The astrocyte-produced growth factor HB-EGF limits autoimmune CNS pathology.

Central nervous system (CNS)-resident cells such as microglia, oligodendrocytes and astrocytes are gaining increasing attention in respect to their contribution to CNS pathologies including multiple sclerosis (MS). Several studies have demonstrated the involvement of pro-inflammatory glial subsets in the pathogenesis and propagation of inflammatory events in MS and its animal models. However, it has only recently become clear that the underlying heterogeneity of astrocytes and microglia can not only drive inflammation, but also lead to its resolution through direct and indirect mechanisms. Failure of these tissue-protective mechanisms may potentiate disease and increase the risk of conversion to progressive stages of MS, for which currently available therapies are limited. Using proteomic analyses of cerebrospinal fluid specimens from patients with MS in combination with experimental studies, we here identify Heparin-binding EGF-like growth factor (HB-EGF) as a central mediator of tissue-protective and anti-inflammatory effects important for the recovery from acute inflammatory lesions in CNS autoimmunity. Hypoxic conditions drive the rapid upregulation of HB-EGF by astrocytes during early CNS inflammation, while pro-inflammatory conditions suppress trophic HB-EGF signaling through epigenetic modifications. Finally, we demonstrate both anti-inflammatory and tissue-protective effects of HB-EGF in a broad variety of cell types in vitro and use intranasal administration of HB-EGF in acute and post-acute stages of autoimmune neuroinflammation to attenuate disease in a preclinical mouse model of MS. Altogether, we identify astrocyte-derived HB-EGF and its epigenetic regulation as a modulator of autoimmune CNS inflammation and potential therapeutic target in MS.

CD200+ fibroblasts form a pro-resolving mesenchymal network in arthritis.

Fibroblasts are important regulators of inflammation, but whether fibroblasts change phenotype during resolution of inflammation is not clear. Here we use positron emission tomography to detect fibroblast activation protein (FAP) as a means to visualize fibroblast activation in vivo during inflammation in humans. While tracer accumulation is high in active arthritis, it decreases after tumor necrosis factor and interleukin-17A inhibition. Biopsy-based single-cell RNA-sequencing analyses in experimental arthritis show that FAP signal reduction reflects a phenotypic switch from pro-inflammatory MMP3+/IL6+ fibroblasts (high FAP internalization) to pro-resolving CD200+DKK3+ fibroblasts (low FAP internalization). Spatial transcriptomics of human joints indicates that pro-resolving niches of CD200+DKK3+ fibroblasts cluster with type 2 innate lymphoid cells, whereas MMP3+/IL6+ fibroblasts colocalize with inflammatory immune cells. CD200+DKK3+ fibroblasts stabilized the type 2 innate lymphoid cell phenotype and induced resolution of arthritis via CD200-CD200R1 signaling. Taken together, these data suggest a dynamic molecular regulation of the mesenchymal compartment during resolution of inflammation.

Immunoglobulin G-dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin-1.

Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.

The complement system drives local inflammatory tissue priming by metabolic reprogramming of synovial fibroblasts.

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.

Locally renewing resident synovial macrophages provide a protective barrier for the joint.

Macrophages are considered to contribute to chronic inflammatory diseases such as rheumatoid arthritis1. However, both the exact origin and the role of macrophages in inflammatory joint disease remain unclear. Here we use fate-mapping approaches in conjunction with three-dimensional light-sheet fluorescence microscopy and single-cell RNA sequencing to perform a comprehensive spatiotemporal analysis of the composition, origin and differentiation of subsets of macrophages within healthy and inflamed joints, and study the roles of these macrophages during arthritis. We find that dynamic membrane-like structures, consisting of a distinct population of CX3CR1+ tissue-resident macrophages, form an internal immunological barrier at the synovial lining and physically seclude the joint. These barrier-forming macrophages display features that are otherwise typical of epithelial cells, and maintain their numbers through a pool of locally proliferating CX3CR1- mononuclear cells that are embedded into the synovial tissue. Unlike recruited monocyte-derived macrophages, which actively contribute to joint inflammation, these epithelial-like CX3CR1+ lining macrophages restrict the inflammatory reaction by providing a tight-junction-mediated shield for intra-articular structures. Our data reveal an unexpected functional diversification among synovial macrophages and have important implications for the general role of macrophages in health and disease.

Quality requirements for MRI simulation in cranial stereotactic radiotherapy: a guideline from the German Taskforce "Imaging in Stereotactic Radiotherapy".

Accurate Magnetic Resonance Imaging (MRI) simulation is fundamental for high-precision stereotactic radiosurgery and fractionated stereotactic radiotherapy, collectively referred to as stereotactic radiotherapy (SRT), to deliver doses of high biological effectiveness to well-defined cranial targets. Multiple MRI hardware related factors as well as scanner configuration and sequence protocol parameters can affect the imaging accuracy and need to be optimized for the special purpose of radiotherapy treatment planning. MRI simulation for SRT is possible for different organizational environments including patient referral for imaging as well as dedicated MRI simulation in the radiotherapy department but require radiotherapy-optimized MRI protocols and defined quality standards to ensure geometrically accurate images that form an impeccable foundation for treatment planning. For this guideline, an interdisciplinary panel including experts from the working group for radiosurgery and stereotactic radiotherapy of the German Society for Radiation Oncology (DEGRO), the working group for physics and technology in stereotactic radiotherapy of the German Society for Medical Physics (DGMP), the German Society of Neurosurgery (DGNC), the German Society of Neuroradiology (DGNR) and the German Chapter of the International Society for Magnetic Resonance in Medicine (DS-ISMRM) have defined minimum MRI quality requirements as well as advanced MRI simulation options for cranial SRT.

Adult alcohol drinking and emotional tone are mediated by neutral sphingomyelinase during development in males.

Alcohol use, abuse, and addiction, and resulting health hazards are highly sex-dependent with unknown mechanisms. Previously, strong links between the SMPD3 gene and its coded protein neutral sphingomyelinase 2 (NSM) and alcohol abuse, emotional behavior, and bone defects were discovered and multiple mechanisms were identified for females. Here we report strong sex-dimorphisms for central, but not for peripheral mechanisms of NSM action in mouse models. Reduced NSM activity resulted in enhanced alcohol consumption in males, but delayed conditioned rewarding effects. It enhanced the acute dopamine response to alcohol, but decreased monoaminergic systems adaptations to chronic alcohol. Reduced NSM activity increased depression- and anxiety-like behavior, but was not involved in alcohol use for the self-management of the emotional state. Constitutively reduced NSM activity impaired structural development in the brain and enhanced lipidomic sensitivity to chronic alcohol. While the central effects were mostly opposite to NSM function in females, similar roles in bone-mediated osteocalcin release and its effects on alcohol drinking and emotional behavior were observed. These findings support the view that the NSM and multiple downstream mechanism may be a source of the sex-differences in alcohol use and emotional behavior.

Skin sodium is increased in male patients with multiple sclerosis and related animal models.

Novel MRI techniques allow a noninvasive quantification of tissue sodium and reveal the skin as a prominent compartment of sodium storage in health and disease. Since multiple sclerosis (MS) immunopathology is initiated in the periphery and increased sodium concentrations induce proinflammatory immune cells, the skin represents a promising compartment linking high sodium concentrations and MS immunopathology. We used a 7-T sodium MRI (23Na-MRI) and inductively coupled plasma mass spectrometry to investigate the skin sodium content in two mouse models of MS. We additionally performed 3-T 23Na-MRI of calf skin and muscles in 29 male relapsing-remitting MS (RRMS) patients and 29 matched healthy controls. Demographic and clinical information was collected from interviews, and disease activity was assessed by expanded disability status scale scoring. 23Na-MRI and chemical analysis demonstrated a significantly increased sodium content in the skin during experimental autoimmune encephalomyelitis independent of active immunization. In male patients with RRMS, 23Na-MRI demonstrated a higher sodium signal in the area of the skin compared to age- and biological sex-matched healthy controls with higher sodium, predicting future disease activity in cranial MRI. In both studies, the sodium enrichment was specific to the skin, as we found no alterations of sodium signals in the muscle or other tissues. Our data add to the recently identified importance of the skin as a storage compartment of sodium and may further represent an important organ for future investigations on salt as a proinflammatory agent driving autoimmune neuroinflammation such as that in MS.

Assessment of myocardial fibrosis in patients with systemic sclerosis using [68Ga]Ga-FAPI-04-PET-CT.

PURPOSE: Myocardial fibrosis (MF) is a factor of poor prognosis in systemic sclerosis (SSc). Direct in-vivo visualization of fibroblast activation as early readout of MF has not been feasible to date. Here, we characterize 68Gallium-labeled-Fibroblast-Activation-Inhibitor-04 ([68Ga]Ga-FAPI-04)-PET-CT as a diagnostic tool in SSc-related MF. METHODS: In this proof-of-concept trial, six SSc patients with and eight without MF of the EUSTAR cohort Erlangen underwent [68Ga]Ga-FAPI-04-PET-CT and cardiac MRI (cMRI) and clinical and serologic investigations just before baseline and during follow-up between January 2020 and December 2020. Myocardial biopsy was performed as clinically indicated. RESULTS: [68Ga]Ga-FAPI-04 tracer uptake was increased in SSc-related MF with higher uptake in SSc patients with arrhythmias, elevated serum-NT-pro-BNP, and increased late gadolinium enhancement (LGE) in cMRI. Histologically, myocardial biopsies from cMRI- and [68Ga]Ga-FAPI-04-positive regions confirmed the accumulation of FAP+ fibroblasts surrounded by collagen deposits. We observed similar but not equal spatial distributions of [68Ga]Ga-FAPI-04 uptake and quantitative cMRI-based techniques. Using sequential [68Ga]Ga-FAPI-04-PET-CTs, we observed dynamic changes of [68Ga]Ga-FAPI-04 uptake associated with changes in the activity of SSc-related MF, while cMRI parameters remained stable after regression of molecular activity and rather indicated tissue damage. CONCLUSIONS: We present first in-human evidence that [68Ga]Ga-FAPI-04 uptake visualizes fibroblast activation in SSc-related MF and may be a diagnostic option to monitor cardiac fibroblast activity in situ.

[Contrast-Enhanced Ultrasound of Fibrolamellar Hepatocellular Carcinoma]. / Kontrastmittelsonografie eines fibrolamellären hepatozellulären Karzinoms.

We present the case of a 24-year-old male patient, who was admitted for endoscopy due to sustained pain in the upper abdomen with nausea and postprandial fullness without vomiting for more than 5 months. In the physical examination, an epigastric induration was found. Endoscopy revealed an external impression of the proximal duodenum. Beyond that, normal findings could be ascertained in gastroscopy and ileo-colonoscopy. Abdominal ultrasound identified a large hypoechoic lesion in the left liver lobe with a sharp delineation. Along the upper mesenteric vessels, enlarged lymphnodes were visible with contact to the proximal duodenum. Contrast-enhanced ultrasound (CE-US) was conducted and revealed the typical perfusion pattern of hepatocellular carcinoma. For further assessment, an ultrasound-guided core-biopsy of the lesion was performed. The histopathological evaluations resulted in the diagnosis of a hepatocellular carcinoma of fibrolamellar subtype.With the present case, we want to illustrate the perfusion pattern of a fibrolamellar hepatocellular carcinoma in contrast-enhanced ultrasound. Even though the tumor tissue is surrounded by lamellar bands of fibrosis with collagen-rich fibers, the perfusion pattern is consistent with the previously known appearance of HCC in CE-US.

Whole-body magnetic resonance imaging plus serological follow-up for early identification of progression in smouldering myeloma patients to prevent development of end-organ damage.

The definition of multiple myeloma (MM) was updated in 2014, with the intent to enable earlier treatment and thereby avoid appearance of end-organ damage at progression from smouldering multiple myeloma (SMM) to MM. The purpose of this study was to investigate to which extent the development of end-organ damage at progression to MM was reduced under the updated guidelines. In this prospective observational cohort study (ClinicalTrials.gov Identifier: NCT01374412), between 2014 and 2020, 96 SMM patients prospectively underwent whole-body magnetic resonance imaging (wb-MRI) and serological follow-up at baseline and every 6 months thereafter. A total of 22 patients progressed into MM during follow-up, of which seven (32%) showed SLiM-criteria only but no end-organ damage. Four (57%) of the seven patients who progressed by SLiM-criteria only progressed with >1 focal lesion (FL) or a growing FL, and three (43%) due to serum free light-chain-ratio ≥100. Fifteen (68%) out of 22 patients who progressed still suffered from end-organ damage at progression. The updated disease definition reduced the proportion of SMM patients suffering from end-organ damage at progression to MM by one third. wb-MRI is an important tool for detection of SMM patients who progress to MM without end-organ damage.

Neutral sphingomyelinase mediates the co-morbidity trias of alcohol abuse, major depression and bone defects.

Mental disorders are highly comorbid and occur together with physical diseases, which are often considered to arise from separate pathogenic pathways. We observed in alcohol-dependent patients increased serum activity of neutral sphingomyelinase. A genetic association analysis in 456,693 volunteers found associations of haplotypes of SMPD3 coding for NSM-2 (NSM) with alcohol consumption, but also with affective state, and bone mineralisation. Functional analysis in mice showed that NSM controls alcohol consumption, affective behaviour, and their interaction by regulating hippocampal volume, cortical connectivity, and monoaminergic responses. Furthermore, NSM controlled bone-brain communication by enhancing osteocalcin signalling, which can independently supress alcohol consumption and reduce depressive behaviour. Altogether, we identified a single gene source for multiple pathways originating in the brain and bone, which interlink disorders of a mental-physical co-morbidity trias of alcohol abuse-depression/anxiety-bone disorder. Targeting NSM and osteocalcin signalling may, thus, provide a new systems approach in the treatment of a mental-physical co-morbidity trias.

Crystal Clots as Therapeutic Target in Cholesterol Crystal Embolism.

RATIONALE: Cholesterol crystal embolism can be a life-threatening complication of advanced atherosclerosis. Pathophysiology and molecular targets for treatment are largely unknown. OBJECTIVE: We aimed to develop a new animal model of cholesterol crystal embolism to dissect the molecular mechanisms of cholesterol crystal (CC)-driven arterial occlusion, tissue infarction, and organ failure. METHODS AND RESULTS: C57BL/6J mice were injected with CC into the left kidney artery. Primary end point was glomerular filtration rate (GFR). CC caused crystal clots occluding intrarenal arteries and a dose-dependent drop in GFR, followed by GFR recovery within 4 weeks, that is, acute kidney disease. In contrast, the extent of kidney infarction was more variable. Blocking necroptosis using mixed lineage kinase domain-like deficient mice or necrostatin-1s treatment protected from kidney infarction but not from GFR loss because arterial obstructions persisted, identifying crystal clots as a primary target to prevent organ failure. CC involved platelets, neutrophils, fibrin, and extracellular DNA. Neutrophil depletion or inhibition of the release of neutrophil extracellular traps had little effects, but platelet P2Y12 receptor antagonism with clopidogrel, fibrinolysis with urokinase, or DNA digestion with recombinant DNase I all prevented arterial occlusions, GFR loss, and kidney infarction. The window-of-opportunity was <3 hours after CC injection. However, combining Nec-1s (necrostatin-1s) prophylaxis given 1 hour before and DNase I 3 hours after CC injection completely prevented kidney failure and infarcts. In vitro, CC did not directly induce plasmatic coagulation but induced neutrophil extracellular trap formation and DNA release mainly from kidney endothelial cells, neutrophils, and few from platelets. CC induced ATP release from aggregating platelets, which increased fibrin formation in a DNase-dependent manner. CONCLUSIONS: CC embolism causes arterial obstructions and organ failure via the formation of crystal clots with fibrin, platelets, and extracellular DNA as critical components. Therefore, our model enables to unravel the pathogenesis of the CC embolism syndrome as a basis for both prophylaxis and targeted therapy.

Siglec-15 on Osteoclasts Is Crucial for Bone Erosion in Serum-Transfer Arthritis.

Siglec-15 is a conserved sialic acid-binding Ig-like lectin, which is expressed on osteoclasts. Deficiency of Siglec-15 leads to an impaired osteoclast development, resulting in a mild osteopetrotic phenotype. The role of Siglec-15 in arthritis is still largely unclear. To address this, we generated Siglec-15 knockout mice and analyzed them in a mouse arthritis model. We could show that Siglec-15 is directly involved in pathologic bone erosion in the K/BxN serum-transfer arthritis model. Histological analyses of joint destruction provided evidence for a significant reduction in bone erosion area and osteoclast numbers in Siglec-15-/- mice, whereas the inflammation area and cartilage destruction was comparable to wild-type mice. Thus, Siglec-15 on osteoclasts has a crucial function for bone erosion during arthritis. In addition, we generated a new monoclonal anti-Siglec-15 Ab to clarify its expression pattern on immune cells. Whereas this Ab demonstrated an almost exclusive Siglec-15 expression on murine osteoclasts and hardly any other expression on various other immune cell types, human Siglec-15 was more broadly expressed on human myeloid cells, including human osteoclasts. Taken together, our findings show a role of Siglec-15 as a regulator of pathologic bone resorption in arthritis and highlight its potential as a target for future therapies, as Siglec-15 blocking Abs are available.

Involvement of radiologists in oncologic multidisciplinary team meetings: an international survey by the European Society of Oncologic Imaging.

OBJECTIVES: Multidisciplinary tumour boards (MTBs) play an increasingly important role in managing cancer patients from diagnosis to treatment. However, many problems arise around the organisation of MTBs, both in terms of organisation-administration and time management. In this context, the European Society of Oncologic Imaging (ESOI) conducted a survey among its members, aimed at assessing the quality and amount of involvement of radiologists in MTBs, their role in it and related issues. METHODS: All members were invited to fill in a questionnaire consisting of 15 questions with both open and multiple-choice answers. Simple descriptive analyses and graphs were performed. RESULTS: A total of 292 ESOI members in full standing for the year 2018 joined the survey. Most respondents (89%) declared to attend MT-Bs, but only 114 respondents (43.9%) review over 70% of exams prior to MTB meetings, mainly due to lack of time due to a busy schedule for imaging and reporting (46.6%). Perceived benefits (i.e. surgical and histological feedback (86.9%), improved knowledge of cancer treatment (82.7%) and better interaction between radiologists and referring clinicians for discussing rare cases (56.9%)) and issues (i.e. attending MTB meetings during regular working hours (71.9%) and lack of accreditation with continuing medical education (CME) (85%)) are reported. CONCLUSIONS: Despite the value and benefits of radiologists' participation in MTBs, issues like improper preparation due to a busy schedule and no counterpart in CME accreditation require efforts to improve the role of radiologists for a better patient care. KEY POINTS: • Most radiologists attend multidisciplinary tumour boards, but less than half of them review images in advance, mostly due to time constraints. • Feedback about radiological diagnoses, improved knowledge of cancer treatment and interaction with referring clinicians are perceived as major benefits. • Concerns were expressed about scheduling multidisciplinary tumour boards during regular working hours and lack of accreditation with continuing medical education.

Combinatorial Drug Treatments Reveal Promising Anticytomegaloviral Profiles for Clinically Relevant Pharmaceutical Kinase Inhibitors (PKIs).

Human cytomegalovirus (HCMV) is a human pathogenic herpesvirus associated with a variety of clinical symptoms. Current antiviral therapy is not always effective, so that improved drug classes and drug-targeting strategies are needed. Particularly host-directed antivirals, including pharmaceutical kinase inhibitors (PKIs), may help to overcome problems of drug resistance. Here, we focused on utilizing a selection of clinically relevant PKIs and determined their anticytomegaloviral efficacies. Particularly, PKIs directed to host or viral cyclin-dependent kinases, i.e., abemaciclib, LDC4297 and maribavir, exerted promising profiles against human and murine cytomegaloviruses. The anti-HCMV in vitro activity of the approved anti-cancer drug abemaciclib was confirmed in vivo using our luciferase-based murine cytomegalovirus (MCMV) animal model in immunocompetent mice. To assess drug combinations, we applied the Bliss independence checkerboard and Loewe additivity fixed-dose assays in parallel. Results revealed that (i) both affirmative approaches provided valuable information on anti-CMV drug efficacies and interactions, (ii) the analyzed combinations comprised additive, synergistic or antagonistic drug interactions consistent with the drugs' antiviral mode-of-action, (iii) the selected PKIs, especially LDC4297, showed promising inhibitory profiles, not only against HCMV but also other α-, ß- and γ-herpesviruses, and specifically, (iv) the combination treatment with LDC4297 and maribavir revealed a strong synergism against HCMV, which might open doors towards novel clinical options in the near future. Taken together, this study highlights the potential of therapeutic drug combinations of current developmental/preclinical PKIs.

Magnetic resonance imaging for brain stereotactic radiotherapy : A review of requirements and pitfalls.

Due to its superior soft tissue contrast, magnetic resonance imaging (MRI) is essential for many radiotherapy treatment indications. This is especially true for treatment planning in intracranial tumors, where MRI has a long-standing history for target delineation in clinical practice. Despite its routine use, care has to be taken when selecting and acquiring MRI studies for the purpose of radiotherapy treatment planning. Requirements on MRI are particularly demanding for intracranial stereotactic radiotherapy, where accurate imaging has a critical role in treatment success. However, MR images acquired for routine radiological assessment are frequently unsuitable for high-precision stereotactic radiotherapy as the requirements for imaging are significantly different for radiotherapy planning and diagnostic radiology. To assure that optimal imaging is used for treatment planning, the radiation oncologist needs proper knowledge of the most important requirements concerning the use of MRI in brain stereotactic radiotherapy. In the present review, we summarize and discuss the most relevant issues when using MR images for target volume delineation in intracranial stereotactic radiotherapy.

Correction to: Assessment of treatment responses in children and adolescents with Ewing sarcoma with metabolic tumor parameters derived from 18F-FDG-PET/CT and circulating tumor DNA.

The author names and family names of the originally published article was inversed. Correct presentation is presented here.

Assessment of treatment responses in children and adolescents with Ewing sarcoma with metabolic tumor parameters derived from 18F-FDG-PET/CT and circulating tumor DNA.

PURPOSE: The purpose of this study was to perform a prospective integrated analysis of 18F-fluorodeoxyglucose (18F-FDG)-positron emission tomography (PET)/computed tomography (CT) and circulating tumor DNA (ctDNA) to assess responses to multimodal chemotherapy in children and adolescents suffering from Ewing sarcoma (EwS). METHODS: A total of 20 patients with histologically confirmed EwS underwent multiple 18F-FDG-PET/CT, performed at the time of each patient's initial diagnosis and after the second and fifth induction chemotherapy block (EWING2008 treatment protocol, NCT00987636). Additional PET examinations were performed as clinically indicated in some patients, e.g., in patients suspected of having progressive or relapsing disease. All 263 18F-FDG-positive lesions in the field of view suggestive of tumor tissue were assessed quantitatively to calculate PET-derived parameters, including whole-body metabolic tumor volume (wb-MTV) and whole-body total lesion glycolysis (wb-TLG), as well as the following data: standardized uptake value (SUV)max and SUVmean. Tumor-specific ctDNA in patient plasma samples was quantified using digital droplet PCR (ddPCR), and the correlations between ctDNA levels and PET-derived parameters were analyzed. Metabolic responses to multimodal chemotherapy as assessed with PET-parameters were compared to biochemical responses as assessed with changes in ctDNA levels. RESULTS: Twenty patients underwent a total of 87 18F-FDG-PET/CT scans, which detected 263 FDG-positive tumor lesions. Significant correlations between SUVmax, SUVmean, wb-MTV and wb-TLG values, and ctDNA levels were observed (all p < 0.0001). All patients suffering from EwS, with histology serving as gold standard, also presented with a positive corresponding ctDNA sample and a positive 18F-FDG-PET/CT examination before initiation of therapy. There were no false-negative results. Evaluation of treatment response after the fifth block of induction chemotherapy showed that the agreement between the metabolic response and biochemical response was 90%, which was statistically significant (Cohen κ = 0.62; p < 0.05). Non-detectable ctDNA after the second block of induction chemotherapy was associated with complete biochemical and metabolic responses after the fifth block of induction chemotherapy in 16/17 patients (94%). During a median follow-up period of 36 months (range: 8-104 months), four patients had tumor relapses, which, in all cases, were accompanied by an increase in plasma ctDNA levels and a positive 18F-FDG-PET/CT. No false-negative results were observed in the study cohort. Complete biochemical and metabolic responses after the fifth block of induction chemotherapy had a high positive predictive value for disease remission during the follow-up period; specifically, the positive predictive value was 88%. CONCLUSION: The combination of 18F-FDG-PET/CT and ctDNA quantification is a very promising noninvasive tool for assessing treatment responses and detecting tumor relapses in children and young adolescents suffering from EwS who are undergoing multimodal chemotherapy.

Aggregated neutrophil extracellular traps resolve inflammation by proteolysis of cytokines and chemokines and protection from antiproteases.

Papillon-Lefèvre syndrome (PLS) is characterized by nonfunctional neutrophil serine proteases (NSPs) and fulminant periodontal inflammation of unknown cause. Here we investigated neutrophil extracellular trap (NET)-associated aggregation and cytokine/chemokine-release/degradation by normal and NSP-deficient human and mouse granulocytes. Stimulated with solid or soluble NET inducers, normal neutrophils formed aggregates and both released and degraded cytokines/chemokines. With increasing cell density, proteolytic degradation outweighed release. Maximum output of cytokines/chemokines occurred mostly at densities between 2 × 107 and 4 × 107 neutrophils/cm3. Assessment of neutrophil density in vivo showed that these concentrations are surpassed during inflammation. Association with aggregated NETs conferred protection of neutrophil elastase against α1-antitrypsin. In contrast, eosinophils did not influence cytokine/chemokine concentrations. The proteolytic degradation of inflammatory mediators seen in NETs was abrogated in Papillon-Lefèvre syndrome (PLS) neutrophils. In summary, neutrophil-driven proteolysis of inflammatory mediators works as a built-in safeguard for inflammation. The absence of this negative feedback mechanism might be responsible for the nonresolving periodontitis seen in PLS.-Hahn, J., Schauer, C., Czegley, C., Kling, L., Petru, L., Schmid, B., Weidner, D., Reinwald, C., Biermann, M. H. C., Blunder, S., Ernst, J., Lesner, A., Bäuerle, T., Palmisano, R., Christiansen, S., Herrmann, M., Bozec, A., Gruber, R., Schett, G., Hoffmann, M. H. Aggregated neutrophil extracellular traps resolve inflammation by proteolysis of cytokines and chemokines and protection from antiproteases.