Investigation of Stent Prototypes for the Eustachian Tube in Human Donor Bodies.

Chronic otitis media is often connected to Eustachian tube dysfunction. As successful treatment cannot be guaranteed with the currently available options, the aim is to develop a stent for the Eustachian tube (ET). Over the course of this development, different prototypes were generated and tested in ex vivo experiments. Four different prototypes of an ET stent and one commercially available coronary stent were implanted in the ET of seven human donor bodies. The position of the stents was verified by cone beam CT. The implanted ETs were harvested, embedded in resin and ground at 200 µm steps. Resulting images of the single steps were used to generate 3D models. The 3D models were then evaluated regarding position of the stent in the ET, its diameters, amount of squeezing, orientation of the axes and other parameters. Virtual reconstruction of the implanted ET was successful in all cases and revealed one incorrect stent placement. The cross-section increased for all metal stents in direction from the isthmus towards the pharyngeal orifice of the ET. Depending on the individual design of the metal stents (open or closed design), the shape varied also between different positions along a single stent. In contrast, the cross-section area and shape remained constant along the polymeric prototype. With the current investigation, insight into the behavior of different prototypes of ET stents was gained, which can help in defining the specifications for the intended ET stent.

Development of an In Vivo Model for Eustachian Tube Dysfunction.

Otitis media is often connected to Eustachian tube dysfunction (ETD). Until now, there was no large animal model available for the examination of new treatment methods such as stents for the Eustachian tube (ET). Thus, the aim of the study was to develop a method to reproducibly induce ETD by injection of fillers and without permanent closure of the ET. Tools for safe injection of hyaluronic acid (HA) in the surrounding of the ET were developed. In ex vivo experiments, HA mixed with Imeron® was injected close to the nasopharyngeal orifice of the ET of blackface sheep. The established depot was visualized using cone beam computer tomography and magnetic resonance imaging, and stents could be placed into the ET. A reliable position of the HA depot was achieved. This method was transferred to in vivo, and middle ear ventilation was investigated by tympanometry. ETD was achieved with amounts of 2.5 mL HA or higher. None of the animals showed any sign of discomfort or complications. The induced ETD lasted for 3 to 13 (maximum observation period) weeks and was also combined with middle ear effusion. A model of ETD based on injection of HA next to the ET was successfully established and is now available to test novel treatment options for ET functionality.

Perfect adaptation of CD8+ T cell responses to constant antigen input over a wide range of affinities is overcome by costimulation.

Reduced T cell responses by contrast antigen stimulation can be rescued by signals from costimulatory receptors.

Quantitative contributions of TNF receptor superfamily members to CD8+ T-cell responses.

T-cell responses to infections and cancers are regulated by co-signalling receptors grouped into the binary categories of co-stimulation or co-inhibition. The co-stimulation TNF receptor superfamily (TNFRSF) members 4-1BB, CD27, GITR and OX40 have similar signalling mechanisms raising the question of whether they have similar impacts on T-cell responses. Here, we screened for the quantitative impact of these TNFRSFs on primary human CD8+ T-cell cytokine production. Although both 4-1BB and CD27 increased production, only 4-1BB was able to prolong the duration over which cytokine was produced, and both had only modest effects on antigen sensitivity. An operational model explained these different phenotypes using shared signalling based on the surface expression of 4-1BB being regulated through signalling feedback. The model predicted and experiments confirmed that CD27 co-stimulation increases 4-1BB expression and subsequent 4-1BB co-stimulation. GITR and OX40 displayed only minor effects on their own but, like 4-1BB, CD27 could enhance GITR expression and subsequent GITR co-stimulation. Thus, different co-stimulation receptors can have different quantitative effects allowing for synergy and fine-tuning of T-cell responses.

Low-molecular-weight heparin use in coronavirus disease 2019 is associated with curtailed viral persistence: a retrospective multicentre observational study.

AIMS: Anticoagulation was associated with improved survival of hospitalized coronavirus disease 2019 (COVID-19) patients in large-scale studies. Yet, the development of COVID-19-associated coagulopathy (CAC) and the mechanism responsible for improved survival of anticoagulated patients with COVID-19 remain largely elusive. This investigation aimed to explore the effects of anticoagulation and low-molecular-weight heparin (LMWH) in particular on patient outcome, CAC development, thromboinflammation, cell death, and viral persistence. METHODS AND RESULTS: Data of 586 hospitalized COVID-19 patients from three different regions of Austria were evaluated retrospectively. Of these, 419 (71.5%) patients received LMWH and 62 (10.5%) received non-vitamin-K oral anticoagulants (NOACs) during hospitalization. Plasma was collected at different time points in a subset of 106 patients in order to evaluate markers of thromboinflammation (H3Cit-DNA) and the cell death marker cell-free DNA (cfDNA). Use of LMWH was associated with improved survival upon multivariable Cox regression (hazard ratio = 0.561, 95% confidence interval: 0.348-0.906). Interestingly, neither LMWH nor NOAC was associated with attenuation of D-dimer increase over time, or thromboinflammation. In contrast, anticoagulation was associated with a decrease in cfDNA during hospitalization, and curtailed viral persistence was observed in patients using LMWH leading to a 4-day reduction of virus positivity upon quantitative polymerase chain reaction [13 (interquartile range: 6-24) vs. 9 (interquartile range: 5-16) days, P = 0.009]. CONCLUSION: Time courses of haemostatic and thromboinflammatory biomarkers were similar in patients with and without LMWH, indicating either no effects of LMWH on haemostasis or that LMWH reduced hypercoagulability to levels of patients without LMWH. Nonetheless, anticoagulation with LMWH was associated with reduced mortality, improved markers of cell death, and curtailed viral persistence, indicating potential beneficial effects of LMWH beyond haemostasis, which encourages use of LMWH in COVID-19 patients without contraindications.

Human CD8+ T Cells Exhibit a Shared Antigen Threshold for Different Effector Responses.

T cells recognizing cognate pMHC Ags become activated to elicit a myriad of cellular responses, such as target cell killing and the secretion of different cytokines, that collectively contribute to adaptive immunity. These effector responses have been hypothesized to exhibit different Ag dose and affinity thresholds, suggesting that pathogen-specific information may be encoded within the nature of the Ag. In this study, using systematic experiments in a reductionist system, in which primary human CD8+ T cell blasts are stimulated by recombinant peptides presented on MHC Ag alone, we show that different inflammatory cytokines have comparable Ag dose thresholds across a 25,000-fold variation in affinity. Although costimulation by CD28, CD2, and CD27 increased cytokine production in this system, the Ag threshold remained comparable across different cytokines. When using primary human memory CD8+ T cells responding to autologous APCs, equivalent thresholds were also observed for different cytokines and killing. These findings imply a simple phenotypic model of TCR signaling in which multiple T cell responses share a common rate-limiting threshold and a conceptually simple model of CD8+ T cell Ag recognition, in which Ag dose and affinity do not provide any additional response-specific information.

Cerebral Venous Drainage in Patients With Space-Occupying Middle Cerebral Artery Infarction: Effects on Functional Outcome After Hemicraniectomy.

Background: Cerebral venous drainage might influence brain edema characteristics and functional outcome of patients with severe ischemic stroke. The purpose of the study was to evaluate whether hypoplasia of transverse sinuses or the internal jugular veins is associated with poor functional outcome in patients with space-occupying middle cerebral artery (MCA) infarction who underwent decompressive surgery. Methods: We performed a retrospective analysis of patients with space-occupying MCA infarction treated with decompressive surgery at our university hospital. The transverse sinuses and the internal jugular veins were evaluated on baseline images and categorized as normal, hypoplastic or occluded. We defined composite variables for ipsilateral, contralateral or any abnormal cerebral venous drainage. We assessed the functional outcome at 12 months with the modified Rankin scale (mRS) score and defined poor functional outcome as mRS scores 5 and 6. Results: We analyzed 88 patients with available baseline imaging data [mean [SD] patient age 53 (±9) years; median[IQR] time to decompressive surgery 31(22-51) h]. At 12 months 44 patients (50%) had a poor outcome. In univariate analysis neither ipsilateral (OR 1.98;95%CI: 0.75-5.40), nor contralateral (OR 1.56;95%CI: 0.59-4.24) or any (OR 1.6; 95%CI: 0.68-3.79) hypoplasia or occlusion of venous drainage were significantly associated with poor functional outcome. In multivariate analyses, higher patient age (OR 1.07;95%CI 1.01-1.14) and baseline stroke severity (OR 3.42;95%CI 1.31-9.40) were independent predictors of poor functional outcome, but not ipsilateral hypoplasia or occlusion of venous drainage (OR 1.31;95%CI 0.47-3.67). Conclusions: The cerebral venous drainage pattern was not significantly associated with poor functional outcome in our cohort of patients with space-occupying MCA infarction who underwent decompressive surgery.

Comparison of T Cell Activities Mediated by Human TCRs and CARs That Use the Same Recognition Domains.

Adoptive T cell therapies have achieved significant clinical responses, especially in hematopoietic cancers. Two types of receptor systems have been used to redirect the activity of T cells, normal heterodimeric TCRs or synthetic chimeric Ag receptors (CARs). TCRs recognize peptide-HLA complexes whereas CARs typically use an Ab-derived single-chain fragments variable that recognizes cancer-associated cell-surface Ags. Although both receptors mediate diverse effector functions, a quantitative comparison of the sensitivity and signaling capacity of TCRs and CARs has been limited due to their differences in affinities and ligands. In this study we describe their direct comparison by using TCRs that could be formatted either as conventional αß heterodimers, or as single-chain fragments variable constructs linked to CD3ζ and CD28 signaling domains or to CD3ζ alone. Two high-affinity TCRs (KD values of ∼50 and 250 nM) against MART1/HLA-A2 or WT1/HLA-A2 were used, allowing MART1 or WT1 peptide titrations to easily assess the impact of Ag density. Although CARs were expressed at higher surface levels than TCRs, they were 10-100-fold less sensitive, even in the absence of the CD8 coreceptor. Mathematical modeling demonstrated that lower CAR sensitivity could be attributed to less efficient signaling kinetics. Furthermore, reduced cytokine secretion observed at high Ag density for both TCRs and CARs suggested a role for negative regulators in both systems. Interestingly, at high Ag density, CARs also mediated greater maximal release of some cytokines, such as IL-2 and IL-6. These results have implications for the next-generation design of receptors used in adoptive T cell therapies.

No benefits of hypothermia in patients treated with hemicraniectomy for large ischemic stroke.

Background Space-occupying middle cerebral artery brain infarcts are associated with the development of brain edema, which may lead to cerebral herniation and death despite early hemicraniectomy. Aims To evaluate the benefit of therapeutic hypothermia in patients with space-occupying cerebral infarction treated with hemicraniectomy within 48 h of stroke onset. Methods Patients aged 18-60 years with space-occupying cerebral infarction treated with hemicraniectomy within 48 h and hypothermia (33-34°C) were selected from a single university hospital between 2001 and 2010 (n = 53). Patients treated with hemicraniectomy alone served as comparison group (n = 58), originating from three randomized controlled trials evaluating the effects of early decompressive surgery (DECIMAL, DESTINY, HAMLET). Primary outcome was the score on the modified Rankin scale at 12 months dichotomized between modified Rankin scale 0-3 and modified Rankin scale 4-6. Secondary outcome measures were modified Rankin scale score 0-4 and survival. Risk ratios were adjusted with Poisson regression. Results Mean patient age was 48 years. Median time from stroke onset to hemicraniectomy was 23.5 h in both treatment groups. Treatment with hypothermia had no effect on the primary outcome (modified Rankin scale 0-3 versus 4-6 (13/53 (25%) versus 24/58 (41%)); adjusted risk ratio 0.66, 95% confidence interval 0.38-1.13). Fewer patients treated with hypothermia had a modified Rankin scale score of 0-4 (21/53 (40%) versus 42/58 (72%); adjusted risk ratio 0.53, 95% confidence interval 0.37-0.76) and fewer patients survived (26/53 (49%) versus 46/58 (79%); adjusted risk ratio 0.60, 95% confidence interval 0.44-0.82). Conclusions In patients with space-occupying cerebral infarction, treatment with hypothermia had no additional benefit on functional outcome compared with treatment with hemicraniectomy alone.

PD-L1 blockade enhances response of pancreatic ductal adenocarcinoma to radiotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is considered a non-immunogenic tumor, and immune checkpoint inhibitor monotherapy lacks efficacy in this disease. Radiotherapy (RT) can stimulate the immune system. Here, we show that treatment of KPC and Pan02 murine PDAC cells with RT and gemcitabine upregulated PD-L1 expression in a JAK/Stat1-dependent manner. In vitro, PD-L1 inhibition did not alter radio- and chemosensitivity. In vivo, addition of anti-PD-L1 to high (12, 5 × 3, 20 Gy) but not low (6, 5 × 2 Gy) RT doses significantly improved tumor response in KPC and Pan02 allografts. Radiosensitization after PD-L1 blockade was associated with reduced CD11b+Gr1+ myeloid cell infiltration and enhanced CD45+CD8+ T-cell infiltration with concomitant upregulation of T-cell activation markers including CD69, CD44, and FasL, and increased CD8:Treg ratio. Depletion of CD8+ T cells abrogated radiosensitization by anti-PD-L1. Blockade of PD-L1 further augmented the effect of high RT doses (12 Gy) in preventing development of liver metastases. Exploring multiple mathematical models reveals a mechanism able to explain the observed synergy between RT and anti-PD-L1 therapy. Our findings provide a rationale for testing the use of immune checkpoint inhibitors with RT in PDAC.

Architecture of a minimal signaling pathway explains the T-cell response to a 1 million-fold variation in antigen affinity and dose.

T cells must respond differently to antigens of varying affinity presented at different doses. Previous attempts to map peptide MHC (pMHC) affinity onto T-cell responses have produced inconsistent patterns of responses, preventing formulations of canonical models of T-cell signaling. Here, a systematic analysis of T-cell responses to 1 million-fold variations in both pMHC affinity and dose produced bell-shaped dose-response curves and different optimal pMHC affinities at different pMHC doses. Using sequential model rejection/identification algorithms, we identified a unique, minimal model of cellular signaling incorporating kinetic proofreading with limited signaling coupled to an incoherent feed-forward loop (KPL-IFF) that reproduces these observations. We show that the KPL-IFF model correctly predicts the T-cell response to antigen copresentation. Our work offers a general approach for studying cellular signaling that does not require full details of biochemical pathways.

Neutrophils: Between host defence, immune modulation, and tissue injury.

Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage.