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Introductionβ-TCP ceramics are bone replacement materials that have recently been tested as a drug delivery system that can potentially be applied to endogenous substances like growth factors found in blood platelets to facilitate positive attributes.MethodsIn this work, we used flow chamber loading to load β-TCP dowels with blood suspensions of platelet-rich plasma (PRP), platelet-poor plasma (PPP), or buffy coat (BC) character. PRP and BC platelet counts were adjusted to the same level by dilution. Concentrations of TGF-β1, PDGF-AB, and IGF-1 from dowel-surrounding culture medium were subsequently determined using ELISA over 5 days. The influence of alginate was additionally tested to modify the release.ResultsConcentrations of TGF-β1 and PDGF-AB increased and conclusively showed a release from platelets in PRP and BC compared to PPP. The alginate coating reduced the PDGF-AB release but did not reduce TGF-β1 and instead even increased TGF-β1 in the BC samples. IGF-1 concentrations were highest in PPP, suggesting circulating levels rather than platelet release as the driving factor. Alginate samples tended to have lower IGF-1 concentrations, but the difference was not shown to be significant.DiscussionThe release of growth factors from different blood suspensions was successfully demonstrated for β-TCP as a drug delivery system with release patterns that correspond to PRP activation after Ca2+-triggered activation. The release pattern was partially modified by alginate coating.

External Quality Assessment schemes (EQAS) are mandatory to ensure quality standards in diagnostic methods and achieve laboratory accreditation. As host institution for two German culture-based bacteriology EQAS (RV-A and RV-B), we investigated the obtained data of 590 up to 720 surveys per year in RV-A and 2,151 up to 2,929 in RV-B from 2006 to 2023. As educational instruments, they function to review applied methodology and are valuable to check for systemic- or method-dependent failures in microbiology diagnostics or guidelines. Especially, containment of multi-resistant bacteria in times of rising antibiotic resistance is one major point to assure public health. The correct identification and reporting of these strains is therefore of high importance to achieve this goal. Moreover, correct antimicrobial susceptibility testing (AST) per se is important for selecting appropriate therapy, to restrict broad-spectrum antibiotics and minimize resistance development. The reports of participating laboratories displayed a high level of correct identification results in both schemes with mostly consistent failure rates around 2.2% (RV-A) and 3.9% (RV-B) on average. In contrast, results in AST revealed increasing failure rates upon modification of AST requirements concerning adherence to standards and subsequent bacterial species-specific evaluation. Stratification on these periods revealed in RV-A a moderate increase from 1.3% to 4.5%, while in RV-B failure rates reached 14% coming from 4.3% on average. Although not mandatory, subsequent AST evaluation and consistent reporting are areas of improvement to benefit public health.

The applicability of electrical impedance tomography (EIT) in birds is unknown. This study aimed to evaluate the use of EIT in anaesthetised chickens in four recumbency positions. Four adult Hyline chickens were anaesthetised with isoflurane in oxygen, and intubated endotracheally for computed tomography (CT). A rubber belt was placed around the coelom caudal to the shoulder joint. A chicken-specific finite element (FE) model, which is essential to generate anatomically accurate functional EIT images for analysis, was constructed based on the CT images obtained at the belt level. Ten additional chickens were anaesthetised with the same protocol. An EIT electrode belt was placed at the same location. The chickens were breathing spontaneously and positioned in dorsal, ventral, right and left lateral recumbency in a randomised order. For each recumbency, raw EIT data were collected over 2 min after 13 min of stabilisation. The data were reconstructed into functional EIT images. EIT variables including tidal impedance variation (TIV), centre of ventilation right to left (CoVRL) and ventral to dorsal (CoVVD), right to left (RL) ratio, impedance change (ΔZ) and eight regional impedance changes including the dorsal, central-dorsal, central-ventral and ventral regions of the right and left regions were analysed. Four breathing patterns (BrP) were observed and categorised based on the expiratory curve. A linear mixed model was used to compare EIT variables between recumbencies. Fisher's exact test was used to compare the frequencies of breathing patterns for each recumbency. The ΔZ observed was synchronous to ventilation, and represented tidal volume of the cranial air sacs as confirmed by CT. Significant differences were found in CoVVD and regional impedance changes between dorsal and ventral recumbencies (P < 0.05), and in CoVRL, RL ratio and regional impedance changes between right and left recumbencies (P < 0.05), which suggested a tendency for the distribution of ventilation to shift towards non-dependent air sacs. No differences were found for TIV and respiratory rate between recumbencies. Recumbency had a significant effect on the frequencies of each of the four BrPs (P = 0.001). EIT can monitor the magnitude and distribution of ventilation of the cranial air sacs in different recumbencies in anaesthetised chickens.

IntroductionWe examined the concurrent language and cognitive abilities in a group of Swedish students with different reading profiles in secondary school, and the retrospective (primary school) and prospective (upper-secondary school) reading skills of each reading profile.MethodsSeventy-nine students participated in data collections in primary (grade 2: age 8), secondary (grade 8: age 14) and upper-secondary school (year 2: age 17). Independent variables included measures of word recognition, and vocabulary and text comprehension in secondary school. Dependent variables included measures of phonemic awareness, verbal fluency, listening comprehension, spelling, verbal working memory and nonverbal reasoning skills in secondary school, and word recognition and reading comprehension in primary and upper-secondary school.ResultsWhen exploring the concurrent language and cognitive abilities of the reading profiles in secondary school, spelling emerged as a weakness and listening comprehension as a strength for students with poor decoding. Students with poor comprehension experienced weaknesses in spelling, and non-verbal reasoning. Students with both poor decoding and comprehension displayed a multi-deficit profile in language and cognition. As regards the retrospective and prospective reading skills, the relative ranking of the reading profiles was rather consistent in both primary and upper-secondary school.DiscussionThe findings suggest that limitations in phonological awareness may not be a prominent feature of secondary school students with poor decoding in more transparent orthographies. From an educational perspective, spoken sources may support learning among students with poor decoding, whereas students with poor comprehension or combined difficulties in decoding and comprehension need support when learning from both spoken and written sources.

IntroductionSelf-efficacy for writing (SEW) and reading ability are some of several factors that may be related to the quality of written text that students produce. The aim of the current study was (1) to explore the variation in SEW and written text quality in L1-Swedish and L2-English among upper secondary students with different reading profiles in L1 (typical reading vs. reading difficulties) and with different study backgrounds (SB1year or SB2years = one or two years of studies of Swedish and English, respectively), and in the next step (2) to explore if individual variations in L1-reading and SEW may explain variation in written text quality.MethodsParticipants were 100 upper secondary students (aged 17–18) with different reading profiles operationalized as typical reading and reading difficulties. Data consisted of screening for word recognition and reading comprehension, text quality results from argumentative L1- and L2-writing tasks, school information on study background in Swedish/English, and students’ responses from an online survey about SEW.ResultsAs to SEW results, an ANOVA revealed significant main effects for reading profile and study background in L1, but in L2 there was only a significant main effect for reading profile. Written text quality results indicated that there was a significant interaction effect between reading profile and study background in L1, indicating that the significant main effect for reading profile on written text quality was influenced by the group of students with reading difficulties and SB1year. There was a significant main effect for reading profile and study background on written text quality in L2. Students with reading difficulties and SB1year were the most vulnerable group, and they had the lowest scores in L1/L2 SEW and written text quality in L1 and L2. Multiple regression results indicated that word recognition and SEW contributed significantly to L1-text quality, and word recognition, reading comprehension, and SEW contributed significantly to L2-text quality. Thus, this study sheds light on the under-researched area of L1/L2 SEW and text quality of students with reading difficulties at the level of upper secondary school.DiscussionPedagogical implications are discussed and highlight the need for writing instruction across subjects in upper secondary school and for extra writing support/scaffolding for students with reading difficulties and shorter study background in the language subjects L1 (Swedish) and L2 (English).

IntroductionElectrical impedance tomography (EIT) allows assessment of ventilation and aeration homogeneity which may be associated with respiratory outcomes in preterm infants.MethodsThis was a secondary analysis to a recent randomized controlled trial in very preterm infants in the delivery room (DR). The predictive value of various EIT parameters assessed 30 min after birth on important respiratory outcomes (early intubation

The goal of this study is to provide a universally applicable procedure for a systematic evaluation of in situ measured data from single sensors regarding quantifying the uncertainty of the measurement results. As determining uncertainty for an environmental parameter also depends on the parameter itself, the focus here will be set on the variable water temperature in the first place. A separate analysis for salinity and other data will follow in later publications. With this first of a series of planned manuscripts on different parameters, we aim at providing a common understanding of how measurement uncertainty on single sensor measurements can be derived. Using an experimental in situ set-up with 6 different standard CTD sensors of two different brands, we created a four month-long, high-quality data set to be used to develop a reliable method for quantifying measurement uncertainties. Although the CTDs were deployed in a mooring in a coastal environment the described method can be extended to other deployment configurations as well. The described procedures have evolved as a stepwise process that takes the different perspectives of the involved authors into account, as well as the special conditions for environmental measurements, which are collected while the observed volume/area is undergoing a constant change. By sharing the ideas with other stakeholders, the basic concept can be extended to other observing programs and to other essential ocean variables.

BackgroundThe decision for withholding and withdrawing of life-sustaining treatments (LSTs) in COVID-19 patients is currently based on a collegial and mainly clinical assessment. In the context of a global pandemic and overwhelmed health system, the question of LST decision support for COVID-19 patients using prognostic biomarkers arises.MethodsIn a multicenter study in 24 French hospitals, 2878 COVID-19 patients hospitalized in medical departments from 26 February to 20 April 2020 were included. In a propensity-matched population, we compared the clinical, biological, and management characteristics and survival of patients with and without LST decision using Student's t-test, the chi-square test, and the Cox model, respectively.ResultsAn LST was decided for 591 COVID-19 patients (20.5%). These 591 patients with LST decision were secondarily matched (1:1) based on age, sex, body mass index, and cancer history with 591 COVID-19 patients with no LST decision. The patients with LST decision had significantly more cardiovascular diseases, such as high blood pressure (72.9 vs. 66.7%, p = 0.02), stroke (19.3 vs. 11.1%, p < 0.001), renal failure (30.4 vs. 17.4%, p < 0.001), and heart disease (22.5 vs. 14.9%, p < 0.001). Upon admission, LST patients were more severely attested by a qSOFA score ≥2 (66.5 vs. 58.8%, p = 0.03). Biologically, LST patients had significantly higher values of D-dimer, markers of heart failure (BNP and NT-pro-BNP), and renal damage (creatinine) (p < 0.001). Their evolutions were more often unfavorable (in-hospital mortality) than patients with no LST decision (41.5 vs. 10.3%, p < 0.001). By combining the three biomarkers (D-dimer, BNP and/or NT-proBNP, and creatinine), the proportion of LST increased significantly with the number of abnormally high biomarkers (24, 41.3, 48.3, and 60%, respectively, for none, one, two, and three high values of biomarkers, trend p < 0.01).ConclusionThe concomitant increase in D-dimer, BNP/NT-proBNP, and creatinine during the admission of a COVID-19 patient could represent a reliable and helpful tool for LST decision. Circulating biomarker might potentially provide additional information for LST decision in COVID-19.

IntroductionSince its first implementation in 1985, intracoronary (ic) electrocardiogram (ECG) has shown ample evidence for its diagnostic value given the higher sensitivity for myocardial ischemia detection in comparison to surface ECG. However, a lack of online systems to quantitatively analyze icECG in real-time prevents its routine use. The present study aimed to develop and validate an autonomous icECG analyzing algorithm.Materials and methodsThis is a retrospective observational study in 100 patients with chronic coronary syndrome. From each patient, a non-ischemic as well as ischemic icECG at the end of a 1-min proximal coronary balloon occlusion was available. An ECG expert as well as the newly developed algorithm for autonomous icECG analysis measured the icECG ST-segment shift in mV for each icECG tracing.ResultsIntraclass correlation coefficient (ICC) demonstrated low variability between the two methods (ICC = 0.968). Using the time point of icECG recording as allocation reference for absent or present myocardial ischemia, ROC-analysis for ischemia detection by the manually determined icECG ST-segment shift showed an area under the curve (AUC) of 0.968 ± 0.021 (p < 0.0001). AUC for the algorithm analysis was 0.967 ± 0.023 (p < 0.0001; p = 0.925 for the difference between the ROC curve AUCs). Time to complete analysis was below 1,000 ms for the autonomous icECG analysis and above 5 min for manual analysis.ConclusionA newly developed autonomous icECG analysing algorithm detects myocardial ischemia with equal accuracy as manual ST-segment shift assessment. The algorithm provides the technical fundament for an analysing system to quantitatively obtain icECG in real-time.

Electrical impedance tomography (EIT) is a non-invasive real-time non-ionising imaging modality that has many applications. Since the first recorded use in 1978, the technology has become more widely used especially in human adult and neonatal critical care monitoring. Recently, there has been an increase in research on thoracic EIT in veterinary medicine. Real-time imaging of the thorax allows evaluation of ventilation distribution in anesthetised and conscious animals. As the technology becomes recognised in the veterinary community there is a need to standardize approaches to data collection, analysis, interpretation and nomenclature, ensuring comparison and repeatability between researchers and studies. A group of nineteen veterinarians and two biomedical engineers experienced in veterinary EIT were consulted and contributed to the preparation of this statement. The aim of this consensus is to provide an introduction to this imaging modality, to highlight clinical relevance and to include recommendations on how to effectively use thoracic EIT in veterinary species. Based on this, the consensus statement aims to address the need for a streamlined approach to veterinary thoracic EIT and includes: an introduction to the use of EIT in veterinary species, the technical background to creation of the functional images, a consensus from all contributing authors on the practical application and use of the technology, descriptions and interpretation of current available variables including appropriate statistical analysis, nomenclature recommended for consistency and future developments in thoracic EIT. The information provided in this consensus statement may benefit researchers and clinicians working within the field of veterinary thoracic EIT. We endeavor to inform future users of the benefits of this imaging modality and provide opportunities to further explore applications of this technology with regards to perfusion imaging and pathology diagnosis.

Equine respiratory physiology might be influenced by the presence of an endotracheal tube (ETT). This experimental, randomized cross-over study aimed to compare breathing pattern (BrP) and ventilation distribution in anesthetized horses spontaneously breathing room air via ETT or facemask (MASK). Six healthy adult horses were anesthetized with total intravenous anesthesia (TIVA; xylazine, ketamine, guaiphenesin), breathing spontaneously in right lateral recumbency, and randomly assigned to ETT or MASK for 30 min, followed by the other treatment for an additional 30 min. During a second anesthesia 1 month later, the treatment order was inversed. Electrical impedance tomography (EIT) using a thoracic electrode belt, spirometry, volumetric capnography, esophageal pressure difference (ΔPoes), venous admixture, and laryngoscopy data were recorded over 2 min every 15 min. Breaths were classified as normal or alternate (sigh or crown-like) according to the EIT impedance curve. A mixed linear model was used to test the effect of treatment on continuous outcomes. Cochran-Mantel-Haenszel analysis was used to test for associations between global BrP and treatment. Global BrP was associated with treatment (p = 0.012) with more alternate breaths during ETT. The center of ventilation right-to-left (CoVRL) showed more ventilation in the non-dependent lung during ETT (p = 0.025). The I:E ratio (p = 0.017) and ΔPoes (p < 0.001) were smaller, and peak expiratory flow (p = 0.009) and physiologic dead space (p = 0.034) were larger with ETT. The presence of an ETT alters BrP and shifts ventilation toward the non-dependent lung in spontaneously breathing horses anesthetized with TIVA.

The climate of the Arabian Desert is not well documented during the past two millennia due to the scarcity of continuous and well-dated terrestrial archives in the region. Reliable interpretation from the climatic records from this region are pivotal for identifying periodicities of inter-annual to multi-decadal variability and trends driven by shifts in position of the Intertropical Convergence Zone (ITCZ) and the strength of the monsoons. A high-resolution multiproxy approach is presented for a ∼3.3 m composite core, GBW, from a karst lake located in Ghayl ba Wazir, southern Yemen. Sedimentary proxies, including particle size distribution, coupled with magnetic susceptibility (MS) and geochemistry (XRF), provide a comprehensive picture of sediment depositional changes that may be linked to climate and environmental variability over the southern Arabian Desert. The chronology of the GBW core is provided by five radiocarbon (14C) dates from terrestrial macrofossils (wood and twigs) extracted from sediment samples and indicates the core extends to ∼900 CE. Our data indicates generally wetter conditions from 930 to 1400 CE corresponding to the “Medieval climate anomaly (MCA)” followed by arid phases during 1,410–1700 CE coinciding with the “Little Ice Age (LIA)”. Evidence for a drier LIA include high authigenic calcium precipitation [Ca/(Al + Fe + Ti)], decreased TOC/TIC values, and gypsum precipitation, whereas the wetter MCA is characterized by higher detrital element ratios (Ti/Al, K/Al, Rb/Sr), and increased TOC/TIC and deposition of finer sediments (EM1). Furthermore, end-member mixing analyses (EMMA) derived from the grain-size distribution (EM2 and EM3) corroborates the deposition of coarser silt sediment through wind erosion and production of carbonate sand during the LIA concurrently with low lake levels under generally dry conditions. Aridity during the LIA is consistent with evidence and theory for weakened boreal summer monsoons during intervals of northern hemisphere cooling.

Background: Microthrombosis and large-vessel thrombosis are the main triggers of COVID-19 worsening. The optimal anticoagulant regimen in COVID-19 patients hospitalized in medical wards remains unknown.Objectives: To evaluate the effects of intermediate-dose vs. standard-dose prophylactic anticoagulation (AC) among patients with COVID-19 hospitalized in medical wards.Methods and results: We used a large French multicentric retrospective study enrolling 2,878 COVID-19 patients hospitalized in medical wards. After exclusion of patients who had an AC treatment before hospitalization, we generated a propensity-score-matched cohort of patients who were treated with intermediate-dose or standard-dose prophylactic AC between February 26 and April 20, 2020 (intermediate-dose, n = 261; standard-dose prophylactic anticoagulation, n = 763). The primary outcome of the study was in-hospital mortality; this occurred in 23 of 261 (8.8%) patients in the intermediate-dose group and 74 of 783 (9.4%) patients in the standard-dose prophylactic AC group (p = 0.85); while time to death was also the same in both the treatment groups (11.5 and 11.6 days, respectively, p = 0.17). We did not observe any difference regarding venous and arterial thrombotic events between the intermediate dose and standard dose, respectively (venous thrombotic events: 2.3 vs. 2.4%, p=0.99; arterial thrombotic events: 2.7 vs. 1.2%, p = 0.25). The 30-day Kaplan–Meier curves for in-hospital mortality demonstrate no statistically significant difference in in-hospital mortality (HR: 0.99 (0.63–1.60); p = 0.99). Moreover, we found that no particular subgroup was associated with a significant reduction in in-hospital mortality.Conclusion: Among COVID-19 patients hospitalized in medical wards, intermediate-dose prophylactic AC compared with standard-dose prophylactic AC did not result in a significant difference in in-hospital mortality.

Transpulmonary driving pressure (DPL) corresponds to the cyclical stress imposed on the lung parenchyma during tidal breathing and, therefore, can be used to assess the risk of ventilator-induced lung injury (VILI). Its measurement at the bedside requires the use of esophageal pressure (Peso), which is sometimes technically challenging. Recently, it has been demonstrated how in an animal model of ARDS, the transpulmonary pressure (PL) measured with Peso calculated with the absolute values method (PL = Paw—Peso) is equivalent to the transpulmonary pressure directly measured using pleural sensors in the central-dependent part of the lung. We hypothesized that, since the PL derived from Peso reflects the regional behavior of the lung, it could exist a relationship between regional parameters measured by electrical impedance tomography (EIT) and driving PL (DPL). Moreover, we explored if, by integrating airways pressure data and EIT data, it could be possible to estimate non-invasively DPL and consequently lung elastance (EL) and elastance-derived inspiratory PL (PI). We analyzed 59 measurements from 20 patients with ARDS. There was a significant intra-patient correlation between EIT derived regional compliance in regions of interest (ROI1) (r = 0.5, p = 0.001), ROI2 (r = −0.68, p < 0.001), and ROI3 (r = −0.4, p = 0.002), and DPL. A multiple linear regression successfully predicted DPL based on respiratory system elastance (Ers), ideal body weight (IBW), roi1%, roi2%, and roi3% (R2 = 0.84, p < 0.001). The corresponding Bland-Altmann analysis showed a bias of −1.4e-007 cmH2O and limits of agreement (LoA) of −2.4–2.4 cmH2O. EL and PI calculated using EIT showed good agreement (R2 = 0.89, p < 0.001 and R2 = 0.75, p < 0.001) with the esophageal derived correspondent variables. In conclusion, DPL has a good correlation with EIT-derived parameters in the central lung. DPL, PI, and EL can be estimated with good accuracy non-invasively combining information coming from EIT and airway pressure.

Carbon Capture and Sequestration (CCS) has been recognized as an important means of mitigating global climate change, but apart from several pilots, it has not yet been successfully implemented on the large scale needed to live up to the expectations as a mitigation method. In Netherlands, the option of CCS has been the subject of debate for a long time, as three unsuccessful projects – two onshore in Barendrecht and the Northern regions, and one offshore near the Port of Rotterdam – demonstrate. Nevertheless, CCS has been accorded an important place in the current Dutch climate policies, being expected to contribute up to 7 Megaton of CO2 reduction. This is reflected in a fresh crop of CCS project plans. For the most, these plans have a long way to go from the drawing board to actual operations due to the technical, economic, legal and societal challenges ahead. In this article we review the status and possibilities of CCS in Netherlands based on an analysis of existing literature in the relevant disciplines. First, a brief overview of the technology options for carbon capture and storage or utilization is given. This is followed by a detailed analysis of the governmental support for CCS, given the vital role that fit-for-purpose legal frameworks and policy instruments will play in CCS deployment. Technical, legal and policy uncertainties translates into factors inhibiting CCS investment and so the paper then presents a CCS investment project to illustrate how such risks affect the business case for CCS. Finally, bearing in mind that societal acceptance has proved to be a major barrier for CCS, both in Netherlands and elsewhere, the conditions that enhance public acceptance of CCS are examined. Our work shows that while CCS is technically a straightforward proposition, its deployment has historically been hindered by the lack of a sound business case and a compelling and stable socio-technical narrative. The main argument in favor of CCS today is that it offers a transition pathway for rapidly and massively reducing CO2 emissions beyond what could be accomplished by alternative methods like electrification and renewable fuels in near future. The introduction of new financial instruments, increased government support and an improvement in social engagement appear to have enhanced the prospects of CCS in Netherlands, but we feel it is premature to assume that this time everything is different.

Constitutive activity of the immune surveillance system detects and kills cancerous cells, although many cancers have developed strategies to avoid detection and to resist their destruction. Cancer immunotherapy entails the manipulation of components of the endogenous immune system as targeted approaches to control and destroy cancer cells. Since one of the major limitations for the antitumor activity of immune cells is the immunosuppressive tumor microenvironment (TME), boosting the immune system to overcome the inhibition provided by the TME is a critical component of oncotherapeutics. In this article, we discuss the main effects of the TME on the metabolism and function of immune cells, and review emerging strategies to potentiate immune cell metabolism to promote antitumor effects either as monotherapeutics or in combination with conventional chemotherapy to optimize cancer management.

Rock falls and landslides plunging into lakes or small reservoirs can result in tsunamis with extreme wave run-ups. The occurrence of these natural hazards in populated areas have encouraged a recent sharp increase of studies that aim to mitigate their impact on human lives and assess infrastructure lost. This paper amalgamates in a novel fashion and at an unprecedented detail in situ historic measurements, geological data and numerical modeling of a rock fall event and associated tsunami wave that occurred in Lake Lovatnet (western Norway) in September 1936. Historical records report an event that released ca. 1 million m3 of rocks and debris from Ramnefjellet Mountain at an altitude of 800 m above Lake Lovatnet. The fragmented material plunged into the lake, causing a tsunami that reached a maximum run-up of 74 m and killed 74 people. In fact, the settlements of Bødal and Nesdal were wiped out as a result of the catastrophic wave. Sediments resulting from the 1936 rock fall and associated tsunami were identified in the subsurface of Lake Lovatnet by shallow geophysical investigations and were retrieved using gravity coring equipment. A set of high resolution physical and geochemical measurements were carried out on the cores with the aim of reproducing a highly detailed reconstruction of this catastrophic event in order to better understand and learn about the processes involved. The cores were retrieved in the northwestern sub-basin of the lake and its chronology was constrained by 210Pb and radiocarbon dating. A specially tailored physically based mathematical model was applied to better understand the tsunami event. Integration of the geophysical record, the sedimentological data and numerical modeling provide a comprehensive background to better understand the effects of such event in a deep fjord-like lacustrine basin and to generate information for better mitigation of similar events elsewhere.

Post-transplant lymphoproliferative disorder (PTLD) is one of the most common malignancies after solid organ or allogeneic stem cell transplantation. Most PTLD cases are B cell neoplasias carrying Epstein-Barr virus (EBV). A therapeutic approach is reduction of immunosuppression to allow T cells to develop and combat EBV. If this is not effective, approaches include immunotherapies such as monoclonal antibodies targeting CD20 and adoptive T cells. Immune checkpoint inhibition (ICI) to treat EBV+ PTLD was not established clinically due to the risks of organ rejection and graft-versus-host disease. Previously, blockade of the programmed death receptor (PD)-1 by a monoclonal antibody (mAb) during ex vivo infection of mononuclear cells with the EBV/M81+ strain showed lower xenografted lymphoma development in mice. Subsequently, fully humanized mice infected with the EBV/B95-8 strain and treated in vivo with a PD-1 blocking mAb showed aggravation of PTLD and lymphoma development. Here, we evaluated vis-a-vis in fully humanized mice after EBV/B95-8 or EBV/M81 infections the effects of a clinically used PD-1 blocker. Fifteen to 17 weeks after human CD34+ stem cell transplantation, Nod.Rag.Gamma mice were infected with two types of EBV laboratory strains expressing firefly luciferase. Dynamic optical imaging analyses showed systemic EBV infections and this triggered vigorous human CD8+ T cell expansion. Pembrolizumab administered from 2 to 5 weeks post-infections significantly aggravated EBV systemic spread and, for the M81 model, significantly increased the mortality of mice. ICI promoted Ki67+CD30+CD20+EBER+PD-L1+ PTLD with central nervous system (CNS) involvement, mirroring EBV+ CNS PTLD in humans. PD-1 blockade was associated with lower frequencies of circulating T cells in blood and with a profound collapse of CD4+ T cells in lymphatic tissues. Mice treated with pembrolizumab showed an escalation of exhausted T cells expressing TIM-3, and LAG-3 in tissues, higher levels of several human cytokines in plasma and high densities of FoxP3+ regulatory CD4+ and CD8+ T cells in the tumor microenvironment. We conclude that PD-1 blockade during acute EBV infections driving strong CD8+ T cell priming decompensates T cell development towards immunosuppression. Given the variety of preclinical models available, our models conferred a cautionary note indicating that PD-1 blockade aggravated the progression of EBV+ PTLD.

Interleukin-15 (IL-15) is crucial for the proliferation and survival of NK and CD8+ T memory cells, and of significant interest in immuno-oncology. Immune cell expansion requires continuous IL-15 exposure above a threshold concentration for an extended period. However, the short t1/2 of IL-15 makes this impossible to achieve after a single injection without a high Cmax and toxicities. The most effective way to deliver IL-15 is continuous intra-venous infusion, but this administration mode is impractical. Efforts have been devoted to developing IL-15 agonists which after a single injection maintain the cytokine in a narrow therapeutic window for a long period. Enigmatically, although the half-life extension technologies used often extend the half-life of a protein to 1 or more weeks, the modified IL-15 agonists studied usually have systemic elimination half-lives of only a few hours and rarely much longer than 1 day. These short half-lives—common to all circulating IL-15 agonists thus far reported—can be explained by a dynamic increase in clearance of the agonists that accompanies target immune cell proliferation. What is needed is an IL-15 agonist that is as effective as continuous intravenous infusion, but with the convenience and acceptance of single injections at 1-week or longer intervals.

Settling marine aggregates, such as zooplankton fecal pellets and marine snow, transport organic matter from the surface ocean to the deep sea and are largely responsible for the ocean’s sequestration of carbon. However, our understanding of the functioning of the biological pump and the distribution of particulate organic matter in the water column often hinge on limited bulk data from sediment traps, large volume filtration or instantaneous snap-shots from in situ optical systems that only see a small part of the particle and aggregate spectra. We evaluated the added value of combining different optical systems to detect a range of organic and inorganic particle types during a case-study in the Cape Blanc upwelling region. Laboratory calibrations showed that one camera system detected large organic aggregates well and in situ data showed that it correlated positively with fluorescence. The other camera was better at detecting small, mainly inorganic particles which were not seen by the first camera and correlated positively with turbidity. The combined deployments of the two optical systems together with fluorescence and turbidity sensors showed potentials for added insights into spatial (depth) and temporal (diurnal) particle dynamics. The case study exemplified the different efficiencies of two camera systems to detect particles of different types in marine waters. From this, the results highlighted the importance of discriminating between qualitative and quantitative ranges of imaging systems, in order to understand the quantitative range of sizes as well as types of particles detected by a given system. This is especially important when optical systems are used to estimate carbon fluxes and particulate organic matter distribution in the water column from vertical profiles of particle size-distribution and abundance.

The Negev Desert in southern Israel hosts a number of late Cenozoic lacustrine and palustrine sedimentary sequences that attest for past wetter conditions in what today constitutes one of the driest deserts on Earth. These sequences are of special importance because the Negev Desert forms part of the Levantine Corridor, which was probably the only continental bridge that enabled initial out-of-Africa expansion of our genus Homo. Yet, the paleoclimatic significance of these sequences still remains unknown, mainly due to their uncertain (late Pliocene to early Pleistocene) age. Here we present a combined sedimentologic, paleontologic and magnetostratigraphic study of one of these sedimentary sequences, the so-called Kuntila Lake sediments, which was carried out at the 30 m-thick Kuntila Gate section in the Nahal Paran basin, southern Israel. Sedimentological evidence and ostracod faunas indicate that these sediments accumulated in a long-lasting lacustrine basin that underwent periodic lake-level variations. Magnetostratigraphic measurements enable the recognition of a normal (N1) and a reverse (R1) polarity zone in the lower and upper halves, respectively, of the Kuntila Gate section. Correlation of N1 to the Olduvai Subchron (1.778–1.945 Ma) appears as the most likely option in view of previously published 10Be ages derived for the uppermost part of the Kuntila Member in nearby sections. The large errors associated with these ages, however, suggest that correlation of N1 to Subchron C2An.1n (2.582–3.032 Ma) is also possible. Although our results do not resolve the age of the Arava Formation, they unequivocally relate the Kuntila Lake sediments with a long period of enhanced climatic variability because the tops of both subchrons are associated with 400 kyr eccentricity maxima. The inferred wetter conditions in the Negev Desert concurred, regardless of the age correlation, with periods of lake expansion in East Africa and clusters of short-lived expansions of the savannah throughout much of the Sahara. This would have facilitated the biogeographic connection between Africa and Eurasia, greening the path for the initial out-of-Africa dispersal of Homo. Further research on the Kuntila Lake sediments will be necessary to better determine the timing, extent and significance of such biogeographic connection.

We completed clinical trials of rhIL-15 by bolus, subcutaneous, and continuous intravenous infusions (CIV). IL-15 administered by CIV at 2 mcg/kg/day yielded a 38-fold increase in 10- day number of circulating NK cells, a 358-fold increase in CD56bright NK cells and a 5.8-fold increase in CD8 T cells. However, IL-15 preparations administered as monotherapy were ineffective, due to actions of immunological checkpoints and due to the lack of tumor specific targeting by NK cells. To circumvent checkpoints, trials of IL-15 in combination with other anticancer agents were initiated. Tumor-bearing mice receiving IL-15 with antibodies to CTLA-4 and PD-L1 manifested marked prolongation of survival compared to mice receiving IL-15 with either agent alone. In translation, a phase I trial was initiated involving IL-15 (rhIL-15), nivolumab and ipilimumab in patients with malignancy (NCT03388632). In rhesus macaques CIV IL-15 at 20 μg/kg/day for 10 days led to an 80-fold increase in number of circulating effector memory CD8 T cells. However, administration of γc cytokines such as IL-15 led to paralysis/depression of CD4 T-cells that was mediated through transient expression of SOCS3 that inhibited the STAT5 signaling pathway. This lost CD4 helper role could be restored alternatively by CD40 agonists. In the TRAMP-C2 prostate tumor model the combination of IL-15 with agonistic anti-CD40 produced additive effects in terms of numbers of TRAMP-C2 tumor specific Spas/SCNC/9H tetramer positive CD8 T cells expressed and tumor responses. A clinical trial is being initiated for patients with cancer using an intralesional anti-CD40 in combination with CIV rhIL-15. To translate IL-15-mediated increases in NK cells, we investigated combination therapy of IL-15 with anticancer monoclonal antibodies including rituximab in mouse models of EL-4 lymphoma transfected with human CD20 and with alemtuzumab (CAMPATH-1H) in a xenograft model of adult T cell leukemia (ATL). IL-15 enhanced the ADCC and therapeutic efficacy of both antibodies. These results provided the scientific basis for trials of IL-15 combined with alemtuzumab (anti-CD52) for patients with ATL (NCT02689453), with obinutuzumab (anti-CD20) for patients with CLL (NCT03759184), and with avelumab (anti-PD-L1) in patients with T-cell lymphoma (NCT03905135) and renal cancer (NCT04150562). In the first trial, there was elimination of circulating ATL and CLL leukemic cells in select patients.

Genome-wide prediction (GWP) has become the state-of-the art method in artificial selection. Data sets often comprise number of genomic markers and individuals in ranges from a few thousands to millions. Hence, computational efficiency is important and various machine learning methods have successfully been used in GWP. Neural networks (NN) and deep learning (DL) are very flexible methods that usually show outstanding prediction properties on complex structured data, but their use in GWP is nevertheless rare and debated. This study describes a powerful NN method for genomic marker data that can easily be extended. It is shown that a one-dimensional convolutional neural network (CNN) can be used to incorporate the ordinal information between markers and, together with pooling and ℓ1-norm regularization, provides a sparse and computationally efficient approach for GWP. The method, denoted CNNGWP, is implemented in the deep learning software Keras, and hyper-parameters of the NN are tuned with Bayesian optimization. Model averaged ensemble predictions further reduce prediction error. Evaluations show that CNNGWP improves prediction error by more than 25% on simulated data and around 3% on real pig data compared with results obtained with GBLUP and the LASSO. In conclusion, the CNNGWP provides a promising approach for GWP, but the magnitude of improvement depends on the genetic architecture and the heritability.

Scientists who observe and distribute oceanographic data require a process to ensure high-quality data. This process includes quality assurance, quality control, quality assessment, standards, and best practices. In this paper, quality assurance is widely regarded as actions taken prior to instrument deployment to improve the probability of generating good data, while quality control is the effort made to examine the resultant data. Herein we focus on quality assurance and strive to guide the oceanographic community by identifying existing quality assurance best practices preferred by the five entities represented by the authors – specifically, the Alliance for Coastal Technology, the AtlantOS project, the Integrated Marine Observing System, the Joint Technical Commission for Oceanographic and Marine Meteorology, and the U.S. IOOS Quality Assurance/Quality Control of Real-Time Oceanographic Data project. The focus has been placed on QA in response to suggestions from the AtlantOS and QARTOD communities. We define the challenges associated with quality assurance, which include a clear understanding of various terms, the overlap in meaning of those terms, establishment of standards, and varying program requirements. Brief, “real-world” case-studies are presented to demonstrate the challenges. Following this is a description of best practices gathered by the authors from hundreds of scientists over the many years or decades the aforementioned entities have been in place. These practices address instrument selection, preparation, deployment, maintenance, and data acquisition. Varying resources and capabilities are considered, and corresponding levels of quality assurance efforts are discussed. We include a comprehensive description of measurement uncertainty with a detailed example of such a calculation. Rigorous estimates of measurement uncertainty are surprisingly complex, necessarily specific, and not provided as often as needed. But they are critical to data users who may have applications not envisioned by the data provider, to ensure appropriate use of the data. The guidance is necessarily generic because of the broad expanse of oceanographic observations. Further, it is platform-agnostic and applies to most deployment scenarios. We identify the recently created Ocean Best Practice System as one means of developing, sharing, documenting, and curating more specific QA processes. Ultimately, our goal here is to foster their development and harmonization.

The large number of markers in genome-wide prediction demands the use of methods with regularization and model comparison based on some hold-out test prediction error measure. In quantitative genetics, it is common practice to calculate the Pearson correlation coefficient (r2) as a standardized measure of the predictive accuracy of a model. Based on arguments from the bias–variance trade-off theory in statistical learning, we show that shrinkage of the regression coefficients (i.e., QTL effects) reduces the prediction mean squared error (MSE) by introducing model bias compared with the ordinary least squares method. We also show that the LASSO and the adaptive LASSO (ALASSO) can reduce the model bias and prediction MSE by adding model variance. In an application of ridge regression, the LASSO and ALASSO to a simulated example based on results for 9,723 SNPs and 3,226 individuals, the best model selected was with the LASSO when r2 was used as a measure. However, when model selection was based on test MSE and coefficient of determination R2 the ALASSO proved to be the best method. Hence, use of r2 may lead to selection of the wrong model and therefore also nonoptimal ranking of phenotype predictions and genomic breeding values. Instead, we propose use of the test MSE for model selection and R2 as a standardized measure of the accuracy.

Lipid metabolism plays a key role in many cellular processes. We show here that regulatory T cells have enhanced lipid storage within subcellular lipid droplets (LD). They also express elevated amounts of both isoforms of diacylglycerol acyl transferase (DGAT1 & 2), enzymes required for the terminal step of triacylglycerol synthesis. In regulatory T-cells (Tregs), the conversion of diacylglycerols to triacylglycerols serves two additional purposes other than lipid storage. First, we demonstrate that it protects T cells from the toxic effects of saturated long chain fatty acids. Second, we show that Triglyceride formation is essential for limiting activation of protein kinase C via free diacyl glycerol moieties. Inhibition of DGAT1 resulted in elevated active PKC and nuclear NFKB, as well as impaired Foxp3 induction in response to TGFβ. Thus, Tregs utilize a positive feedback mechanism to promote sustained expression of Foxp3 associated with control of LD formation.

The oceans play a key role in global issues such as climate change, food security, and human health. Given their vast dimensions and internal complexity, efficient monitoring and predicting of the planet’s ocean must be a collaborative effort of both regional and global scale. A first and foremost requirement for such collaborative ocean observing is the need to follow well-defined and reproducible methods across activities: from strategies for structuring observing systems, sensor deployment and usage, and the generation of data and information products, to ethical and governance aspects when executing ocean observing. To meet the urgent, planet-wide challenges we face, methods across all aspects of ocean observing should be broadly adopted by the ocean community and, where appropriate, should evolve into “Ocean Best Practices.” While many groups have created best practices, they are scattered across the Web or buried in local repositories and many have yet to be digitized. To reduce this fragmentation, we introduce a new open access, permanent, digital repository of best practices documentation (oceanbestpractices.org) that is part of the Ocean Best Practices System (OBPS). The new OBPS provides an opportunity space for the centralized and coordinated improvement of ocean observing methods. The OBPS repository employs user-friendly software to significantly improve discovery and access to methods. The software includes advanced semantic technologies for search capabilities to enhance repository operations. In addition to the repository, the OBPS also includes a peer reviewed journal research topic, a forum for community discussion and a training activity for use of best practices. Together, these components serve to realize a core objective of the OBPS, which is to enable the ocean community to create superior methods for every activity in ocean observing from research to operations to applications that are agreed upon and broadly adopted across communities. Using selected ocean observing examples, we show how the OBPS supports this objective. This paper lays out a future vision of ocean best practices and how OBPS will contribute to improving ocean observing in the decade to come.

Background: High frequency oscillatory ventilation (HFOV) is considered a lung protective ventilation mode in preterm infants only if lung volume is optimized. However, whilst a “high lung volume strategy” is advocated for HFOV in preterm infants this strategy is not precisely defined. It is not known to what extent lung recruitment should be pursued to provide lung protection. In this study we aimed to determine the relationship between the magnitude of lung volume optimization and its effect on gas exchange and lung injury in preterm lambs.Methods: 36 surfactant-deficient 124–127 d lambs commenced HFOV immediately following a sustained inflation at birth and were allocated to either (1) no recruitment (low lung volume; LLV), (2) medium- (MLV), or (3) high lung volume (HLV) recruitment strategy. Gas exchange and lung volume changes over time were measured. Lung injury was analyzed by post mortem pressure-volume curves, alveolar protein leakage, gene expression, and histological injury score.Results: More animals in the LLV developed a pneumothorax compared to both recruitment groups. Gas exchange was superior in both recruitment groups compared to LLV. Total lung capacity tended to be lower in the LLV group. Other parameters of lung injury were not different.Conclusions: Lung recruitment during HFOV optimizes gas exchange but has only modest effects on lung injury in a preterm animal model. In the HLV group aiming at a more extensive lung recruitment gas exchange was better without affecting lung injury.

Colonization of newborn piglets with beneficial and ubiquitous microorganisms in combination with colostral passive immunity is the prerequisite for development of immunity and gut maturation. In this study living strains of Clostridium perfringens type A (CpA) and non-pathogenic Escherichia (E.) coli strains harvested from healthy piglets were administered to piglets prior to first colostrum intake in order to prevent disease caused by pathogenic variants of the same bacterial species by competitive exclusion. In addition, it was investigated whether these potential beneficial colonizers were able to prevent harmful effects of infection with Cystoisospora (C.) suis as a primary invasive pathogen. In a first trial, half of the piglets from four litters were treated with a bacterial cocktail consisting of two E. coli and four CpA strains immediately after birth on two consecutive days, while the other half of the litters served as control group. In a second trial, piglets were treated following the protocol of the first trial, and additionally all piglets were infected 4 h after the end of littering with ~1,000 sporulated oocysts of a C. suis laboratory strain. General health, body weight development, fecal consistency and, in the second trial, oocyst excretion were monitored from birth until weaning. No adverse effects of the cocktail on the health status were observed. Treated piglets of the first trial showed a higher average daily weight gain until weaning. In the second trial, no significant differences were found with respect to average daily weight gain, fecal consistency, amount, and duration of oocyst excretion assessed in daily samples. In treatment group 51.1% and in the control group 38.5% of the fecal samples were positive for oocysts in autofluorescence. The average duration of oocyst excretion was longer in treatment group (7.7 days) than in control group (5.6 days). Application of bacterial cocktail could not effectively minimize disease symptoms caused by C. suis. There was a trend toward an increase in severity of disease symptoms in treated pigs, suggesting that the synergism between CpA and C. suis was independent of the bacterial strains, but is exclusively dominated by the pathogenic effect of C. suis.

During an immune response, naïve CD4+ T cells proliferate and generate a range of effector, memory, and regulatory T cell subsets, but how these processes are co-ordinated remains unclear. A traditional model suggests that memory cells use mitochondrial respiration and are survivors from a pool of previously proliferating and glycolytic, but short-lived effector cells. A more recent model proposes a binary commitment to either a memory or effector cell lineage during a first, asymmetric cell division, with each lineage able to undergo subsequent proliferation and differentiation. We used improved fixation and staining methods with imaging flow cytometry in an optimized in vitro system that indicates a third model. We found that cell fates result from stochastic decisions that depend on GITR co-stimulation and which take place before any cell division. Effector cell commitment is associated with mTORC2 signaling leading to uropodium development, while developing memory cells lose mitochondria, have a nuclear localization of NFκB and depend on TGFβ for their survival. Induced, T helper subsets and foxp3+ regulatory T cells were found in both the effector and memory cell lineages. This in vitro model of T cell differentiation is well suited to testing how manipulation of cytokine, nutrient, and other components of the microenvironment might be exploited for therapeutic purposes.

We present a novel and readily accessible method facilitating cellular time-resolved imaging of transplanted pancreatic islets. Grafting of islets to the mouse ear pinna allows non-invasive, in vivo longitudinal imaging of events in the islets and enables improved acquisition of experimental data and use of fewer experimental animals than is possible using invasive techniques, as the same mouse can be assessed for the presence of islet infiltrating cells before and after immune intervention. We have applied this method to investigating therapeutic protection of beta cells through the well-established use of anti-CD3 injection, and have acquired unprecedented data on the nature and rapidity of the effect on the islet infiltrating T cells. We demonstrate that infusion of anti-CD3 antibody leads to immediate effects on islet infiltrating T cells in islet grafts in the pinna of the ear, and causes them to increase their speed and displacement within 20 min of infusion. This technique overcomes several technical challenges associated with intravital imaging of pancreatic immune responses and facilitates routine study of beta islet cell development, differentiation, and function in health and disease.

IntroductionNon-adherence to immunosuppressive medication is regarded as an important factor for graft rejection and loss after successful renal transplantation. Yet, results on prevalence and relationship with psychosocial parameters are heterogeneous. The main aim of this study was to investigate the association of immunosuppressive medication non-adherence and psychosocial factors.MethodsIn 330 adult renal transplant recipients (≥12 months posttransplantation), health-related quality of life, depression, anxiety, social support, and subjective medication experiences were assessed, and their associations with patient-reported non-adherence was evaluated.Results33.6% of the patients admitted to be partially non-adherent. Non-adherence was associated with younger age, poorer social support, lower mental, but higher physical health-related quality of life. There was no association with depression and anxiety. However, high proportions of clinically relevant depression and anxiety symptoms were apparent in both adherent and non-adherent patients.ConclusionIn the posttransplant follow-up, kidney recipients with lower perceived social support, lower mental and higher physical health-related quality of life, and younger age can be regarded as a risk group for immunosuppressive medication non-adherence. In follow-up contacts with kidney transplant patients, physicians may pay attention to these factors. Furthermore, psychosocial interventions to optimize immunosuppressive medication adherence can be designed on the basis of this information, especially including subjectively perceived physical health-related quality of life and fostering social support seems to be of importance.

The differentiation and effector functions of both the innate and adaptive immune system are inextricably linked to cellular metabolism. The features of metabolism which affect both arms of the immune system include metabolic substrate availability, expression of enzymes, transport proteins, and transcription factors which control catabolism of these substrates, and the ability to perform anabolic metabolism. The control of lipid metabolism is central to the appropriate differentiation and functions of T lymphocytes, and ultimately to the maintenance of immune tolerance. This review will focus on the role of fatty acid (FA) metabolism in T cell differentiation, effector function, and survival. FAs are important sources of cellular energy, stored as triglycerides. They are also used as precursors to produce complex lipids such as cholesterol and membrane phospholipids. FA residues also become incorporated into hormones and signaling moieties. FAs signal via nuclear receptors and their channeling, between storage as triacyl glycerides or oxidation as fuel, may play a role in survival or death of the cell. In recent years, progress in the field of immunometabolism has highlighted diverse roles for FA metabolism in CD4 and CD8 T cell differentiation and function. This review will firstly describe the sensing and modulation of the environmental FAs and lipid intracellular signaling and will then explore the key role of lipid metabolism in regulating the balance between potentially damaging pro-inflammatory and anti-inflammatory regulatory responses. Finally the complex role of extracellular FAs in determining cell survival will be discussed.

Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children. Despite intensive research in recent decades the prognosis for patients with metastatic or relapsed diseases has hardly improved. New therapeutic concepts in anti-tumor therapy aim to modulate the patient’s immune system to increase its aggressiveness or targeted effects toward tumor cells. Besides surgery, radiotherapy and chemotherapy, immune activation by direct application of cytokines, antibodies or adoptive cell therapy are promising approaches. In the last years, adoptive transfer of natural killer (NK) cells came into the focus of translational medicine, because of their high cytotoxic potential against transformed malignant cells. A main challenge of NK cell therapy is that it requires a high amount of functional NK cells. Therefore, ex vivo NK cell expansion protocols are currently being developed. Many culturing strategies are based on the addition of feeder or accessory cells, which need to be removed prior to the clinical application of the final NK cell product. In this study, we addressed feeder cell-free expansion methods using common γ-chain cytokines, especially IL-15 and IL-21. Our results demonstrated high potential of IL-15 for NK cell expansion, while IL-21 triggered NK cell maturation and functionality. Hence, we established a two-phase expansion protocol with IL-15 to induce an early NK cell expansion, followed by short exposure to IL-21 that boosted the cytotoxic activity of NK cells against RMS cells. Further functional analyses revealed enhanced degranulation and secretion of pro-inflammatory cytokines such as interferon-γ and tumor necrosis factor-α. In a proof of concept in vivo study, we also observed a therapeutic effect of adoptively transferred IL-15 expanded and IL-21 boosted NK cells in combination with image guided high precision radiation therapy using a luciferase-transduced RMS xenograft model. In summary, this two-phased feeder cell-free ex vivo culturing protocol combined efficient expansion and high cytolytic functionality of NK cells for treatment of radiation-resistant RMS.

Significant progress has been made in recent years toward realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also the established NK cell line NK-92 is being developed for adoptive immunotherapy, and general safety of infusion of irradiated NK-92 cells has been established in phase I clinical trials with clinical responses observed in some of the cancer patients treated. To enhance their therapeutic utility, NK-92 cells have been modified to express chimeric antigen receptors (CARs) composed of a tumor-specific single chain fragment variable antibody fragment fused via hinge and transmembrane regions to intracellular signaling moieties such as CD3ζ or composite signaling domains containing a costimulatory protein together with CD3ζ. CAR-mediated activation of NK cells then bypasses inhibitory signals and overcomes NK resistance of tumor cells. In contrast to primary NK cells, CAR-engineered NK-92 cell lines suitable for clinical development can be established from molecularly and functionally well-characterized single cell clones following good manufacturing practice-compliant procedures. In preclinical in vitro and in vivo models, potent antitumor activity of NK-92 variants targeted to differentiation antigens expressed by hematologic malignancies, and overexpressed or mutated self-antigens associated with solid tumors has been found, encouraging further development of CAR-engineered NK-92 cells. Importantly, in syngeneic mouse tumor models, induction of endogenous antitumor immunity after treatment with CAR-expressing NK-92 cells has been demonstrated, resulting in cures and long-lasting immunological memory protecting against tumor rechallenge at distant sites. Here, we summarize the current status and future prospects of CAR-engineered NK-92 cells as off-the-shelf cellular therapeutics, with special emphasis on ErbB2 (HER2)-specific NK-92 cells that are approaching clinical application.