalysis of 59 published data sets, the 2-high-threshold model (2HTM) fit the data better than an unequal variance signal detection model in about half of the cases. Three recognition experiments with experimental bias manipulation were conducted that yielded linear ROCs and a better fit of the 2HTM in all cases. On the basis of actual data and a simulation, the authors argue that both models are at least equally valid as measurement tools and can perhaps be integrated theoretically.

Utilization of covariation knowledge in source monitoring: no evidence for implicit processes.

In three experiments, a "hidden covariation" (Lewicki, in Journal of Experimental Psychology: Learning, Memory, and Cognition, 12, 135-146, 1986) of nonsalient stimulus attributes and the source of stimulus information was established to test whether implicit knowledge about this correlation influences source memory judgments. The source monitoring framework (Johnson, Hashtroudi, and Lindsay, in Psychological Bulletin, 114, 3-28, 1993) postulates heuristic and strategic judgment processes in source attributions. A multinomial model analysis disentangled memory and guessing processes. While there were large strategic guessing biases involving explicit knowledge in all experiments, there was no evidence for the use of implicit covariation knowledge. Only participants who were later able to verbalize the covariation had shown corresponding biases during the source memory test, suggesting that implicit covariation knowledge plays no prominent role in the reconstruction processes in source monitoring.