H3K27M neoepitope vaccination in diffuse midline glioma induces B and T cell responses across diverse HLA loci of a recovered patient.

H3K27M, a driver mutation with T and B cell neoepitope characteristics, defines an aggressive subtype of diffuse glioma with poor survival. We functionally dissect the immune response of one patient treated with an H3K27M peptide vaccine who subsequently entered complete remission. The vaccine robustly expanded class II human leukocyte antigen (HLA)-restricted peripheral H3K27M-specific T cells. Using functional assays, we characterized 34 clonally unique H3K27M-reactive T cell receptors and identified critical, conserved motifs in their complementarity-determining region 3 regions. Using detailed HLA mapping, we further demonstrate that diverse HLA-DQ and HLA-DR alleles present immunogenic H3K27M epitopes. Furthermore, we identified and profiled H3K27M-reactive B cell receptors from activated B cells in the cerebrospinal fluid. Our results uncover the breadth of the adaptive immune response against a shared clonal neoantigen across multiple HLA allelotypes and support the use of class II-restricted peptide vaccines to stimulate tumor-specific T and B cells harboring receptors with therapeutic potential.

Cerebrospinal fluid cfDNA sequencing for classification of central nervous system glioma.

PURPOSE: Primary central nervous system (CNS) gliomas can be classified by characteristic genetic alterations. In addition to solid tissue obtained via surgery or biopsy, cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) is an alternative source of material for genomic analyses. EXPERIMENTAL DESIGN: We performed targeted next-generation sequencing (NGS) of CSF cfDNA in a representative cohort of 85 patients presenting at two neurooncological centers with suspicion of primary or recurrent glioma. Copy-number variation (CNV) profiles, single nucleotide variants (SNVs), and small insertions/ deletions (indels) were combined into a molecular-guided tumor classification. Comparison with the solid tumor was performed for 38 cases with matching solid tissue available. RESULTS: Cases were stratified into four groups: glioblastoma (n = 32), other glioma (n = 19), non-malignant (n = 17) and non-diagnostic (n = 17). We introduced a molecular-guided tumor classification, which enabled identification of tumor entities and/ or cancer specific alterations in 75.0 % (n = 24) of glioblastoma and 52.6 % (n = 10) of other glioma cases. The overlap between CSF and matching solid tissue was highest for CNVs (26-48 %) and SNVs at pre-defined gene loci (44 %), followed by SNVs/ indels identified via uninformed variant calling (8-14 %). A molecular-guided tumor classification was possible for 23.5 % (n = 4) of non-diagnostic cases. CONCLUSIONS: We developed a targeted sequencing workflow for CSF cfDNA as well as a strategy for interpretation and reporting of sequencing results based on a molecular-guided tumor classification in glioma.

INTERCEPT H3: a multicenter phase I peptide vaccine trial for the treatment of H3-mutated diffuse midline gliomas.

INTRODUCTION: Diffuse midline gliomas (DMG) are universally lethal central nervous system tumors that carry almost unanimously the clonal driver mutation histone-3 K27M (H3K27M). The single amino acid substitution of lysine to methionine harbors a neoantigen that is presented in tumor tissue. The long peptide vaccine H3K27M-vac targeting this major histocompatibility complex class II (MHC class II)-restricted neoantigen induces mutation-specific immune responses that suppress the growth of H3K27M+ flank tumors in an MHC-humanized rodent model. METHODS: INTERCEPT H3 is a non-controlled open label, single arm, multicenter national phase 1 trial to assess safety, tolerability and immunogenicity of H3K27M-vac in combination with standard radiotherapy and the immune checkpoint inhibitor atezolizumab (ATE). 15 adult patients with newly diagnosed K27M-mutant histone-3.1 (H3.1K27M) or histone-3.3 (H3.3K27M) DMG will be enrolled in this trial. The 27mer peptide vaccine H3K27M-vac will be administered concomitantly to standard radiotherapy (RT) followed by combinatorial treatment with the programmed death-ligand 1 (PD-L1) targeting antibody ATE. The first three vaccines will be administered bi-weekly (q2w) followed by a dose at the beginning of recovery after RT and six-weekly administrations of doses 5 to 11 thereafter. In a safety lead-in, the first three patients (pts. 1-3) will be enrolled sequentially. PERSPECTIVE: H3K27M-vac is a neoepitope targeting long peptide vaccine derived from the clonal driver mutation H3K27M in DMG. The INTERCEPT H3 trial aims at demonstrating (1) safety and (2) immunogenicity of repeated fixed dose vaccinations of H3K27M-vac administered with RT and ATE in adult patients with newly diagnosed H3K27M-mutant DMG. TRIAL REGISTRATION: NCT04808245.

A H3K27M-targeted vaccine in adults with diffuse midline glioma.

Substitution of lysine 27 to methionine in histone H3 (H3K27M) defines an aggressive subtype of diffuse glioma. Previous studies have shown that a H3K27M-specific long peptide vaccine (H3K27M-vac) induces mutation-specific immune responses that control H3K27M+ tumors in major histocompatibility complex-humanized mice. Here we describe a first-in-human treatment with H3K27M-vac of eight adult patients with progressive H3K27M+ diffuse midline glioma on a compassionate use basis. Five patients received H3K27M-vac combined with anti-PD-1 treatment based on physician's discretion. Repeat vaccinations with H3K27M-vac were safe and induced CD4+ T cell-dominated, mutation-specific immune responses in five of eight patients across multiple human leukocyte antigen types. Median progression-free survival after vaccination was 6.2 months and median overall survival was 12.8 months. One patient with a strong mutation-specific T cell response after H3K27M-vac showed pseudoprogression followed by sustained complete remission for >31 months. Our data demonstrate safety and immunogenicity of H3K27M-vac in patients with progressive H3K27M+ diffuse midline glioma.

The Natural Product ß-Escin Targets Cancer and Stromal Cells of the Tumor Microenvironment to Inhibit Ovarian Cancer Metastasis.

The high mortality of OvCa is caused by the wide dissemination of cancer within the abdominal cavity. OvCa cells metastasize to the peritoneum, which is covered by mesothelial cells, and invade into the underlying stroma, composed of extracellular matrices (ECM) and stromal cells. In a study using a three-dimensional quantitative high-throughput screening platform (3D-qHTS), we found that ß-escin, a component of horse chestnut seed extract, inhibited OvCa adhesion/invasion. Here, we determine whether ß-escin and structurally similar compounds have a therapeutic potential against OvCa metastasis. Different sources of ß-escin and horse chestnut seed extract inhibited OvCa cell adhesion/invasion, both in vitro and in vivo. From a collection of 160 structurally similar compounds to ß-escin, we found that cardiac glycosides inhibited OvCa cell adhesion/invasion and proliferation in vitro, and inhibited adhesion/invasion and metastasis in vivo. Mechanistically, ß-escin and the cardiac glycosides inhibited ECM production in mesothelial cells and fibroblasts. The oral administration of ß-escin inhibited metastasis in both OvCa prevention and intervention mouse models. Specifically, ß-escin inhibited ECM production in the omental tumors. Additionally, the production of HIF1α-targeted proteins, lactate dehydrogenase A, and hexokinase 2 in omental tumors was blocked by ß-escin. This study reveals that the natural compound ß-escin has a therapeutic potential because of its ability to prevent OvCa dissemination by targeting both cancer and stromal cells in the OvCa tumor microenvironment.

[Acute ischemic stroke and elevated troponin: Diagnostic work-up and therapeutic consequences]. / Akuter ischämischer Schlaganfall und Troponinerhöhung: Diagnostische Abklärung und therapeutische Konsequenzen.

Elevated troponin values are frequently detected in patients with acute ischemic stroke, requiring adequate diagnostic work-up due to the high cardiac mortality after stroke. Since dual platelet inhibition can cause secondary intracerebral hemorrhage careful consideration of invasive coronary intervention is mandatory. Based on three case reports, this review article presents a diagnostic algorithm taking into account latest findings from the literature.

Microplastic burden in Daphnia is aggravated by elevated temperatures.

Contamination of freshwater habitats with microplastic is threatening particularly filter-feeders within the aquatic community. Using Daphnia magna and Daphnia pulex as models, the effects of food supply and temperature on the ingestion of polystyrene spheres (diameter 1 µm, concentration of 200 ng*ml-1) was analysed. The ingestion rates of microplastic beads were increased in conditions of low food and high temperatures, reflecting the complex regulation patterns of the water current generated by the animals' thoracic limbs. Maximal enrichment of 1160 times the concentration in the ambient medium was observed within one hour. Analyses of the impact of microplastic ingestion on physiological parameters used the carbohydrate concentration as an indicator for the animals' metabolic state. Exposure to the microplastic beads for three days in the presence or absence of Desmodesmus subspicatus did not affect the animals' glycogen reserves beyond the response to the prevailing food and temperature conditions. Projecting the insights from laboratory experiments to the habitat situation, increased burdens of microplastic particles can be expected in filtering zooplankton organisms in warm water and scarce supply of food, like the clear-water phase of lakes in the summer.

Changes in Demographic and Diagnostic Spectra of Patients with Neurological Symptoms Presenting to an Emergency Department During the COVID-19 Pandemic: A Retrospective Cohort Study.

OBJECTIVE: To analyse the characteristics of patients with neurological complaints seeking evaluation in an interdisciplinary emergency department (ED) during the rise of the COVID-19 pandemic in Germany. METHODS: In this retrospective study, data on the number of ED presentations due to neurological complaints in weeks 1-15/2020 were collected. In addition, hospital chart data of patients referred for neurological evaluation during weeks 12-15/2020 when the pandemic began impacting on public life in Germany were analysed regarding demographic information, chief complaints, modes of presentation and disposition and ED discharge diagnosis. Both data sets were compared to respective periods from 2017. RESULTS: During the surge of COVID-19, we found a significant decrease of the total number of neurological ED patients by 47.6%. Comparing weeks 12-15 of 2017 and 2020, we found a decrease in the number of patients of <30 years (p<0.001) and an increase of those 70 years (p<0.001). A higher proportion of patients were admitted to escalated care (p=0.03), and fewer patients were discharged against medical advice (p<0.001). In addition, the ratio of less acute diagnoses (eg, benign headaches) declined significantly. CONCLUSION: Our findings suggest that the pandemic has contributed to a - potentially transient - reframing of laypeople's perception of urgency and necessity for emergency presentation. The establishment and promotion of health-care structures and services like telemedical consultations and the creation of safe ED environments will be essential to enable adequate delivery of care in potential future waves of the pandemic.

Prolonged activated partial thromboplastin time after prophylactic-dose unfractionated heparin in the post-operative neurosurgical setting: case series and management recommendations.

Primary brain tumors, both benign and malignant, pose a high risk of perioperative venous thromboembolism (VTE) due to the development of a prothrombotic state. Perioperative pharmacologic thromboprophylaxis with subcutaneous (SC) unfractionated heparin (UFH) has significantly reduced VTE associated morbidity. Recent reports suggest an association between prolonged activated partial thromboplastin time (aPTT) due to prophylactic SC UFH and increased bleeding risk. We present three patients with normal baseline coagulation parameters in whom pharmacologic thromboprophylaxis with SC UFH resulted in a marked prolongation of the aPTT, leading to adverse outcomes in two patients. These cases demonstrate the uncertain kinetics of SC UFH and effect on aPTT, suggesting the significance of routine aPTT monitoring in high-risk settings. Given the wide variation in presentations of therapeutic or supratherapeutic values of aPTT in the perioperative neurosurgical setting, we propose a practical standardized approach to the evaluation and management of aPTT prolongation following prophylactic SC UFH administration.

Mesothelial Cell HIF1α Expression Is Metabolically Downregulated by Metformin to Prevent Oncogenic Tumor-Stromal Crosstalk.

The tumor microenvironment (TME) plays a pivotal role in cancer progression, and, in ovarian cancer (OvCa), the primary TME is the omentum. Here, we show that the diabetes drug metformin alters mesothelial cells in the omental microenvironment. Metformin interrupts bidirectional signaling between tumor and mesothelial cells by blocking OvCa cell TGF-ß signaling and mesothelial cell production of CCL2 and IL-8. Inhibition of tumor-stromal crosstalk by metformin is caused by the reduced expression of the tricarboxylic acid (TCA) enzyme succinyl CoA ligase (SUCLG2). Through repressing this TCA enzyme and its metabolite, succinate, metformin activated prolyl hydroxylases (PHDs), resulting in the degradation of hypoxia-inducible factor 1α (HIF1α) in mesothelial cells. Disruption of HIF1α-driven IL-8 signaling in mesothelial cells by metformin results in reduced OvCa invasion in an organotypic 3D model. These findings indicate that tumor-promoting signaling between mesothelial and OvCa cells in the TME can be targeted using metformin.

Women's Attitudes Toward Self-Monitoring of Their Pregnancy Using Noninvasive Electronic Devices: Cross-Sectional Multicenter Study.

BACKGROUND: Pregnancy can be distressing, particularly if expectant mothers are worried about the well-being of their fetus. Consequently, the desire for reassurance and frequent fetal monitoring is often pronounced. Smart wearable devices and telemedicine are promising tools that could assist women in self-monitoring their pregnancy at home, hence disburdening emergency departments (EDs). They present the possibility to clarify the need for urgent care remotely and offer tighter pregnancy monitoring. However, patients' acceptance of such new technologies for fetal monitoring has not yet been explored extensively. OBJECTIVE: This survey aimed to elucidate the attitudes of women toward self-monitoring of their pregnancy using noninvasive electronic devices. The technical details of the proposed devices were not specified. METHODS: A cross-sectional multicenter study was conducted at the departments of obstetrics of the University Hospitals of Heidelberg and Leipzig, Germany. All patients seen in the obstetrics clinic who were above 18 years were offered participation. We designed a survey questionnaire including validated instruments covering population characteristics, issues in current and past pregnancies, as well as attitudes toward self-monitoring of pregnancy with smart devices. RESULTS: A total of 509 pregnant women with no previous experience in telemedicine participated. Only a small minority of 5.9% (29/493) regarded self-monitoring with wearable devices as an alternative to consulting their physicians. Along these lines, only 7.7% (38/496) strongly believed they would visit the ED less often if such devices were readily available. However, if the procedure were combined with a Web-based telemetric physician consult, 13.5% (66/487) would be highly motivated to use the devices. Furthermore, significantly more women regarded it as an alternative prior to seeing a doctor when they perceived a decline in fetal movements (P<.001). Interestingly, women with university degrees had a higher propensity to engage in pregnancy self-monitoring compared with women without one (37% vs 23%; P=.001). Of the participants, 77.9% (381/489) would like smart wearable devices to measure fetal heart sounds, and 62.6% (306/489) wished to use the devices on their own. Feedback from a doctor or midwife was also very important in their choice of such devices (61.8%, 301/487 wished feedback). The intended frequency of use differed vastly among women, ranging from 13.8% (65/471) who would like to use such a device several times per day to 31.6% (149/471) who favored once per week at most. CONCLUSIONS: Our results point to a skeptical attitude toward pregnancy self-monitoring among pregnant women. Nevertheless, many women are open to using devices for pregnancy monitoring in parallel to consulting their physician. The intention to use such devices several times daily or weekly, expressed by more than half of the participants, highlights the potential of such technologies.

A Web-Based Survey Assessing the Attitudes of Health Care Professionals in Germany Toward the Use of Telemedicine in Pregnancy Monitoring: Cross-Sectional Study.

BACKGROUND: The demand for fetal monitoring and constant reassurance is high in pregnant women. Consequently, pregnant women use various health apps and are more likely to visit emergency departments due to subjective but nonurgent complaints. However, electronic health (eHealth) and mobile health (mHealth) solutions are rarely used to prevent nonurgent emergency consultations. To implement modern care solutions, a better understanding of the attitudes, fears, and hopes of health care professionals toward eHealth and mHealth is needed. OBJECTIVE: The aim of this study was to investigate the attitudes of health care professionals in obstetrics toward telemedicine. METHODS: A quantitative Web-based survey on health care professionals in obstetrics in Germany was conducted. The participants included nurses, midwives, and physicians of all age groups and job positions working in hospitals that provide various levels of health care. The questionnaire comprised 24 questions about the characteristics of the study population, views about emergency consultations in obstetrics, attitude toward telemedicine, job satisfaction, and sleeping behavior. RESULTS: In total, 244 health care professionals participated in the Web-based survey. In general, health care professionals were skeptical (170/233, 72.9%) about the use of telemedicine in obstetrics; however, 55.8% (130/233) recognized its potential. Moreover, 72% (62/86) of physicians were optimistic in using apps for pregnancy monitoring, whereas 36.1% (47/130) of nonphysicians (P<.001) were not. Significantly, more nonphysicians rejected such developments (75/130, 57.7% rejected) compared with physicians (24/86, 28%; P<.001). We also found that obstetricians with more than 10 years of work-experience are more skeptical; however, approximately 49% (18/37) of them believed that telemedicine could reduce nonurgent emergency consultations, whereas 73.2% (106/145) of obstetricians with less than 5 years of experience (P=.01) thought otherwise. Our survey revealed a high job satisfaction and a prevalence of regular sleeping problems of 45.9% (91/198) among health care professionals in obstetrics. Surprisingly, both job satisfaction and sleeping problems were independent from the number of night shifts per month (P=.77 and P=.99, respectively). Yet, 56.6% (112/198) of the survey participants thought they would be happier with their job if they had to work fewer night shifts per month. CONCLUSIONS: Our study reveals an ambivalent attitude toward the use of telemedicine among health care professionals in obstetrics in Germany at the moment. Efforts to promote the use of telemedicine should focus on nurses and midwives because these groups are the most skeptical. By contrast, particularly young physicians recognize the potential of apps in patient care and would like to use such technology in pregnancy monitoring.

Allergies - A T cells perspective in the era beyond the TH1/TH2 paradigm.

Allergic diseases have emerged as a major health care burden, especially in the western hemisphere. They are defined by overshooting reactions of an aberrant immune system to harmless exogenous stimuli. The TH1/TH2 paradigm assumes that a dominance of TH2 cell activation and an inadequate TH1 cell response are responsible for the development of allergies. However, the characterization of additional T helper cell subpopulations such as TH9, TH17, TH22, THGM-CSF and their interplay with regulatory T cells suggest further layers of complexity. This review summarizes state-of-the-art knowledge on T cell diversity and their induction, while revisiting the TH1/TH2 paradigm. With respect to these numerous contributors, it offers a new perspective on the pathogenesis of asthma, allergic rhinitis (AR) and atopic dermatitis (AD) incorporating recent discoveries in the field of T cell plasticity.

Proteomics reveals the effects of sustained weight loss on the human plasma proteome.

Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill-defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed by a year of weight maintenance. Using mass spectrometry-based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual-specific protein levels with wide-ranging effects of losing weight on the plasma proteome reflected in 93 significantly affected proteins. The adipocyte-secreted SERPINF1 and apolipoprotein APOF1 were most significantly regulated with fold changes of -16% and +37%, respectively (P < 10-13), and the entire apolipoprotein family showed characteristic differential regulation. Clinical laboratory parameters are reflected in the plasma proteome, and eight plasma proteins correlated better with insulin resistance than the known marker adiponectin. Nearly all study participants benefited from weight loss regarding a ten-protein inflammation panel defined from the proteomics data. We conclude that plasma proteome profiling broadly evaluates and monitors intervention in metabolic diseases.

Ultra-deep and quantitative saliva proteome reveals dynamics of the oral microbiome.

BACKGROUND: The oral cavity is home to one of the most diverse microbial communities of the human body and a major entry portal for pathogens. Its homeostasis is maintained by saliva, which fulfills key functions including lubrication of food, pre-digestion, and bacterial defense. Consequently, disruptions in saliva secretion and changes in the oral microbiome contribute to conditions such as tooth decay and respiratory tract infections. Here we set out to quantitatively map the saliva proteome in great depth with a rapid and in-depth mass spectrometry-based proteomics workflow. METHODS: We used recent improvements in mass spectrometry (MS)-based proteomics to develop a rapid workflow for mapping the saliva proteome quantitatively and at great depth. Standard clinical cotton swabs were used to collect saliva form eight healthy individuals at two different time points, allowing us to study inter-individual differences and interday changes of the saliva proteome. To accurately identify microbial proteins, we developed a method called "split by taxonomy id" that prevents peptides shared by humans and bacteria or between different bacterial phyla to contribute to protein identification. RESULTS: Microgram protein amounts retrieved from cotton swabs resulted in more than 3700 quantified human proteins in 100-min gradients or 5500 proteins after simple fractionation. Remarkably, our measurements also quantified more than 2000 microbial proteins from 50 bacterial genera. Co-analysis of the proteomics results with next-generation sequencing data from the Human Microbiome Project as well as a comparison to MALDI-TOF mass spectrometry on microbial cultures revealed strong agreement. The oral microbiome differs between individuals and changes drastically upon eating and tooth brushing. CONCLUSION: Rapid shotgun and robust technology can now simultaneously characterize the human and microbiome contributions to the proteome of a body fluid and is therefore a valuable complement to genomic studies. This opens new frontiers for the study of host-pathogen interactions and clinical saliva diagnostics.

Parallel Accumulation-Serial Fragmentation (PASEF): Multiplying Sequencing Speed and Sensitivity by Synchronized Scans in a Trapped Ion Mobility Device.

In liquid chromatography-mass spectrometry (LC-MS)-based proteomics, many precursors elute from the column simultaneously. In data-dependent analyses, these precursors are fragmented one at a time, whereas the others are discarded entirely. Here we employ trapped ion mobility spectrometry (TIMS) on an orthogonal quadrupole time-of-flight (QTOF) mass spectrometer to remove this limitation. In TIMS, all precursor ions are accumulated in parallel and released sequentially as a function of their ion mobility. Instead of selecting a single precursor mass with the quadrupole mass filter, we here implement synchronized scans in which the quadrupole is mass positioned with sub-millisecond switching times at the m/z values of appropriate precursors, such as those derived from a topN precursor list. We demonstrate serial selection and fragmentation of multiple precursors in single 50 ms TIMS scans. Parallel accumulation-serial fragmentation (PASEF) enables hundreds of MS/MS events per second at full sensitivity. Modeling the effect of such synchronized scans for shotgun proteomics, we estimate that about a 10-fold gain in sequencing speed should be achievable by PASEF without a decrease in sensitivity.

Dual melanocortin-4 receptor and GLP-1 receptor agonism amplifies metabolic benefits in diet-induced obese mice.

We assessed the efficacy of simultaneous agonism at the glucagon-like peptide-1 receptor (GLP-1R) and the melanocortin-4 receptor (MC4R) for the treatment of obesity and diabetes in rodents. Diet-induced obese (DIO) mice were chronically treated with either the long-acting GLP-1R agonist liraglutide, the MC4R agonist RM-493 or a combination of RM-493 and liraglutide. Co-treatment of DIO mice with RM-493 and liraglutide improves body weight loss and enhances glycemic control and cholesterol metabolism beyond what can be achieved with either mono-therapy. The superior metabolic efficacy of this combination therapy is attributed to the anorectic and glycemic actions of both drugs, along with the ability of RM-493 to increase energy expenditure. Interestingly, compared to mice treated with liraglutide alone, hypothalamic Glp-1r expression was higher in mice treated with the combination therapy after both acute and chronic treatment. Further, RM-493 enhanced hypothalamic Mc4r expression. Hence, co-dosing with MC4R and GLP-1R agonists increases expression of each receptor, indicative of minimized receptor desensitization. Together, these findings suggest potential opportunities for employing combination treatments that comprise parallel MC4R and GLP-1R agonism for the treatment of obesity and diabetes.