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Copper tungsten oxide films are deposited with the help of reactive high power impulse magnetron sputtering (HiPIMS) in an argon/oxygen gas mixture. Two magnetrons, one equipped with a tungsten target and the other with a copper target, are employed. The HiPIMS discharge is operated with a repetition frequency of f=100 Hz. Pulse widths of 100 and 20 µs separated by 25 µs are chosen for the tungsten and copper target, respectively. Films deposited on two different glass substrates [soda lime glass and fluorine doped tin oxide (FTO) coated glass] are characterized by energy dispersive x-ray spectroscopy, x-ray photoelectron spectroscopy, x-ray diffraction, Raman spectroscopy, and ellipsometry. Photoelectrochemical activity was investigated by linear voltammetry. The composition and crystal structure of as-deposited and annealed films are found to depend on the deposition conditions. Annealed films deposited on FTO glass are composed of WO3 and CuWO4 or Cu2WO4 crystal phases. Films deposited on soda lime glass are subject to sodium diffusion into the films during annealing and the formation of Na2W2O7 and Na2W4O13 phases.
This article sheds light on the obstacles that women face as members of the government by answering the questions: How does the sex of ministers shape the way MPs’ assess the quality of their work? And, how does this relationship differ depending on the political ideology of MPs? We argue that legislators assess the competencies of women ministers differently after the activation of gender stereotypes, but that the way they react depends on the ideological orientation of their party. We investigate this topic in a real-word context using a unique survey experiment with German and Austrian MPs. The evidence reveals that, while MPs belonging to right-wing parties perceive women in the executive as less competent than men ministers, their colleagues from left-wing parties actually assess them more favorably. These findings highlight the persistence of old myths about women’s lacking political skills and the emergence of new ones about women’s superior ability to govern.
Introduction: Adiposity and excessive weight are on the rise in western industrialized countries. In cases where conservative measures fail and surgical interventions are not (yet) desired, gastric balloon therapy has proven to be a safe and reversible endoscopic method. Methods: Aside from weight progression under gastric balloon therapy and by using MRI, our research paper describes the behavior of different abdominal body fat compartments at the beginning and at the end of the gastric balloon therapy. Additionally, the volume of the left liver lobe as well as the fill volume and performance of the gastric balloon were analyzed over the duration of treatment. For assessing potential impacts of weight reduction on the muscle mass, we determined the area of the m. psoas on a comparable cross-sectional area at the beginning and at the end of the therapy. Results: We were able to verify a significant reduction of the layer of subcutaneous fat, adipose capsule of the kidney, and intra-abdominal fatty tissue during the therapy. The volume of the left liver lobe was shrinking in addition to a muscle loss during the balloon therapy. The volume of the gastric balloon remained stable (not hyperinflation). There were variable gas bubbles in the gastric balloon. Conclusion: The gastric balloon is a temporary and successful option for weight reduction by reducing body fat, liver volume, but also muscle mass.
Hintergrund
Migräne ist eine hochprävalente Erkrankung, die bei betroffenen Patient*innen eine hohe Belastung sowohl durch die Kopfschmerzen an sich als auch durch die Einschränkung ihres Sozial- und Berufslebens hervorruft. Die Therapie besteht aus einer Akuttherapie der Kopfschmerzattacken sowie einer prophylaktischen Therapie zur Reduktion der Kopfschmerzfrequenz und -schwere. In der Prophylaxe stehen mit Antikörpern gegen das Calcitonin-gene-related-peptide (CGRP) und dessen Rezeptor erstmalig für die Migräne entwickelte gezielte prophylaktische Therapien zur Verfügung. Es stellt sich jedoch hierbei die Frage, ob CGRP-Antikörper lediglich symptomatisch in der Peripherie des trigemino-vaskulären-Systems wirken oder auch im zentralen Nervensystem die zugrundeliegenden pathophysiologischen Mechanismen beeinflussen, was einer krankheitsmodifizierenden Wirkung entspräche. Ziel unserer Studie war es, die Nullhypothese einer rein symptomatischen Wirkung gegen die Alternativhypothese einer Krankheitsmodifikation und somit zentralnervösen Wirkung, zu prüfen, indem bei Patient*innen mit episodischer Migräne der nozizeptive Blinkreflex vor und nach der Behandlung mit CGRP-Antikörpern untersucht wurde.
Methoden
22 Patient*innen mit episodischer Migräne (21 Frauen, 46,2 ± 13,8 Jahre alt) und 22 alters- und geschlechts-gematchte Kontrollen wurden im Rahmen dieser prospektiven Beobachtungsstudie eingeschlossen. Sie erhielten einen umfassenden Fragebogen zur Erhebung demografischer Charakteristika sowie der Kopfschmerzanamnese. Es erfolgte eine Messung des Blinkreflexes (10 Durchgänge à 6 Stimuli) vor (V0) und 3 Monate (V3) nach der Behandlung mit CGRP-Antikörpern (Kontrollen wurden einmalig gemessen). Im Rahmen der Messung wurden wiederholt schmerzhafte Stimuli supraorbital appliziert, die direkte Rückschlüsse auf die zentralnervöse Erregbarkeit des Hirnstamms als pathophysiologisch zentralen Mechanismus im Rahmen der Migräneentstehung zulassen. Die Area-under-the-curve (AUC) der R2-Komponente der Muskelsummenaktionspotentiale des Blinkreflexes sowie das Habituationsverhalten (Regressionskoeffizient über mehrere Blöcke) der stimulierten sowie nicht-stimulierten Seite wurden über 10 Blöcke hinweg evaluiert (primärer Endpunkt). Es wurde jeweils zuerst ein Test auf globale Veränderungen durchgeführt, der dann durch post-hoc-Analysen weiter spezifiziert wurde.
Ergebnisse
Alle Patient*innen zeigten eine signifikante Reduktion der Kopfschmerztage/Monat (V0: 12,4±3,3, V3: 6,6 ± 4,9) nach Beginn der Behandlung mit einem CGRP-/Rezeptorantikörper. Auf der stimulierten Seite reduzierte sich die AUC signifikant in den Blöcken eins, zwei sowie acht (Fglobal=5,86, p<0,001; block 1: R2a_s: -28%, p<0,001). Auf der nicht-stimulierten Seite zeigten sich Block eins, zwei, drei, acht sowie zehn als signifikant reduziert (Fglobal=8,22, p<0,001, block 1: R2a_ns: -22%, p=0,003). Die Veränderung der Habituation erwies sich in den Blöcken sechs, sieben, acht und zehn auf der nicht-stimulierten Seite als signifikant (Fglobal=3,07, p<0,001; block 6: R2h_ns: r=-1,36, p=0,007). Weder die AUC noch die Habituation des ersten Messtermins (V0) korrelierte mit dem späteren klinischen Ansprechen, sodass kein Prädiktor für das Therapieansprechen detektiert werden konnte (binär logistische Regression; alle Prädiktoren p>0,05).
Diskussion & Zusammenfassung
Die Ergebnisse dieser Studie zeigen, dass die dreimonatige Therapie mit CGRP-Antikörpern die Erregbarkeit des Hirnstamms als Antwort auf wiederholte schmerzhafte Stimuli bei Patient*innen mit Migräne normalisiert und liefert somit Hinweise für ein krankheitsmodifizierendes Potenzial. Veränderungen der Habituation korrelierten signifikant mit der Verringerung der Kopfschmerz-Frequenz, weitere Studien sind jedoch nötig, um zu eruieren, ob Parameter als Prädiktor geeignet sind um eine Voraussage über das Therapieansprechen und das Risiko einer Verschlechterung nach Beendigung der Therapie zu ermöglichen.
Representative epidemiologic data on the average volume of the parotid gland in a large population-based MRI survey is non-existent. Within the Study of Health in Pomerania (SHIP), we examined the parotid gland in 1725 non-contrast MRI-scans in T1 weighted sequence of axial layers. Thus, a reliable standard operating procedure (Intraclass Correlation Coefficient > 0.8) could be established. In this study, we found an average, single sided parotid gland volume of 27.82 cm3 (95% confidence interval (CI) 27.15 to 28.50) in male and 21.60 cm3 (95% CI 21.16 to 22.05) in female subjects. We observed positive associations for age, body mass index (BMI), as well as male sex with parotid gland size in a multivariate model. The prevalence of incidental tumors within the parotid gland regardless of dignity was 3.94% in the Northeast German population, slightly higher than assumed. Further epidemiologic investigations regarding primary salivary gland diseases are necessary.
Hematophagous leeches express a broad variety of secretory factors in their salivary glands; among them are hirudins, inhibitors of blood coagulation, and decorsins/ornatins, inhibitors of platelet aggregation. Here, we describe the identification and molecular and functional characterization of putative hirudins and decorsins/ornatins in two leech species of American origin, Limnobdella mexicana and Haementeria vizottoi. The leech species represent two orders of leeches, the proboscis-bearing Rhynchobdellida and the non-proboscis-bearing Arhynchobdellida. Members of the hirudin superfamily, such as hirudins or decorsins/ornatins, are described for the first time in the genus Haementeria. Both species expressed very potent inhibitors of platelet aggregation, but only the putative hirudins of L. mexicana displayed high thrombin-inhibitory potency, whereas the putative hirudin of H. vizottoi turned out to be a hirudin-like factor. The results of our study provide new insights into the evolutionary background of the blood-sucking lifestyle in leeches.
Although the common pathology of Alzheimer’s disease (AD) and white matter hyperintensities (WMH) is disputed, the gene TREML2 has been implicated in both conditions: its whole-blood gene expression was associated with WMH volume and its missense variant rs3747742 with AD risk. We re-examined those associations within one comprehensive dataset of the general population, additionally searched for cross-relations and illuminated the role of the apolipoprotein E (APOE) ε4 status in the associations. For our linear regression and linear mixed effect models, we used 1949 participants from the Study of Health in Pomerania (Germany). AD was assessed using a continuous pre-symptomatic MRI-based score evaluating a participant’s AD-related brain atrophy. In our study, increased whole-blood TREML2 gene expression was significantly associated with reduced WMH volume but not with the AD score. Conversely, rs3747742-C was significantly associated with a reduced AD score but not with WMH volume. The APOE status did not influence the associations. In sum, TREML2 robustly associated with WMH volume and AD-related brain atrophy on different molecular levels. Our results thus underpin TREML2’s role in neurodegeneration, might point to its involvement in AD and WMH via different biological mechanisms, and highlight TREML2 as a worthwhile target for disentangling the two pathologies.
The predominantly vegetative propagating duckweeds are of growing commercial interest. Since clonal accessions within a respective species can vary considerably with respect to their physiological as well as biochemical traits, it is critical to be able to track the clones of species of interest after their characterization. Here, we compared the efficacy of five different genotyping methods for Spirodela polyrhiza, a species with very low intraspecific sequence variations, including polymorphic NB-ARC-related loci, tubulin-gene-based polymorphism (TBP), simple sequence repeat variations (SSR), multiplexed ISSR genotyping by sequencing (MIG-seq), and low-coverage, reduced-representation genome sequencing (GBS). Four of the five approaches could distinguish 20 to 22 genotypes out of the 23 investigated clones, while TBP resolved just seven genotypes. The choice for a particular method for intraspecific genotyping can depend on the research question and the project budget, while the combination of orthogonal methods may increase the confidence and resolution for the results obtained.
Target Mechanisms of the Cyanotoxin Cylindrospermopsin in Immortalized Human Airway Epithelial Cells
(2022)
Cylindrospermopsin (CYN) is a cyanobacterial toxin that occurs in aquatic environments worldwide. It is known for its delayed effects in animals and humans such as inhibition of protein synthesis or genotoxicity. The molecular targets and the cell physiological mechanisms of CYN, however, are not well studied. As inhalation of CYN-containing aerosols has been identified as a relevant route of CYN uptake, we analyzed the effects of CYN on protein expression in cultures of immortalized human bronchial epithelial cells (16HBE14o−) using a proteomic approach. Proteins whose expression levels were affected by CYN belonged to several functional clusters, mainly regulation of protein stability, cellular adhesion and integration in the extracellular matrix, cell proliferation, cell cycle regulation, and completion of cytokinesis. With a few exceptions of upregulated proteins (e.g., ITI inhibitor of serine endopeptidases and mRNA stabilizer PABPC1), CYN mediated the downregulation of many proteins. Among these, centrosomal protein 55 (CEP55) and osteonectin (SPARC) were significantly reduced in their abundance. Results of the detailed semi-quantitative Western blot analyses of SPARC, claudin-6, and CEP55 supported the findings from the proteomic study that epithelial cell adhesion, attenuation of cell proliferation, delayed completion of mitosis, as well as induction of genomic instability are major effects of CYN in eukaryotic cells.
Background: Little is known about how substance use affects health-related quality of life (HRQOL) in depressed individuals. Here, associations between alcohol consumption and HRQOL in hospital and ambulatory care patients with past-year depressive symptoms are analyzed. Method: The sample consisted of 590 participants (26.8% non-drinkers) recruited via consecutive screenings. Individuals with alcohol use disorders were excluded. HRQOL was assessed with the Veterans Rand 12-item health survey (VR-12). Multivariable fractional polynomials (MFP) regression analyses were conducted (1) to test for non-linear associations between average daily consumption and HRQOL and (2) to analyze associations between alcohol consumption and the physical and mental health component summaries of the VR-12 and their subdomains. Results: Alcohol consumption was positively associated with the physical health component summary of the VR-12 (p = 0.001) and its subdomains general health (p = 0.006), physical functioning (p < 0.001), and bodily pain (p = 0.017), but not with the mental health component summary (p = 0.941) or any of its subdomains. Average daily alcohol consumption was not associated with HRQOL. Conclusion: Alcohol consumption was associated with better physical HRQOL. Findings do not justify ascribing alcohol positive effects on HRQOL. Data indicate that non-drinkers may suffer from serious health disorders. The results of this study can inform the development of future alcohol- and depression-related interventions.
Simple Summary
The treatment of high-risk neuroblastoma patients with anti-GD2 antibodies has improved survival, and it is an established treatment strategy; however, many patients still experience a late relapse. One disadvantage of passive immunotherapy is the absence of a memory response. Therefore, developing an active immunotherapy leading to a sustained immune response may provide a solution and prevent the occurrence of late relapses following anti-GD2 antibody therapy. Here, we describe the first-in-man compassionate use of the ganglidiomab vaccine following passive immunotherapy with an anti-GD2 antibody (dinutuximab beta) in seven neuroblastoma patients. The vaccine was well-tolerated, and all patients not pre-treated by haploidentical transplantation developed vaccine-specific immune responses.
Abstract
(1) Background: High-risk neuroblastoma (HR-NB) is associated with a poor prognosis despite a multimodal high-intensity treatment regimen, including immunotherapy with anti-GD2 monoclonal antibodies (mAb). Here, we investigated the effects of an anti-idiotypic vaccine based on the mAb ganglidiomab that structurally mimics GD2. (2) Methods: Patients with HR-NB treated with anti-GD2 mAb dinutuximab beta and who achieved complete remission after frontline or salvage therapy were offered the vaccine (0.5 mg ganglidiomab adsorbed to Alhydrogel®). Side effects (CTCAE v4.03) and immune responses were determined on each visit. We also evaluated the time to relapse or progression until the last follow-up. (3) Results: Seven HR-NB patients (five frontlines, two relapsed) received 6–22 subcutaneous injections every two weeks. Six of the seven patients showed an immune response. The non-responding patient had a haploidentical stem cell transplantation as part of the previous treatment. No fever, pain, neuropathy, or toxicities ≥ grade 3 occurred during or post-treatment. All immunized patients did not experience relapses or progressions of their neuroblastoma. (4) Conclusions: This is the first-in-man use of the ganglidiomab vaccine, which was well-tolerated, and all patients not pre-treated by haploidentical transplantation developed vaccine-specific immune responses. These findings provide an important basis for the design of prospective clinical trials.
Eicosanoids are lipid mediators generated from arachidonic acid with pro- and anti-inflammatory properties. Despite these lipid mediators being known for decades, quantitative determination in biological samples is still challenging due to low abundance, instability, the existence of regio- and stereoisomers, and a wide polarity range that hampers chromatographic separation. In this study, we developed a supercritical fluid chromatography mass spectrometry (SFC-MS) platform for the quantification of relevant eicosanoids. Application of a chiral amylose-based column and modifier combination of 2-propanol/acetonitrile offered separation and sufficient resolution of 11 eicosanoids (5-, 12-, 15-HETE, PGB1, LTB4, t-LTB4, 20-OH-LTB4, PGE2, PGD2, PGF2α, TxB2) with baseline separation of isobaric analytes within 12 min. The method was validated in terms of range (78–2500 ng/mL), linearity, accuracy, precision, and recovery according to EMA guidelines. Finally, we confirmed the method’s applicability by quantifying eicosanoid levels in human primary blood cells. In conclusion, we present a validated SFC-MS method for the determination of relevant eicosanoids in biological samples with a wide range of polarity while maintaining baseline separation of isobars, which allows coupling to a single quadrupole mass detector.
A New Laboratory Workflow Integrating the Free Light Chains Kappa Quotient into Routine CSF Analysis
(2022)
We performed this cohort study to test whether further analysis of intrathecal inflammation can be omitted if the free light chain kappa (FLCκ) quotient is within the reference range in the corresponding quotient diagram. FLCκ concentrations were measured in serum and cerebrospinal fluid (CSF) samples. The intrathecal fraction (IF) of FLCκ was calculated in relation to the hyperbolic reference range. 679 patient samples were used as a discovery cohort (DC). The sensitivity and negative predictive value (NPV) of the FLCκ-IF for the detection of an intrathecal humoral immune response (CSF-specific OCB and/or IF IgG/A/M > 0%) was determined. Based on these data, a diagnostic algorithm was developed and prospectively validated in an independent validation cohort (VC, n = 278). The sensitivity of the FLCκ-IF was 98% in the DC and 97% in the VC with a corresponding NPV of 99%. The use of the FLCκ-IF as a first line analysis would have reduced the Ig and OCB analysis by 62% in the DC and 74% in the VC. The absence of a FLCκ-IF predicts the absence of a humoral intrathecal immune response with a very high NPV of 99%. Thus, integration of our proposed algorithm into routine CSF laboratory analysis could help to reduce analytical efforts.
Amine transaminases (ATAs) are powerful biocatalysts for the stereoselective synthesis of chiral amines. However, wild-type ATAs usually show pH optima at slightly alkaline values and exhibit low catalytic activity under physiological conditions. For efficient asymmetric synthesis ATAs are commonly used in combination with lactate dehydrogenase (LDH, optimal pH: 7.5) and glucose dehydrogenase (GDH, optimal pH: 7.75) to shift the equilibrium towards the synthesis of the target chiral amine and hence their pH optima should fit to each other. Based on a protein structure alignment, variants of (R)-selective transaminases were rationally designed, produced in E. coli, purified and subjected to biochemical characterization. This resulted in the discovery of the variant E49Q of the ATA from Aspergillus fumigatus, for which the pH optimum was successfully shifted from pH 8.5 to 7.5 and this variant furthermore had a two times higher specific activity than the wild-type protein at pH 7.5. A possible mechanism for this shift of the optimal pH is proposed. Asymmetric synthesis of (R)-1-phenylethylamine from acetophenone in combination with LDH and GDH confirmed that the variant E49Q shows superior performance at pH 7.5 compared to the wild-type enzyme.
Background: Colorectal carcinoma (CRC) is a heterogeneous disease, and differences in outcomes have been reported among patients diagnosed with the same disease stage. Prognostic and predictive biomarkers provide information for patient risk stratification and guide treatment selection. Although numerous studies have analyzed the effects of systemic inflammatory factors on CRC outcomes, clinical significance remains to be elucidated. In particular, the treatment strategy of colon cancer patients is different from that of rectal cancer due to outcome and recurrence differences. The identification of patients with a poor prognosis who might benefit from intensive treatment approaches is clinically necessary. Methods: This study aimed to evaluate the value of different blood-based markers and assess the significance of our newly developed inflammatory-nutrition-related biomarker (NCR = BMI × albumin/CRP) in patients with colon cancer. A two-stage design was used with 212 patients with colon cancer (CC) in the discovery cohort (n = 159) and in an external validation cohort (n = 53). Results: A lower preoperative NCR level was significantly correlated with a worse prognosis, sidedness, undifferentiated histology, nodal involvement, and advanced UICC stage. We compared the NCR with other established prognostic indices and showed that the NCR is a more reliable indicator of a poor prognosis for patients with CC. Patients with low NCR levels experienced a significantly shorter Overall Survival (OS) than patients with high levels. Multivariate analysis confirmed preoperative NCR levels as an independent predictor for overall survival with a hazard ratio of 3.3 (95% confidence interval 1.628–6.709, p < 0.001). Finally, we confirmed the predictive value of the NCR in an independent validation cohort and confirmed NCR as an independent prognostic factor for OS. Conclusion: Taken together, we discovered a new prognostic index (NCR) based on BMI, albumin, and CRP levels as an independent prognostic predictor of OS in patients with colon cancer. In all UICC stages, our newly developed NCR marker is able to distinguish patients with better and worse prognoses. We, therefore, propose that NCR may serve as a supplement to the TNM staging system to optimize the risk stratification in CC patients towards personalized oncology. In particular, NCR can be used in clinical trials to stratify patients with UICC II and III tumors and help better select patients who might benefit from adjuvant treatment.
Background
The alignment of large numbers of protein sequences is a challenging task and its importance grows rapidly along with the size of biological datasets. State-of-the-art algorithms have a tendency to produce less accurate alignments with an increasing number of sequences. This is a fundamental problem since many downstream tasks rely on accurate alignments.
Results
We present learnMSA, a novel statistical learning approach of profile hidden Markov models (pHMMs) based on batch gradient descent. Fundamentally different from popular aligners, we fit a custom recurrent neural network architecture for (p)HMMs to potentially millions of sequences with respect to a maximum a posteriori objective and decode an alignment. We rely on automatic differentiation of the log-likelihood, and thus, our approach is different from existing HMM training algorithms like Baum–Welch. Our method does not involve progressive, regressive, or divide-and-conquer heuristics. We use uniform batch sampling to adapt to large datasets in linear time without the requirement of a tree. When tested on ultra-large protein families with up to 3.5 million sequences, learnMSA is both more accurate and faster than state-of-the-art tools. On the established benchmarks HomFam and BaliFam with smaller sequence sets, it matches state-of-the-art performance. All experiments were done on a standard workstation with a GPU.
Conclusions
Our results show that learnMSA does not share the counterintuitive drawback of many popular heuristic aligners, which can substantially lose accuracy when many additional homologs are input. LearnMSA is a future-proof framework for large alignments with many opportunities for further improvements.
Aim
The aim of this study was to evaluate whether extraction thresholds in persons with severe periodontitis have changed between 2000 and 2010 and whether potential shifts have contributed to the reported decrease in tooth extractions in German adults over the last decades.
Materials and Methods
Data from two German population-based cohort studies in Northeast Germany (Studies of Health in Pomerania; SHIP-START [baseline 1997–2001; 11-year follow-up] and SHIP-TREND [baseline 2008–2012; 7-year follow-up]) were used. In SHIP-START (SHIP-TREND), 522 (478) participants with severe periodontitis according to the CDC/AAP case definition were included. Patterns of maximum probing depth (PD) and maximum clinical attachment level (CAL) for retained and extracted teeth were compared between SHIP-START and SHIP-TREND participants.
Results
No major differences in patterns of baseline maximum CAL of retained or extracted teeth were detected between SHIP-START and SHIP-TREND. Extraction thresholds were identified at the baseline at maximum CAL ≥6 and ≥9 mm. Tooth-level incidence rates for extraction for baseline maximum CAL of 6 mm were comparable between SHIP-START and SHIP-TREND (17.1 vs. 15.9 events per 1000 person-years).
Conclusions
After a decade, teeth in persons with severe periodontitis were still undergoing extraction with minor or moderate attachment loss. A change in extraction pattern did not contribute to the higher tooth retention rate.
Background
Understanding how SARS-CoV-2 affects respiratory centres in the brainstem may help to preclude assisted ventilation for patients in intensive care setting. Viral invasion appears unlikely, although autoimmunity has been implicated, the responsible antigens remain unknown. We previously predicted the involvement of three epitopes within distinct brainstem proteins: disabled homolog 1 (DAB1), apoptosis-inducing-factor-1 (AIFM1), and surfeit-locus-protein-1 (SURF1).
Methods
Here, we used microarrays to screen serum from COVID-19 patients admitted to intensive care and compared those with controls who experienced mild course of the disease.
Findings
The results confirm the occurrence of IgG and IgM antibodies against the hypothesised epitopes in COVID-19 patients. Importantly, while IgM levels were similar in both groups, IgG levels were significantly elevated in severely ill patients compared to controls, suggesting a pathogenic role of IgG.
Interpretation
The newly discovered anti-neuronal antibodies might be promising markers of severe disease and the targeted peptide epitopes might be used for targeted immunomodulation. Further work is needed to determine whether these antibodies may play a role in long-COVID.
Funding
AF, CF and PR received support from the German Research Foundation (grants FL 379/22-1, 327654276-SFB 1315, FR 4479/1-1, PR 1274/8-1). SH, DR, and DB received support from the Ministry of Economy, State of Mecklenburg Western Pomerania, Germany (grant COVIDPROTECT: “Optimisation of diagnostic and therapeutic pathways for COVID-19 patients in MV”). SH received support from the Research Group Molecular Medicine University of Greifswald (FVMM, seed funding FOVB-2021-01). AV received support from the Else Kröner Fresenius Foundation and the Alzheimer Research Initiative.
Treatment of high-risk neuroblastoma (NB) patients with the anti-GD2 antibody (Ab) dinutuximab beta (DB) improves survival by 15%. Ab-dependent cellular cytotoxicity (ADCC) is the major mechanism of action and is primarily mediated by NK cells. Since IL-2 co-treatment did not show a therapeutic benefit but strongly induced Treg, we investigated here a DB-based immunotherapy combined with the immunocytokine FAP-IL-2v, which comprises a fibroblast activation protein α (FAP)-specific Ab linked to a mutated IL-2 variant (IL-2v) with abolished binding to the high-affinity IL-2 receptor, thus stimulating NK cells without induction of Treg. Effects of FAP-IL-2v on NK cells, Treg and ADCC mediated by DB, as well as FAP expression in NB, were investigated by flow cytometry, calcein-AM-based cytotoxicity assay and RT-PCR analysis. Moreover, the impact of soluble factors released from tumor cells on FAP expression by primary fibroblasts was assessed. Finally, a combined immunotherapy with DB and FAP-IL-2v was evaluated using a resistant syngeneic murine NB model. Incubation of leukocytes with FAP-IL-2v enhanced DB-specific ADCC without induction of Treg. FAP expression on NB cells and myeloid-derived suppressor cells (MDCS) in tumor tissue was identified. A tumor-cell-dependent enhancement in FAP expression by primary fibroblasts was demonstrated. Combination with DB and FAP-IL-2v resulted in reduced tumor growth and improved survival. Analysis of tumor tissue revealed increased NK and cytotoxic T cell numbers and reduced Treg compared to controls. Our data show that FAP-IL-2v is a potent immunocytokine that augments the efficacy of DB against NB, providing a promising alternative to IL-2.
Platelet adhesion and spreading at the sites of vascular injury is vital to hemostasis. As an integral part of the innate immune system, platelets interact with opsonized bacterial pathogens through FcγRIIA and contribute to host defense. As mechanoscavangers, platelets actively migrate and capture bacteria via cytoskeleton-rich, dynamic structures, such as filopodia and lamellipodia. However, the role of human platelet FcγRIIA in cytoskeleton-dependent interaction with opsonized bacteria is not well understood. To decipher this, we used a reductionist approach with well-defined micropatterns functionalized with immunoglobulins mimicking immune complexes at planar interfaces and bacteriamimetic microbeads. By specifically blocking of FcγRIIA and selective disruption of the platelet cytoskeleton, we show that both functional FcγRIIA and cytoskeleton are necessary for human platelet adhesion and haptotaxis. The direct link between FcγRIIA and the cytoskeleton is further explored by single-particle tracking. We then demonstrate the relevance of cytoskeleton-dependent differential mobilities of FcγRIIA on bacteria opsonized with the chemokine platelet factor 4 (PF4) and patient-derived anti-PF4/polyanion IgG. Our data suggest that efficient capture of opsonized bacteria during host-defense is governed by mobility dynamics of FcγRIIA on filopodia and lamellipodia, and the cytoskeleton plays an essential role in platelet morphodynamics at biological interfaces that display immune complexes.